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Human masseter motor unit behaviour McMillan, Anne Sinclair 1989

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HUMAN MASSETER MOTOR UNIT BEHAVIOUR by ANNE SINCLAIR MCMILLAN B.D.S. with commendation, The University of Dundee, 1980 F.D.S.,R.C.P.S., Royal College of Physicians and Surgeons of Glasgow, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Oral Biology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1989 ©Anne S i n c l a i r McMillan, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writ ten permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT There i s a dearth of knowledge on the functional organization of the anatomically complex human masseter muscle. Limited physiological studies suggest a functional organization which may d i f f e r s i g n i f i c a n t l y from human limb muscles. The present studies aimed to examine the putative r e l a t i o n s h i p between structure and function i n the human masseter muscle as a basis for understanding function and dysfunction i n human jaw muscles. In the f i r s t experiment single motor unit (SMU) a c t i v i t y was recorded from pairs of recording s i t e s d i s t r i b u t e d throughout the masseter muscle. In each case SMU a c t i v i t y at a chosen location was used as a reference to search for synchronized SMU a c t i v i t y at another selected s i t e . The locations of the needle t i p s were estimated i n 3-dimensions (3-D) by means of an o p t i c a l system, then transferred to 3-D reconstructions derived from Magnetic Resonance images. This approach permitted c a l c u l a t i o n of the li n e a r distances between v e r i f i e d muscle recording s i t e s . The mean separation of the s i t e s from which synchronous SMU a c t i v i t y could be recorded was 8.8±3.4mm. The putative t e r r i t o r i e s had a preferred orientation i n the antero-posterior axis. Motor unit t e r r i t o r i e s were larger than described previously, and appeared to be related to anatomical compartments. The second experiment involved recording a c t i v i t y from s t e r e o t a c t i c a l l y mapped masseter SMUs. In each case, the lowest ii sustainable f i r i n g frequency (LSFF) was reached by slow increases and decreases i n voluntary f i r i n g rate, followed by sustained f i r i n g at the lowest possible rate. Pulse-discrimination and d i g i t a l sampling of consecutive inter-spike i n t e r v a l s (ISIs) were then used to measure LSFF for 2-6 separate occlusal and postural tasks to which each unit contributed. There were s i g n i f i c a n t differences between mean ISIs for the tasks performed by most units, which suggests descending drive to masseter units i s highly task-dependent. There were also regional differences i n unit task s p e c i f i c i t i e s . In the t h i r d paradigm, r e f l e x SMU a c t i v i t y was recorded from units i n the masseter muscle and the i n f e r i o r head of the l a t e r a l pterygoid muscle. Bipolar electrodes fixed to the gingiva near the maxillary canine delivered single pulses of 1ms duration at sub-noxious l e v e l s of int e n s i t y . At constrained f i r i n g frequencies (10, 15Hz), pulses were injected sequentially, with increasing delays, a f t e r preselected spikes. More profound i n h i b i t i o n occurred i n units f i r i n g at 10 than 15Hz. There were s i g n i f i c a n t differences i n masseter i n h i b i t o r y responses when the unit task varied. Reflex i n h i b i t i o n i n masseter and l a t e r a l pterygoid SMUs i s highly frequency-dependent, and also task-dependent i n masseter units. The fourth study involved recording a c t i v i t y from SMUs i n the masseter muscle. A midline load c e l l was fixed to the i n c i s o r teeth and aligned either perpendicular (P) or 3 0 degrees anterior iii (A) to the occlusal plane, without a l t e r i n g jaw po s i t i o n . A r i g i d spike-triggered averaging (STA) paradigm was used to extract the contribution of ind i v i d u a l SMUs to the o v e r a l l force at load c e l l orientations P and A. Spikes preceded or followed by an i n t e r v a l of less than 100ms were rejected p r i o r to averaging. At background b i t e forces from 0.06-8N, the isometric forces apparently developed by ind i v i d u a l units varied randomly with load c e l l orientations, (P range 36.2±19.6mN; A range 38.2±28.4mN). A l l units could be f i r e d slowly with varying degrees of muscle coactivation, i n some instances without contact on the load c e l l . The use of STA as a method for determining SMU tension i n the masseter muscle appears to be task-dependent and i n the presence of coactivation may be inappropriate. The findings c o l l e c t i v e l y indicate the heterogeneous nature of SMU behaviour i n the human masseter muscle which i s consistent with i n t e r n a l muscle compartments based on anatomical features and functional behaviour. There thus appear to be both physiological and anatomical substrates for d i f f e r e n t i a l motor control of selected regions of the human masseter muscle. iv TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS V LIST OF FIGURES ix LIST OF TABLES x i i i ACKNOWLEDGEMENT xiv INTRODUCTION 1 1. REVIEW OF THE LITERATURE 2 A. ANATOMY OF THE MAJOR JAW MUSCLES 2 I. Masseter 4 II. Temporalis 7 II I . Medial Pterygoid 9 IV. Lateral Pterygoid 11 V. Digastric 13 VI. Functional Implications of Pinnation 15 VII. Summary 17 B. FIBRE TYPING OF THE MAJOR JAW MUSCLES 19 I. Masseter 21 II. Temporalis 2 3 II I . Medial Pterygoid 25 IV. Lateral Pterygoid 2 6 V. Digastric 26 VI. Implications of Muscle Fibre D i s t r i b u t i o n . . 27 VII. Summary 2 8 C. MOTOR UNIT BEHAVIOUR OF THE JAW MUSCLES 3 0 I. Motor Unit Recording Techniques 3 0 a. Bipolar 31 b. Monopolar 31 c. Concentric 3 2 d. Single-Fibre 33 e. Macro-EMG 3 3 f. Summary 3 4 II. Motor Unit T e r r i t o r y 3 5 a. Motor Unit D i s t r i b u t i o n 3 6 b. Compartmentalization of Muscles 37 I. Neuromuscular 37 II. S e r i a l 39 c. Electrophysiological Studies 40 d. Functional Implications 42 e. Summary 44 III. Motor Unit A c t i v i t y 4 6 a. Motor Unit Recruitment 4 8 b. Interaction of Motor Unit F i r i n g Rate and Recruitment 52 c. Summary. 56 v PAGE IV. Reflex Behaviour... 57 a. Animal Studies 60 b. Human Studies 62 c. Summary 64 V. Motor Unit Twitch Tension 66 a. Spike-Triggered Averaging Technique... 67 b. Biomechanical Linkage of the Jaw 70 c. E f f e c t s of Muscle Architecture 72 d. E f f e c t s of Muscle Coactivation 76 e. Summary 77 11. STATEMENT OF THE PROBLEM 79 GENERAL METHODS 82 A. SINGLE MOTOR UNIT RECORDING 8 2 I. Technique 82 II. E f f e c t of Bandpass-Filtering 84 B. STEREOTACTIC LOCATION OF NEEDLE ELECTRODE RECORDING SITES 91 I. Morphological Application 91 II. Magnetic Resonance Imaging Technique 93 III. Development of a Stereotactic Method 94 IV. The Position of Needle Electrode Tip Relative to Anatomical Landmarks 95 V. Error of Measurement 100 VI. Application i n vivo 103 SPECIFIC STUDIES (Methods and Results) 1 0 9 A. TERRITORIAL MAPPING OF MOTOR UNITS IN THE MASSETER MUSCLE. . 109 I. Single Motor Unit Recording Technique 109 II. Selection of Muscle Recording Sites I l l III. Location of Electrode Recording Sites I l l IV. Data Analysis and Display 113 V. Results 115 a. D i s t r i b u t i o n of Triggering Electrode Sites within the Muscle 115 b. Distances between Paired Needle Electrode Recording Sites 119 c. Orientation of Putative Motor Unit T e r r i t o r i e s 119 B. MOTOR UNIT TASK PROFILES IN DIFFERENT REGIONS OF THE MASSETER MUSCLE 119 1. Single Motor Unit Recording Method 119 a. Technique 119 b. Task P r o f i l e s 120 c. Inter-Spike Interval Measurement 12 2 vi PAGE II. Location of Electrode Recording Sites 124 II I . Data Analysis and Display 124 IV. Results 125 a. D i s t r i b u t i o n of Recording Sites within the Muscle 125 b. Relationship of Single Motor Unit Task P r o f i l e s to S p e c i f i c Regions of the Muscle 12 5 I. Single Motor Unit Waveform Character 12 5 II . Unit Activation Strategies with and without Tooth Contact 128 III. S p e c i f i c Unit A c t i v a t i o n Strategies 13 0 c. Task-related Single Motor Unit Inter-Spike Intervals 13 3 C. MOTOR UNIT INHIBITORY REFLEX BEHAVIOUR IN THE MASSETER AND LATERAL PTERYGOID MUSCLES 141 I. Single Motor Unit Recording technique 141 a. Masseter 141 b. Lateral Pterygoid 141 II. Intraoral Stimulation 143 III. Reflex Paradigm 144 IV. Data Analysis and Display 14 6 V. Results 146 a. E f f e c t of F i r i n g Frequency 14 6 I. Masseter 14 6 II . Lateral Pterygoid 150 b. E f f e c t s of Different Motor Unit Activation Strategies on Reflex I n h i b i t i o n 155 D. TWITCH TENSION MEASUREMENTS OF MOTOR UNITS IN THE MASSETER MUSCLE 160 I. Force Transducer C a l i b r a t i o n 160 II. Bite-Force and Single Motor Unit Recordings 165 III. Data Sampling... 169 IV. Analysis of Biteforce 170 V. Biomechanical Analysis 172 VI. Results 173 a. Spike-Triggered Measured Tensions 173 b. Spike-Triggered Torque Measurements... 175 c. E f f e c t s of Muscle Coactivation 175 D I S C U S S I O N 180 1. GENERAL METHODS 180 A. SINGLE MOTOR UNIT RECORDING 180 B. STEREOTACTIC LOCATION OF THE NEEDLE ELECTRODE RECORDING SITES 182 vii PAGE I I . S P E C I F I C S T U D I E S 186 A. MOTOR UNIT TERRITORIES IN THE MASSETER MUSCLE 186 B. MOTOR UNIT TASK PROFILES IN DIFFERENT REGIONS OF THE MASSETER MUSCLE 193 C. MOTOR UNIT INHIBITORY REFLEX BEHAVIOUR IN THE MASSETER AND LATERAL PTERYGOID MUSCLES 2 08 D. MOTOR UNIT TWITCH TENSIONS IN THE MASSETER MUSCLE 218 I I I . GENERAL CONCLUSIONS 224 I V . FUTURE D I R E C T I O N S 229 BIBLIOGRAPHY 231 viii LIST OF FIGURES PAGE Figure 1 Single motor unit recording technique i n the masseter muscle 83 Figure 2 Ef f e c t s of bandpass-filtering a single motor unit EMG signal 85 Figure 3 Measurement of needle electrode i n s e r t i o n along a medio-lateral t r a j e c t o r y within the masseter muscle. 87 Figure 4 Changes i n a single motor unit EMG signal according to the pos i t i o n of the electrode recording t i p 88 Figure 5 Variations i n voltage r a t i o s of four single motor unit EMG signals with changes i n the pos i t i o n of the electrode 90 Figure 6 A reference g r i d attached to a b i t e - f o r k . . . 96 Figure 7 Relationship of the reference g r i d to o r o - f a c i a l landmarks and the Magnetic Resonance imaging plane of section 98 Figure 8 Ax i a l spin echo Magnetic Resonance image... 99 Figure 9 Metrograph and Magnetic Resonance-generated reference squares 101 Figure 10 Validation of ste r e o t a c t i c needle electrode placement 104 Figure 11 Location of a needle electrode recording s i t e within the ri g h t masseter muscle 106 Figure 12 Changes i n single motor unit waveform shape due to needle electrode movement within the masseter muscle 107 Figure 13 Technique for single motor unit t e r r i t o r i a l mapping i n the masseter muscle 110 Figure 14 Masseter muscle recording regions 112 ix PAGE Figure 15 The location of two paired needle electrode recording t i p s within the masseter muscle 114 Figure 16 D i s t r i b u t i o n by region of 32 single motor unit t r i g g e r i n g s i t e s i n the r i g h t masseter muscle 116 Figure 17 Distance between paired recording s i t e s i n the r i g h t masseter muscle . 117 Figure 18 Axes of orientation of single motor unit t e r r i t o r i e s i n the masseter muscle 118 Figure 19 Single motor unit inter-spike i n t e r v a l measurement at the lowest sustainable f i r i n g frequency 123 Figure 20 D i s t r i b u t i o n by region of 50 single motor units recorded i n the right masseter muscle 126 Figure 21 Consistency of a single motor unit waveform for d i f f e r e n t a c t i v a t i o n strategies 127 Figure 22 D i s t r i b u t i o n by muscle region of dental and dental/non-dental single masseter motor units 129 Figure 23 Motor unit task p r o f i l e s i n d i f f e r e n t regions of the masseter muscle 131 Figure 24 Single motor unit recording technique i n the l a t e r a l pterygoid muscle 142 Figure 25 EMG response from a single l a t e r a l pterygoid motor unit when a controlled i n t r a o r a l e l e c t r i c a l stimulus i s delivered 145 Figure 26 Raster display of spikes and inter-spike i n t e r v a l s for a single masseter motor unit f i r i n g at a controlled prestimulus f i r i n g frequency of 10±2Hz 147 Figure 27 Raster display of spikes and inter-spike i n t e r v a l s for a single masseter motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 15±2Hz 148 x PAGE Figure 28 Peristimulus time histograms for three masseter single motor units 149 Figure 29 Raster display of spikes and inter-spike i n t e r v a l s for a single l a t e r a l pterygoid motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 10±2Hz 152 Figure 30 Raster display of spikes and inter-spike i n t e r v a l s for a single l a t e r a l pterygoid motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 15±2Hz.... 153 Figure 31 Peristimulus time histograms for three l a t e r a l pterygoid single motor units 154 Figure 32 Raster displays of spikes and inter-spike i n t e r v a l s for a single masseter motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 10±2Hz, activated by three d i f f e r e n t tasks, and subject to constant i n t r a o r a l e l e c t r i c a l s t i m u l i 156 Figure 33 Peristimulus time histograms for a single masseter motor unit activated by three d i f f e r e n t tasks 159 Figure 34 Method of applying loads to the force transducer 161 Figure 35 C a l i b r a t i o n of the biteforce-transducer.... 162 Figure 3 6 Asymmetrical loading of the transducer 163 Figure 37 I n c i s a l b i teforce transducer 166 Figure 38 A mid-sagittal p r o f i l e of the s k u l l showing the projection of the r i g h t a r t i c u l a r condyle, the r i g h t masseter muscle, the f i r s t molar and i n c i s o r teeth, the functional occlusal plane, and the two possible orientations, P and A, of the force transducer 168 Figure 39 An example of a spike-triggered force recording 171 xi PAGE F i g u r e 40 Spike-triggered averages of the load c e l l output (STMTs) recorded at transducer orientations P and A 174 F i g u r e 41 Peak STMTs measured at transducer orientation P plotted against peak STMTs measured at orientation A, for 24 single motor units 17 6 F i g u r e 42 STTORs calculated from P-STMTs plotted against STTORs calculated from A-STMTs, for 24 single motor units 177 F i g u r e 43 STMTs for a single motor unit recorded at transducer orientations P and A with and without antagonistic muscle co-activation 178 F i g u r e 44 Internal anatomy of the human masseter muscle 190 F i g u r e 45 Raster display of spikes and inter-spike i n t e r v a l s for a single masseter motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 10±2Hz, with no exteroceptive s t i m u l i 213 xii LIST OF TABLES PAGE T a b l e 1. Motor unit a c t i v a t i o n strategies 121 T a b l e 11. Single motor unit behavioural p r o f i l e s i n d i f f e r e n t regions of the masseter muscle... 135 T a b l e 111. Human muscle motor unit mean inter-spike i n t e r v a l s recorded at the lowest sustainable f i r i n g frequency 200 xiii ACKNOWLEDGEMENT I wish to express my thanks to my supervisor Dr Alan Hannam, whose i n s p i r a t i o n and s c i e n t i f i c acumen made t h i s project such a rewarding accomplishment. I would l i k e to thank Drs B.H. Bressler and A.A. Eisen for t h e i r counsel during the development of the thesis proposal and at various stages during the project. I am indebted to Ms Joy Scott and Mr Bruce S i n c l a i r who so generously shared t h e i r computing expertise. I am grateful to Dr Donald Brunette and Ms Lisa Khan for t h e i r s t a t i s t i c a l advice. Special thanks to my l o y a l band of experimental subjects for t h e i r unstinting support and enthusiasm. The author was supported f i n a n c i a l l y by a Medical Research Council Fellowship and an I.W. Killam Predoctoral Fellowship. The project was supported by the Medical Research Council. xiv INTRODUCTION The motor unit (MU) represents the basic functional quantum of muscle action and as such, knowledge of i t s functional and anatomical organization i s p i v o t a l to the understanding of the neural control of m o t i l i t y . Motor unit t e r r i t o r i e s i n many human and animal muscles are quite extensive, with s i g n i f i c a n t intermingling of MUs being usual. This f a c i l i t a t e s smooth summation of asynchronously f i r i n g units as muscle force increases. In the masseter muscle however, t e r r i t o r i e s are reported to be small, r e l a t i v e l y homogenous e n t i t i e s which may be related to functional or anatomical compartments, or both. I t i s however l i k e l y that human masseter MU t e r r i t o r i e s have been underestimated due to inherent weaknesses in the i n d i r e c t experimental techniques employed i n t h i s complex muscle. In the human masseter muscle there are seeming va r i a t i o n s i n voluntary and r e f l e x SMU behaviour although how t h i s relates to present putative theories of compartmentalization i s uncertain. It does nonetheless indicate a potential capacity for regional s p e c i a l i z a t i o n which has important implications for the neural control of the muscle. The uncertainty underlying functional and anatomical interactions i n the human masseter also impacts on studies aimed at measuring mechanical contributions of MUs to t o t a l muscle force, because putative v a r i a t i o n s i n regional muscle 1 behaviour may d i s t o r t the r e s u l t s . I t would therefore be useful to develop means of investigating MU t e r r i t o r y and voluntary and r e f l e x MU behaviour i n known regions of the human masseter muscle i n order to c l a r i f y the rela t i o n s h i p between anatomical form and function, and ultimately to comment on the neural control of t h i s complex muscle. 1. Review of the Literature A. ANATOMY OF THE MAJOR JAW MUSCLES A muscle has been described as consisting of an array of fi b r e s organized i n a geometrical pattern between two simple or complex surfaces of connective tiss u e (Gans and Bock, 1965). The organization of muscle f i b r e s i s p i v o t a l to the way i n which muscles function (Gans and de Vree, 1987) . I f the muscle i s small i t s constituent f i b r e s may l i e i n p a r a l l e l arrays r e l a t i v e to the muscle l i n e of action, and be enveloped i n connective tissue epimysium to form a discrete e n t i t y (Loeb and Gans, 1986). But most muscles are more complex and even at the macroscopic l e v e l they are divided by connective tissue perimysium into subunits or " f a s c i c l e s " which i n turn are surrounded by connective tissue endomysium (Loeb and Gans, 1986). The inter n a l architecture of these f a s c i c l e s i s frequently complex (Baron and Debussy, 1979; Gaspard, 1974). Within f a s c i c l e s , muscle f i b r e s may l i e in p a r a l l e l , or i n a pinnate pattern (Gans and Bock, 1965; Loeb and Gans, 1986). Examples of p a r a l l e l muscles are the human soleus, 2 biceps, and thyrohyoid muscles (Gans, 1982) . Muscle f i b r e s i n the cat biventer c e r v i c i s , splenius, and semitendinosus muscles appear to l i e i n p a r a l l e l , but they are separated at various points along the path from t h e i r o r i g i n to i n s e r t i o n by connective tissue tendinous i n s c r i p t i o n s which divide these muscles into s e r i a l compartments (Armstrong et a l , 1988; Richmond and Armstrong, 1988; Bodine et a l , 1982). In pinnate muscles the f i b r e s are inserted obliquely into a number of connective t i s s u e tendons or aponeuroses, and thus l i e at an angle to the d i r e c t i o n of force generation (Gans and Bock, 1965). There are various degrees of pinnation: i n the unipinnate flexor p o l l i c i s longus muscle fi b r e s l i e at constant angles between the surfaces of o r i g i n and i n s e r t i o n ; i n the bipinnate rectus femoris muscle f i b r e s contact on both sides of connective tiss u e tendons; i n the multipinnate d e l t o i d and masseter muscles f i b r e s run in alternating directions between interleaved tendinous sheets (Gans, 1982). Muscle force generation i s reported to be affected by internal muscle architecture (Gans and de Vree, 1987; Weijs and H i l l e n , 1985). Muscle cross-sectional s i z e i s important i n the generation of muscle force (Weijs and H i l l e n , 1985) . The t o t a l cross-section of a l l f i b r e s of a muscle at a known muscle length has been described as the physiological cross-sectional area ( P C S ) (Weber, 1846). The PCS of a muscle i s markedly increased by muscle f i b r e pinnation, thus the potential for force generation i s increased (Gans and de Vree, 1987; Gans, 1982; Gans and Bock, 1965). 3 The anatomy of a muscle and i t s in t e r n a l a r c h i t e c t u r a l design appear to have important implications for the motor performance of muscles therefore a description of jaw muscle macroscopic anatomy w i l l be presented, with p a r t i c u l a r emphasis on inter n a l architecture. 1. Masseter The human masseter muscle i s a thick rectangular muscle which arises from the zygomatic arch and inserts into the l a t e r a l aspect of the mandibular ramus. The muscle i s comprised macroscopically of at least two major parts (Du Brul, 1980). A s u p e r f i c i a l part arises as strong tendinous f i b r e s from the lower border of the zygomatic arch which extend downwards and backwards to the angle of the mandible where they ins e r t as tendinous and fleshy attachments. The fi b r e s of the deep part of the muscle aris e from the entire length of the zygomatic arch, and extend down v e r t i c a l l y to inse r t on to the ramus as far superiorly as the coronoid process. The deep part of the muscle tends to fuse with the superior f i b r e s of the temporalis muscle. A n t e r i o r l y the s u p e r f i c i a l and deep parts merge, whereas p o s t e r i o r l y they are separated by loose connective t i s s u e . Baron and Debussy (1979) have located up to f i v e f a s c i c l e s within the muscle and suggest that the muscle may be separated into three major parts rather than the two based on gross anatomical features. The muscle i s multipinnate, and has f i v e major, obliquely orientated connective tissue tendons which appear to separate the muscle into putative 4 compartments (Schumacher, 1982; 1961). The PCS of the masseter muscle i s greater than that of the medial and l a t e r a l pterygoid muscles but less than the temporalis muscle (Hannam and Wood, 1989; Weijs and H i l l e n , 1985). The PCS varies s i g n i f i c a n t l y with c r a n i o f a c i a l type being greatest i n subjects with brachiocephalic s k u l l s , short f a c i a l heights, and small gonial angles (Weijs and H i l l e n , 1984b). The masseter muscle f i b r e length (2.22cm) and weight are less than the temporalis muscle but greater than the medial and l a t e r a l pterygoid muscles (Weijs and H i l l e n , 1985). The muscle i s a powerful jaw elevator due to i t s f i b r e orientation r e l a t i v e to the functional occlusal plane (Hannam and Wood, 1989). The deep part of the muscle i s also involved i n jaw retrusion and l a t e r a l jaw movements (Brand and Isselhard, 1986; Du Brul, 1980). In non-human primates the masseter muscle has both s u p e r f i c i a l and deep layers (Schumacher, 1982). The pinnate pattern and connective tiss u e tendon d i s t r i b u t i o n are s i m i l a r to those in humans. However, the connection between the f i b r e s of the deep masseter and temporalis muscle i s much more extensive and takes the form of a separate muscle e n t i t y : the zygomaticomandibularis muscle (Schumacher, 1982). The f i b r e s of the primate masseter muscle are more v e r t i c a l l y aligned than i n humans, therefore jaw protrusion i s more limited. The masseter muscle of carnivores i s multipinnate with f i v e connective tissue tendons arranged i n a s i m i l a r form to humans (Schumacher, 1982). The muscle i s divided into a s u p e r f i c i a l layer with f i b r e s running obliquely backwards from the zygomatic arch, 5 a central layer with v e r t i c a l f i b r e orientation, and a deep layer where the f i b r e s almost l i e horizontal (Schumacher, 1982). In herbivores the masseter muscle i s also multipinnate with up to f i v e i n t e r n a l connective tiss u e septa, as in humans (Schumacher, 1982). Herring et a l (1989) have suggested anatomical compartmentalization within the pig masseter muscle based on the presence and d i s t r i b u t i o n of these connective t i s s u e tendons and aponeuroses. However, the spectrum of f i b r e orientations within the pig masseter muscle prevents d e f i n i t i o n of discrete compartments based on f i b r e orientation alone (Herring et a l , 1989). Nonetheless, anatomical compartments tend to be formed by obliquely orientated connective tissue tendon s t r i p s i n the anterior part of the muscle, and by p a r a s a g i t t a l connective tissue planes i n the posterior part (Herring, 1980). In the anterior part of the muscle f i b r e s are orientated v e r t i c a l l y and are u t i l i z e d in jaw closure, whereas in the posterior region the f i b r e s have a more horizontal alignment and are involved i n l a t e r a l jaw movements and anterior t r a n s l a t i o n of the condyle (Herring et a l , 1989; 1979; Herring and Wineski, 1986; Herring, 1980; Strom et a l , 1986). The multipinnate rabbit masseter muscle i s comprised of major s u p e r f i c i a l and deep parts, and l i k e the pig masseter appears to be anatomically compartmentalized by means of connective tissue aponeuroses (Weijs and Dantuma, 1981) . The s u p e r f i c i a l part i s subdivided into medial and l a t e r a l areas and the deep part into anterior and posterior areas (Weijs and Dantuma, 1981). The PCS 6 of the rabbit masseter muscle i s the largest of a l l rabbit jaw muscles (Weijs and Dantuma, 1981). The masseter muscle i s p a r t i c u l a r l y well-developed i n rodents, where i t has the largest PCS of a l l rodent jaw muscles (Hiiamae, 1971; Schumacher, 1982). Although the rat masseter i s divided into two major parts, the s u p e r f i c i a l one i s large and unipinnate whereas i n the deep part the f i b r e orientation i s more p a r a l l e l (Hiiamae, 1971). Neuromuscular spindles are common i n the masseter muscle of humans, non-human primates, carnivores, herbivores, and rodents (Dmytruk, 1974; Smith and Macarian, 1967). 11. Temporalis The human temporalis muscle i s fan-shaped and arises from a wide area over the temporal fossa on the l a t e r a l surface of the s k u l l , from the f r o n t a l process of the zygoma, and from the region of the infratemporal crest (Du Brul, 1980). Additional f i b r e s a r i s e from an aponeurosis attached to the inner surface of the temporal f a s c i a . The muscle f i b r e s converge between the zygomatic arch and the l a t e r a l surface of the s k u l l to i n s e r t on to the coronoid process of the mandible, and into a f l a t tendinous region which extends from the coronoid process superiorly into the muscle body (Du Brul, 1980; Schumacher, 1982). Fibres from the temporal aspect of the muscle ins e r t mesially into t h i s tendinous region, and f i b r e s a r i s i n g from the temporal f a s c i a i n s e r t into the l a t e r a l aspect. The anterior f i b r e s of the muscle are orientated 7 v e r t i c a l l y , whereas i n the mid part the f i b r e s become increasingly oblique i n orientation and f i n a l l y h o r i z o n t a l l y placed i n the posterior region of the muscle (Du Brul, 1980). The muscles' bipinnate f i b r e arrangement i s less complex than that of the masseter and medial pterygoid muscles. There are s u p e r f i c i a l and deep connective tiss u e tendon sheets which i n s e r t i n the region of the coronoid process and appear to demarcate putative anatomical compartments (Du Brul, 1980; Schumacher, 1982). Two major f a s c i c l e s have been discerned within the muscle (Baron and Debussy, 1979). The PCS and weight of the temporalis muscle are greater than a l l other jaw muscles, and unlike the masseter and medial pterygoid muscles the PCS i s not affected appreciably by differences i n c r a n i o f a c i a l form (Weijs and H i l l e n , 1984a; Weijs and H i l l e n , 1984b). The muscle f i b r e s are longer than those of other masticatory muscles (Weijs and H i l l e n , 1984a). The role of the temporalis muscle i s to elevate and retrude the mandible (Brand and Isselbrand, 1986). In non-human primates the s u p e r f i c i a l layer of the temporalis muscle i s more strongly developed, the PCS i s larger, and the pinnation pattern i s more complex than i n humans (Schumacher, 1982) . The temporalis muscle of carnivores has a s u p e r f i c i a l and deep layer attached to a large in t e r n a l tendon (Schumacher, 1982) . The PCS i s much greater than i n a l l other carnivore jaw muscles (Schumacher, 1982). 8 In herbivores the temporalis muscle i s separated into s u p e r f i c i a l and deep parts by an inter n a l connective tiss u e tendon (Schumacher, 1982; Strom et a l , 1986; Weijs and Dantuma, 1981). In rabbits the s u p e r f i c i a l part of the muscle i s less well developed than the deep part (Weijs and Dantuma, 1981). The PCS of the rabbit temporalis muscle i s greater than the l a t e r a l pterygoid and d i g a s t r i c muscles but smaller than the masseter and medial pterygoid muscles, whereas i t s muscle f i b r e length i s si m i l a r to that of the masseter and l a t e r a l pterygoid muscles (Weijs and Dantuma, 1981). The development of the rodent temporalis muscle has been reported to be s i m i l a r to that of the herbivore, with s u p e r f i c i a l and deep muscle layers, and f i b r e orientations which become progressively more horizontal i n the posterior region of the muscle (Schumacher, 1982) . An alte r n a t i v e form of separation into anterior and posterior parts has been observed i n the rat temporalis muscle (Hiiamae, 1971). Neuromuscular spindles have been demonstrated i n the temporalis muscle of humans, non-human primates, herbivores, carnivores and rodents (Dmytruk, 1974; Smith and Macarian, 1967). 111. Medial pterygoid The human medial pterygoid muscle i s considered to be the anatomical and functional counterpart of the masseter muscle (Du Brul, 1980). I t arises from the pterygoid fossa and the medial aspect of the l a t e r a l pterygoid plate. The muscle i s inserted into 9 the medial aspect of the angle of the mandibular ramus. Baron and Debussy (1979) have detected three f a s c i c l e s within the muscle. The muscle has a complex pinnation pattern incorporating s i x intern a l connective tissue tendons, some of which posses multiple tendon ends (Schumacher, 1982; 1961). The muscle PCS varies with c r a n i o f a c i a l type, and i s greater than the l a t e r a l pterygoid muscle but i t i s smaller than the temporalis and masseter muscles (Weijs and H i l l e n , 1984a,b). Its muscle f i b r e s are shorter (1.39cm) than those of the l a t e r a l pterygoid, temporalis and masseter muscles (Weijs and H i l l e n , 1984a). The complex architecture of t h i s muscle permits the development of large forces in a small space, and also allows d i f f e r e n t i a l i n t e r n a l s h i f t i n g of layers of muscle f i b r e s (Schumacher, 1982). The muscle i s a mandibular elevator although i t i s biomechanically less e f f e c t i v e than the masseter muscle as i t i s less a n t e r i o r l y i n c l i n e d r e l a t i v e to the functional occlusal plane (Hannam and Wood, 1989). In non-human primates the medial pterygoid muscle has a complex multipinnate structure. Seven connective tiss u e tendons have been described which i n turn convolute to form a secondary pinnation pattern (Schumacher, 1982). In primates, the more v e r t i c a l f i b r e orientation of the muscle permits jaw r e t r a c t i o n (Schumacher, 1982). The medial pterygoid muscles of carnivores and herbivores are multipinnate. Five tendons have been described in the medial pterygoids of carnivores and four i n herbivores. In the rabbit 10 medial pterygoid the PCS i s greater than that of the l a t e r a l pterygoid and temporalis muscles but less than the masseter muscle (Weijs and Dantuma, 1981). The muscle f i b r e s are shorter than other rabbit jaw muscles. In rodents, f i v e connective t i s s u e tendons have been demonstrated, although the pinnation pattern i s less complex than in herbivores (Schumacher, 1982). Within the rat medial pterygoid, Hiiamae (1971) found p a r t i a l s p l i t t i n g of the muscle by connective tiss u e aponeuroses. Neuromuscular spindles have been found i n the medial pterygoid muscle of humans, non-human primates, carnivores, and herbivores (Dmytruk, 1974; Smith and Macarian, 1967). IV. Lateral pterygoid The human l a t e r a l pterygoid muscle has two heads: the larger, i n f e r i o r head arises from the l a t e r a l aspect of the l a t e r a l pterygoid plate; the superior head arises medial to the infratemporal crest on the infratemporal surface of the greater wing of the sphenoid bone (Du Brul, 1980). The anterior f i b r e s of the superior head intermingle with the deep f i b r e s of the temporalis muscle at t h e i r o r i g i n on the roof of the infratemporal fossa (Widmalm et a l , 1987). Both heads of the l a t e r a l pterygoid muscle attach to a fovea on the anterior surface of the condylar neck, with only part of the superior head i n s e r t i n g i n the temporomandibular j o i n t a r t i c u l a r d i s c and capsule (Du Brul, 1 9 8 0 ; Widmalm et a l , 1987). The two heads of the muscle are separated by a variable gap a n t e r i o r l y but tend to fuse around the j o i n t a r t i c u l a t i o n . The fi b r e s within the muscle l i e i n p a r a l l e l arrays (Schumacher, 1980). Widmalm et a l (1987) have suggested that the i n f e r i o r head may be multipinnate at i t s o r i g i n , but Schumacher (1980) did not detect any tendons within the muscle. Baron and Debussy (1979) have described only one f a s c i c l e i n each of the two heads of the muscle. The l a t e r a l pterygoid has a smaller PCS than other jaw muscles although i t s weight i s greater than that of the medial pterygoid muscle (Weijs and H i l l e n , 1984a). Like the temporalis muscle i t s PCS i s not affected by c r a n i o f a c i a l type (Weijs and H i l l e n , 1984a,b). The l a t e r a l pterygoid muscle f i b r e s are longer (2.42cm) than those of the medial pterygoid but shorter than those of the temporalis muscle (Weijs and H i l l e n , 1984a). A small number of neuromuscular spindles have been demonstrated i n both fo e t a l and adult human l a t e r a l pterygoid muscles ( G i l l , 1971). The i n f e r i o r head of the muscle permits t r a n s l a t i o n of the condyle, thus i t acts i n jaw protrusion (Du Brul, 198 0) . The i n f e r i o r head has also been implicated i n jaw opening (Widmalm et a l , 1987), i . e . i t may act as a synergist or antagonist to jaw closi n g muscles. The superior head i s active during tooth clenching (Du Brul, 1980). There are two heads present i n the l a t e r a l pterygoid of non-human primates, although both heads are smaller than t h e i r human counterparts (Schumacher, 1982). McNamara (1973) considers the primate l a t e r a l pterygoid to be two d i s t i n c t muscles, despite s i m i l a r muscle o r i g i n and insertions to the human muscle. In 12 biomechanical terms the superior head i s suited to jaw closure, whereas the f i b r e orientation i n the i n f e r i o r head i s more appropriate for jaw opening i n both human and non-human primates (Grant, 1973; McNamara, 1973). In carnivores, the l a t e r a l pterygoid i s small and sometimes absent (Schumacher, 1982). The f i b r e s are normally p a r a l l e l within the muscle. The l a t e r a l pterygoid of herbivores has two areas of o r i g i n s i m i l a r to man, and apart from small tendon ends has no inter n a l tendons (Schumacher, 1982) . In the pig the superior head i s attached to the a r t i c u l a r disc, and the i n f e r i o r head to the condylar neck (Strom et a l , 1986). The muscle f i b r e s of the rabbit l a t e r a l pterygoid are longer than i n other rabbit jaw elevator muscles (Weijs and Dantuma, 1981). The p a r a l l e l - f i b r e d l a t e r a l pterygoid muscle i n rodents i s sometimes larger than that i n herbivores (Schumacher, 1982), although i n rats i t i s present only as a small band of muscle fi b r e s (Hiiamae, 1971). V. Dig a s t r i c The human d i g a s t r i c muscle consists of two fleshy b e l l i e s connected by a round intermediate tendon. The posterior b e l l y arises from the mastoid notch of the temporal bone; the anterior b e l l y has tendinous and fleshy attachments to the d i g a s t r i c fossa on the i n f e r i o r aspect of the mandible near the midline; the intermediate tendon i s fixed to the hyoid bone by a loop of c e r v i c a l f a s c i a and extends p o s t e r i o r l y as f a r as the sternomastoid muscle (Du Brul, 1980; Widmalm et a l , 1988). The posterior b e l l y i s mainly round i n cross-section and tapers a n t e r i o r l y into the tendon (Du Brul, 1980) . The anterior b e l l y i s smaller than the posterior b e l l y and has i t s largest cross-sectional area i n the region d i s t a l to the mandibular second premolar and mesial to the second molar (Widmalm et a l , 1988). The f i b r e s of the muscle l i e i n p a r a l l e l within each b e l l y . No neuromuscular spindles have been found in human or non-human primate d i g a s t r i c muscles (Dmytruk, 1974). The two b e l l i e s acting i n concert a s s i s t i n jaw opening when the hyoid bone i s fixed by the infrahyoid muscles (Du Brul, 1980; Scott and Dixon, 1972). The muscle also a s s i s t s i n jaw retrusion (Brand and Isselhard, 1986). With the jaws approximated, the muscle elevates the hyoid bone during such acts as swallowing (Scott and Dixon, 1972). The human d i g a s t r i c muscle i s the only muscle of mastication not t o t a l l y innervated by the mandibular d i v i s i o n of the trigeminal nerve. The posterior b e l l y i s supplied by a branch of the f a c i a l nerve. In the rabbit, the d i g a s t r i c muscle i s s i n g l e - b e l l i e d and unipinnate (Muhl, 1982; Weijs and Dantuma, 1981). This form of pinnation enhances the range of action of the rabbit d i g a s t r i c muscle (Muhl, 1982). The muscle has the smallest PCS of the jaw muscles, and has the longest f i b r e length (1.28cm) (Weijs and Dantuma, 1981). 14 The rat d i g a s t r i c has the appearance of two muscles because of the discontinuity of f i b r e s within i t s two b e l l i e s (Hiiamae, 1971). VI. Functional Implications of Pinnation Differences i n muscle f i b r e architecture have major influences upon muscle function (Gans and Bock, 1965; Gans, 1982; Herring et a l , 1979). P a r a l l e l - f i b r e d muscles are reported to act i n translatory movements, whereas pinnate muscles rotate about t h e i r o r i g i n thereby a l t e r i n g the angle of muscle f i b r e pinnation as they contract (Gans, 1982). The arrangement of muscle f i b r e s i n p a r a l l e l appears to permit maximum excursion of the muscle in s e r t i o n with e f f i c i e n t force production during contraction (Gans and Bock, 1965). However, Gans and Bock (1965) consider t h e o r e t i c a l l y that a pinnate muscle should have a greater range of excursion than a p a r a l l e l - f i b r e d muscle provided the muscles have s i m i l a r f i b r e lengths. A number of advantages have been c i t e d for pinnate muscles: a greater number of f i b r e s may be inserted into a tendon; a pinnate muscle may be located i n a r e s t r i c t e d anatomical space; connective t i s s u e tendons may contract i n d i f f e r e n t directions dependent on d i f f e r e n t i a l muscle f i b r e contraction; and, pinnate arrangements may compensate for muscle f i b r e loss with age thus retaining force generating capacity i n atrophic muscles (Gans, 1982; Gans and Bock, 1965). The functional interpretation of muscle pinnation patterns may 15 be ascribed to two broadly c o n f l i c t i n g hypotheses: 1) that a l l f i b r e s of a muscle act i n concert, and 2) that muscle f i b r e s contract d i f f e r e n t i a l l y (Herring et a l , 1979). In a multipinnate muscle, s e l e c t i v e contraction of muscle f i b r e groups could conceivably produce a force i n more than one d i r e c t i o n . However, t h i s would require the r e s t r i c t i o n of i n d i v i d u a l motor units (MU) to discrete regions of the muscle. In many muscles, MU f i b r e s are d i s t r i b u t e d widely and cross from one f a s c i c l e to another (Dubner et a l , 1978; English and Weeks, 1984; English and Letbetter, 1982; Gray, 1973), however i n some pinnate muscles, f i b r e s of in d i v i d u a l MUs are r e s t r i c t e d i n t h e i r d i s t r i b u t i o n (Gans, 1982). Herring et a l (1989, 1979) have described the r e s t r i c t i o n of single motor unit (SMU) f i b r e s within i n d i v i d u a l f a s c i c l e s of the multipinnate pig masseter muscle. The multiple connective t i s s u e tendons i n pinnate muscles also appear to segment these muscles into putative compartments (Herring et a l , 1979; Schumacher, 1961; 1982; Weijs and Dantuma, 1981). Overlaid on t h i s anatomical compartmentalization, functional heterogeneity has been demonstrated i n the pig masseter muscle whereby d i f f e r e n t i a l a c t i v a t i o n during mastication permits the muscle to a l t e r i t s l i n e of action according to the p a r t i c u l a r jaw movement being performed during the chewing task (Herring et a l , 1979; 1989) . In the rabbit masseter muscle, d i f f e r e n t i a l a c t i v i t y has been described i n d i f f e r e n t anatomical compartments separated by connective septa (Weijs and van der Wielen Drent, 1983; Weijs and Dantuma, 1981). During mastication, on the jaw working side a wave of a c t i v i t y extends from the deep to the s u p e r f i c i a l parts of the muscle, whereas the converse occurs on the balancing side (Weijs and Dantuma, 1981). English (1985) investigated the p o s s i b i l i t y that a si m i l a r d i v i s i o n of labour to masseter muscle may be found i n the complex pinnated cat limb muscles. The complex l a t e r a l gastrocnemius was compared with the s t r u c t u r a l l y more simple medial gastrocnemius, p l a n t a r i s , and soleus muscles, but there was no evidence to support the d i v i s i o n of labour th e s i s . Functional heterogeneity has been ascribed to the human masseter (Belser and Hannam, 1986; Eriksson et a l , 1984) and temporalis muscles (Wood, 1986). Masseter MUs appear to be capable of a c t i v a t i o n by more than one strategy which suggests changes i n muscle l i n e s of action during function (Eriksson et a l , 1984). However, the relat i o n s h i p of these d i f f e r e n t i a l jaw muscle a c t i v i t y patterns to putative anatomical compartments within the human jaw muscles i s presently unclear. V l l . Summary The major jaw muscles have a complex architecture when compared with the limb and trunk muscles which possess a wide spectrum of ar c h i t e c t u r a l arrangements. The jaw elevator muscles have a multipinnate pattern which serves to increase the muscle cross-section, and thus the potential for force generation. In addition, putative anatomical compartments have been suggested within these muscles based on the presence of multiple internal 17 connective tissue septa. The d i s p o s i t i o n of connective tissue sheets and the r e l a t i o n of muscle f i b r e s to them i s important to the function of these muscles because of the poten t i a l for d i f f e r e n t i a l movement of muscle layers. However, t h i s capacity for regional a c t i v i t y i s dependent on the s p a t i a l r e l a t i o n s h i p of MUs within the muscle. D i f f e r e n t i a l a c t i v i t y has been described within d i f f e r e n t regions of the rabbit and pig masseter muscles which suggests putative functional compartmentalization, however the extent to which t h i s occurs i n the human masseter muscle i s uncertain. At present the functional s i g n i f i c a n c e of complex muscle architecture i n the human jaw elevator muscles i s poorly understood. 18 B. FIBRE TYPING OF THE MAJOR JAW MUSCLES Different types of muscle f i b r e s can be characterized by histochemical techniques which reveal s t r u c t u r a l proteins and a c t i v i t i e s of metabolic enzymes inherent to the f i b r e s . More recently, immunocytochemical determination by immunofluorescent l a b e l l i n g of s t r u c t u r a l proteins has been described (Balice-Gordon and Thomson, 1988; Goldnick and Hodgson, 1986). Histochemical staining for oxidative po t e n t i a l i s considered inaccurate because the metabolic enzymes are unstable (Burke, 1981; Gollnick and Hodgson, 1986). However, a method based on m y o f i b r i l l a r actomyosin adenosine triphosphatase (ATPase) r e a c t i v i t y patterns i s considered to be more r e l i a b l e (Burke, 1981; Gollnick and Hodgson, 1986) . In normal human muscle the standard ATPase reaction at pH9.4 can be used to discern two major types of f i b r e s , v i z . type 1 and type 11 f i b r e s . Three subtypes of type 11 f i b r e s (Types 11A, 11B, 11C) can be i d e n t i f i e d by modification of the standard ATPase reaction (Dubowitz and Brooke, 1973). There are a number of systems of nomenclature used to describe f i b r e types, however they are not r e a d i l y interchangeable because of differences between muscles and species, i n addition to differences i n the histochemical methods of f i b r e typing. A c l a s s i f i c a t i o n based on reviews by Close (1973) and Burke (1981) w i l l be described to aid comparison of histochemical studies and functional correlations which w i l l be reported : 19 Types of Fibre i n Mammalian Skeletal Muscle C l a s s i f i c a t i o n s Ogata & Mori, 1964 white medium red Stein & Padykula, 1962 A B C Engel, 1962; 1970 11 1 11 Romanul, 19 64 1 111 11 Padykula & Gauthier, 1967 white intermediate red Edstrom & Kugelberg, 1968; A C B Henneman & Olson, 1965; Olson & Swett, 1966 Barnard et a l , 1971 FG:fast- SO:slow- FOG:fast twitch twitch twitch white intermediate red Brooke & Kaiser, 1970 11B 1 HA & 11C Corresponding motor unit type Burke et a l , 1973 FF FR S The histochemical properties of muscle f i b r e s d i f f e r between species and also between the limb and jaw muscles (Luschei and Goldberg, 1981; Taylor e l , 1973). Within the jaw muscles of humans major groups of muscle f i b r e s of the same histochemical f i b r e type have been described (Stalberg et a l , 1986) . This i s i n sharp contrast to the mosaic-like arrays of d i f f e r e n t f i b r e types reported to be present i n most normal limb and trunk muscles (Johnson et a l , 1973 ; Stalberg et a l , 1986) . Muscle f i b r e diameter i s frequently related to the histochemical f i b r e type, with type 11B diameter > 11A > 1 i n many animals (Burke, 1981). There are, however, exceptions to t h i s pattern of f i b r e diameters i n human muscles: i n some muscles of females, type 1 f i b r e s are larger than type 11 f i b r e s . In jaw muscles profound differences have been observed between the diameters of type 1 and 11 f i b r e s , and both types are smaller than t h e i r limb counterparts (Eriksson and 20 Thornell, 1983; Ringqvist, 1973a). S i g n i f i c a n t numbers of ATPase intermediate f i b r e s have also been described i n jaw muscles, i n contrast to t h e i r paucity i n limb and trunk muscles (Ringqvist et a l , 1982; Ringqvist, 1973b). Differences i n histochemical p r o f i l e s of muscle f i b r e s have been correlated with t h e i r functional properties (Burke et a l , 1981; Burke et a l , 1971; Edstrom and Kugelberg, 1968). D i s t i n c t groupings of MUs have been described, based upon t h e i r c o n t r a c t i l e c h a r a c t e r i s t i c s , v i z . slow (S) or fast (F) , and re s i s t a n t to fatigue (R) , e a s i l y fatigued (F) or r e s i s t a n t to fatigue (R) . Three classes of MUs have been named according to these c r i t e r i a : "S", slow and fatigue r e s i s t a n t ; "FF", fast and r e a d i l y fatigued; and, "FR", fast and fatigue r e s i s t a n t . Burke (1981) has described a general rela t i o n s h i p between t h i s c l a s s i f i c a t i o n of MU types and the histochemical c l a s s i f i c a t i o n of Brooke and Kaiser (1970) [type 1 , 11A, 11B]. In order to improve our understanding of the relat i o n s h i p between motor unit f i b r e d i s t r i b u t i o n and function i n the masticatory system, the histochemical p r o f i l e s of jaw muscles w i l l be reviewed. 1. Masseter In young, adults humans, type 1 f i b r e s appear to be predominant i n almost a l l regions of the muscle, ranging from 62-72% of the t o t a l f i b r e content (Eriksson and Thornell, 1983; Vignon et a l , 1980). The anterior deep part of the muscle consists 21 of more than 70% type 1 f i b r e s . The posterior s u p e r f i c i a l part of the muscle however, has approximately equal proportions of type 1 and 11 f i b r e s , with type 11B f i b r e s concentrated mainly i n t h i s part of the muscle. Type 1 f i b r e s appear to be larger than type 11B f i b r e s except i n the middle part of the muscle (Eriksson and Thornell, 1983; Ringqvist, 1973a). The cross-sectional f i b r e area of type 1 f i b r e s ranges from 70% i n the posterior s u p e r f i c i a l part of the muscle to 88% i n the anterior deep part, whereas type 11 f i b r e s range from 7 to 21% i n these regions. ATPase intermediate and type 11C f i b r e s represent approximately 9% of the t o t a l f i b r e population. In an adult population with a much greater age range (2 0-87 years), Vignon et a l (1980) demonstrated s l i g h t l y more type 11 than type 1 f i b r e s i n the s u p e r f i c i a l part of the muscle. ATPase intermediate f i b r e s represented 5% of the population in t h i s region. Type 11 f i b r e s were consistently smaller than type 1 f i b r e s . Within the same study, the masseter muscles of a small number of subjects aged less than 13 years were examined. The respective proportions of type 1 and type 11 f i b r e s were si m i l a r i n the s u p e r f i c i a l part of the muscle. Type 11 f i b r e s were smaller than type 1 f i b r e s , and both types were smaller than t h e i r adult counterparts. The cat masseter muscle comprises mainly of type A f i b r e s with few type C f i b r e s (Taylor et a l , 1973), i n contrast to the human masseter muscle. In the pig masseter muscle, Herring et a l (1979) found more type 11 than type 1 f i b r e s , with 65% type 11 f i b r e s i n 22 the anterior part of the muscle and 77% i n the posterior part. In the non-human primate the r e l a t i v e quantities of f i b r e types appear to l i e somewhere between those of the human and the cat. In the monkey masseter muscle, Clark and Luschei (1981) described s l i g h t l y more type FG than type SO f i b r e s with few type FOG f i b r e s , although the data from various muscle parts was pooled. A comparative study of anterior and posterior regions of the non-human primate masseter described type SO as the dominant f i b r e i n the anterior s u p e r f i c i a l part of the muscle, p a r t i c u l a r l y i n females (Maxwell et a l , 1979). There were s i g n i f i c a n t l y more type FG and FOG f i b r e s i n the posterior s u p e r f i c i a l region of the muscle. Type FG f i b r e s were of a larger diameter than type SO f i b r e s i n males, whereas type FG f i b r e s were s i m i l a r or smaller than type SO f i b r e s i n females. This finding d i f f e r e d from the f i b r e diameters found i n cats, where type A f i b r e s were consistently larger than type C f i b r e s (Taylor et a l , 1973). 11. Temporalis In the anterior s u p e r f i c i a l part of the muscle i n young, adults humans, type 1 and 11B f i b r e s appear to be present i n approximately equal proportions, v i z . 47% and 4 5 % respectively, whereas i n the posterior s u p e r f i c i a l part there are more type 11B f i b r e s (57%) than type 1 f i b r e s (40%) [Eriksson and Thornell, 1983]. The deep part of the muscle i s comprised mainly of type 1 f i b r e s (81%). ATPase intermediate and type 11C f i b r e s constitute a s i m i l a r proportion to that found i n the masseter muscle. Type 23 11B f i b r e s are smaller than type 1 f i b r e s throughout the muscle (Eriksson and Thornell, 1983; Vignon et a l , 1980). In the posterior s u p e r f i c i a l part of the muscle, the cross-sectional f i b r e area i s 52% for type 1 f i b r e s compared with 9 0% i n the deep part of the muscle. In the anterior s u p e r f i c i a l part, cross-sectional f i b r e areas appear to be s i m i l a r for both type 1 and type 11 f i b r e s . Vignon et a l (1980) found type 11 f i b r e s to be predominant, however the s p e c i f i c location of the biopsy s i t e was uncertain and could conceivably have been the posterior s u p e r f i c i a l region of the muscle. In a small sample of muscles obtained from subjects aged less than 13 years, s l i g h t l y more type 11 than type 1 f i b r e s were present (Vignon et a l , 1980). In a series of adult denture wearers, Ringqvist ( 1 9 7 4 ) noted that type 11 f i b r e s were smaller than type 1 f i b r e s , and i n some instances these f i b r e s were atrophied. Smaller proportions of type 11 f i b r e s were recorded i n subjects with denture d e f i c i e n c i e s compared with subjects with natural dentitions, and may conceivably have been due to changes i n functional demand (Ringqvist, 1974). In the cat temporalis muscle the majority of f i b r e s were reported to be type A, with no s i g n i f i c a n t regional vari a t i o n s (Taylor et a l , 1973). However, variatio n s i n f i b r e composition have been described i n the temporalis muscle of the non-human primate (Clark and Luschei, 1981; Maxwell et a l , 1979). There are more type SO f i b r e s i n the anterior part of the muscle of both 24 males and females, whereas i n the posterior part there are more type FG f i b r e s . i l l . Medial Pterygoid In the anterior part of the muscle of young, adult humans, type 1 f i b r e s are reported to be the dominant f i b r e type (64%) [Eriksson and Thornell, 1983]. Similar numbers of type 1 and type 11B f i b r e s (44% of each type) are present i n the posterior part of the muscle. ATPase intermediate and type 11C f i b r e s comprise a s i m i l a r proportion of f i b r e s to the masseter and temporalis muscles. Type 1 f i b r e s have a larger diameter than type 11B f i b r e s i n the anterior part of the muscle. Type 1 f i b r e s represent 79% of the cross-sectional f i b r e area i n the anterior part of the muscle and 52% i n the posterior part compared with 16 and 20% respectively for type 11 f i b r e s i n these regions. Vignon et a l (1980) described the muscle f i b r e content as 56% type 11 compared with 37% type 1 and 7% ATPase intermediate f i b r e s , although the s p e c i f i c dispositions of the sample s i t e s within the muscles were unknown. Type 11 f i b r e s were consistently smaller than type 1 f i b r e s . In the cat medial pterygoid the dominant f i b r e was type A (Taylor et a l , 1973), whereas i n the non-human primate the f i b r e content was almost exclusively type SO (Clark and Luschei, 1981). IV. Lateral Pterygoid Type 1 f i b r e s are reported to be the predominant f i b r e type 25 within the muscle (Eriksson et a l , 1981). Up to 70% of the f i b r e s are of the type 1 variety, i n some cases with whole f a s c i c l e s homogenous i n terms of f i b r e type. Type 11B f i b r e s are dominant i n the superior head of the muscle whereas type 1 f i b r e s are dominant i n the i n f e r i o r head. No type 11A f i b r e s were detected in a series of muscle specimens examined by Eriksson et a l (1981). Type 1 f i b r e s were s i g n i f i c a n t l y larger than type 11 f i b r e s . In adolescents s i m i l a r proportions of type 1 and type 11 f i b r e s were observed (Vignon et a l , 1980). Fibres were smaller than those of adult muscle specimens, but type 11 f i b r e s were nonetheless smaller than type 1 f i b r e s . In the non-human primate type SO f i b r e s have been described as the dominant f i b r e type, with no detectable type FOG f i b r e s (Clark and Luschei, 1981). V. Dig a s t r i c Although the anterior and posterior b e l l i e s of the muscle are of d i f f e r e n t embryological o r i g i n , t h e i r f i b r e compositions are d i s t i n c t from the other jaw muscles and more akin to those of trunk and limb muscles (Eriksson et a l , 1982). Type 1, 11A and 11B f i b r e s appear to be present i n s i m i l a r numbers, and are d i s t r i b u t e d evenly throughout the muscle. ATPase intermediate f i b r e s are scarce. Fibre diameters are s i m i l a r for each f i b r e type, although f i b r e diameters are s l i g h t l y larger i n the anterior b e l l y compared with the posterior b e l l y of the muscle. In the d i g a s t r i c muscle of the non-human primate there are 26 s l i g h t l y more type SO than FG f i b r e s (Clark and Luschei, 1981). VI. Implications of Muscle Fibre Type D i s t r i b u t i o n Type 1 f i b r e s appear to be dominant i n the masseter, temporalis, and medial and l a t e r a l pterygoid muscles, p a r t i c u l a r i n c e r t a i n regions of the muscles. This "fibre-type" grouping which seems to be normal i n jaw muscles would be considered pathological i f i t occurred i n the limb or trunk muscles-where i t would be i n d i c a t i v e of denervation and reinnervation (Schwartz et a l , 1976). As MUs are comprised of muscle f i b r e s o f the same histochemical type, i t suggests that MUs in jaw muscles may not be d i s t r i b u t e d throughout the muscle, instead r e s t r i c t e d to cer t a i n regions of the muscle. With the lack of morphological methods available to systematically examine the location of d i f f e r e n t MUs i n l i v e human subjects, i t i s d i f f i c u l t to determine whether groups of muscle f i b r e s of the same type do i n fact belong to the same MU. In animals, glycogen depletion studies may be performed to determine the location of MUs. Herring et a l (1979) have presented work i n the pig masseter muscle which appears to show muscle regions with d i f f e r e n t histochemical compositions. The non-primate temporalis muscle i s also considered to be histochemically compartmentalized (Clark and Luschei, 1981). However, Stalberg et a l (1986) consider that groups o f f i b r e s of consonant histochemical type i n the jaw muscles are most l i k e l y composed of d i f f e r e n t MUs. The dominance of type 1 f i b r e i n s p e c i f i c regions of the jaw muscles does not i n i t s e l f appear to confer "compartmentalization". On the basis of the preponderance of type 1 f i b r e s i n the jaw muscles, one would expect to f i n d a s i g n i f i c a n t number of functional type S MUs. However, i n terms of contraction c a p a b i l i t y the masseter appears to behave as a fast muscle (Yemm, 1977), thus there i s a c o n f l i c t between histochemical findings and functional behaviour. VI. Summary In the major human jaw muscles, with the exception of the d i g a s t r i c muscles, there appear to be s i g n i f i c a n t groups of muscle f i b r e s of the same histochemical type which contrasts with the spectrum of f i b r e types present i n normal human limb muscles. In human jaw elevator muscles and the l a t e r a l pterygoid muscle, type 11 f i b r e s are much smaller than type 1 f i b r e s , i n cont r a d i s t i n c t i o n to that usually found i n trunk and limb muscles. Type 1 f i b r e s appear to be the dominant species i n the masseter, temporalis and medial and l a t e r a l pterygoid muscles. In addition, ATPase-intermediate f i b r e s appear to be r e l a t i v e l y common whereas i n the limb muscles these f i b r e s are rare. Grouping of the fi b r e s of the same type i n various regions of the jaw muscles appears to be a normal finding i n contrast to limb and trunk muscles where i t i s in d i c a t i v e of denervation and reinnervation. The extent to which the f i b r e patterns may be described as histochemical "compartmentalization" i s at present uncertain, and not compelling based on the available evidence. The spe c i a l histochemical 28 features of f i b r e s i n the jaw muscles may be related to special functional demands of the masticatory system. 29 C. MOTOR UNIT BEHAVIOUR IN THE JAW MUSCLES 1. Motor Unit Recording Techniques When the f i b r e s of a MU are activated, they contract almost synchronously, and the action potentials summate so that a compound action potential (CAP) can be recorded. Electromyographic (EMG) recording from MUs are usually made with needle electrodes. The signal obtained by the needle electrode i s the temporal and s p a t i a l summation of action potentials from the MU fib r e s (Stalberg & Antoni, 1980). The c h a r a c t e r i s t i c s of the MU CAP may vary s i g n i f i c a n t l y depending on the anatomical arrangement of the muscle f i b r e s of the MU, the pos i t i o n of the electrode r e l a t i v e to the f i b r e s , the type of electrode used, the e l e c t r i c a l c h a r a c t e r i s t i c s of the recording equipment, the temperature of the muscle, the p a r t i c u l a r muscle, and the age of the subject (Daube, 1978; Gath & Stalberg, 1975; Lenman & Ritchie, 1987; Stalberg & Tr o n t e l j , 1979). Various features of the MU CAP may be measured, including i t s peak-to-peak amplitude, the number of phases, the duration, the number of turns, and the area (Nandedkar & Sanders, 1989; Stalberg & T r o n t e l j , 1979). These features are useful i n the diagnosis of a number of muscle disorders (Daube, 1978; Stalberg & T r o n t e l j , 1979). In addition, various techniques involving d i f f e r e n t types of needle electrodes have been developed to improve the s e l e c t i v i t y of MU recordings and to permit single f i b r e recordings (Ekstedt & Stalberg, 1973; Gath & Stalberg, 30 1976). The major types of needle electrodes w i l l be reviewed as the recording c h a r a c t e r i s t i c s of these electrodes vary quite markedly. a. Bipolar A bipolar needle electrode usually consists of a s t a i n l e s s s t e e l cannula containing two fine wires of platinum or s t a i n l e s s s t e e l whose t i p s are bared. The cannula i s often of larger diameter than the standard concentric needle electrode. The poten t i a l difference between the wire t i p s i s recorded, with the body of the cannula acting as a ground. Action potentials recorded from f i b r e s located equidistant from the two electrodes are cancelled, thus the bipolar configuration permits highly s e l e c t i v e recordings (Stalberg & T r o n t e l j , 1979). b. Monopolar Monopolar electrodes are t h i n s t a i n l e s s s t e e l needles insulated with Teflon except at t h e i r fine point. The recording t i p i s usually 0.2-0.4mm in length. Because the needle t i p i s sharp, i t s i n s e r t i o n causes less discomfort than other needles. Voltage changes are recorded between the electrode and a second reference electrode which may be situated on the skin surface or a needle situated i n the subcutaneous tissues (Lenman & Ritchie, 1987) . A separate surface electrode i s required as a ground. The monopolar electrode i s considered to be less e l e c t r i c a l l y stable and thus n o i s i e r than other electrode types (Kimura, 1983). The size of the recording t i p i s r e l a t i v e l y large when compared with other needles such as the single f i b r e or concentric electrodes, therefore when these needles are placed at the same distance from a MU f i b r e the monopolar needle w i l l record an action potential of smaller amplitude because i t averages i s o p o t e n t i a l l i n e s over a greater tiss u e area (Gath & Stalberg, 1976). However, the s e l e c t i v i t y of the monopolar needle can be improved s i g n i f i c a n t l y by bandpass-filtering of low frequency components of the EMG signal (Gath & Stalberq, 1976; Stalberg & T r o n t e l j , 1979). c. Concentric Concentric (coaxial) needles consist of an insulated s t a i n l e s s s t e e l or platinum wire inserted into the shaft of a s t e e l cannula. The shaft of the cannula and the wire are bared at t h e i r t i p s . The leading-off surface of the int e r n a l wire i s e l l i p t i c a l i n shape with a recording surface of 150/xm x 600/xm. A p o t e n t i a l difference i s recorded between the wire t i p and the shaft of the cannula. A separate ground electrode i s required. The CAPs recorded from the concentric electrode are considered to a r i s e from approximately 10 MU f i b r e s i n a normal muscle (Stalberg & Antoni, 1980). Based on t h i s finding, the radius of uptake of the needle has been estimated to be approximately 1mm (Stalberg, 1980). I t i s possible to record from single MU f i b r e s with a concentric needle but the d i s p a r i t y i n si z e between the small f i b r e and the larger leading-off surface of the needle diminishes 32 the amplitude of the recorded action p o t e n t i a l compared with that obtained with needles incorporating a small area of pick-up (Ekstedt & Stalberg, 1973; Stalberg & T r o n t e l j , 1979). d. Single-Fibre The technique of single f i b r e EMG (SFEMG) was introduced by Ekstedt & Stalberg (1963), and involves the use of a needle electrode with a very small leading-off surface capable of highly s e l e c t i v e recordings. The electrode i s a s t a i n l e s s s t e e l cannula (approximately 0.6mm i n diameter) with 1-14 insulated 25nm wires inserted through the shaft and taken out of a side port l-3mm d i s t a l to the needle t i p then embedded i n epoxy r e s i n (Stalberg & T r o n t e l j , 1979). The cannula i s used as a ground. Because of the small recording surface of the insulated wire, the area of pick-up i s estimated to be of radius 300/zm (Stalberg, 1980) . In a normal muscle, 1-2 MU f i b r e s are within t h i s pick-up area (Stalberg & Antoni, 1980). e. Macro-EMG Most needle electrodes record from only a small number of fib r e s of a MU (Lenman & Ritchie, 1987; Stalberg, 1980). If a single f i b r e action potential recorded by a s i n g l e - f i b r e electrode i s used to t r i g g e r an averager, surface electrodes are capable of recording from the whole MU although they are often situated remotely from the MU t e r r i t o r y (Stalberg, 1980). "Macro-EMG" permits an electrode to record from a whole MU with the electrode 33 in a better p o s i t i o n r e l a t i v e to the MU f i b r e s than i s possible with a surface electrode technique. The macro electrode comprises a 50mm st a i n l e s s s t e e l cannula with the t i p of a 25jum wire exposed through a side-port at a distance of 10mm from the t i p of the cannula. Potential differences are recorded between the cannula and a reference concentric electrode situated subcutaneously. Single f i b r e action potentials recorded at the small recording surface are used to t r i g g e r an averager. The averaged signal represents a summation of f i b r e potentials from a SMU (Stalberg, 1980). f. Summary Most needle electrodes record CAPs from groups of MU f i b r e s located i n proximity to the recording t i p . More foc a l single f i b r e recordings can, however, be obtained by needles with small leading-off surfaces. Although the area of pick-up of needle electrodes i s quite variable between d i f f e r e n t types, i n most instances more s e l e c t i v e recordings can be performed by bandpass-f i l t e r i n g of the EMG si g n a l . The monopolar electrode, although reputed to be less e l e c t r i c a l l y stable than other needles, appears to cause le a s t discomfort during i n s e r t i o n which i s an advantage during multiple recording sessions. 34 11. Motor Unit T e r r i t o r y When a muscle contracts during a weak voluntary e f f o r t , the ef f e c t i s smooth and integrated. The eveness of contraction has been attributed to a wide d i s t r i b u t i o n and extensive overlapping of MUs (Buchthal and Schmalbruch, 1980; Edstrom and Kugelberg, 19 68) . There i s ample evidence that the f i b r e s of many MUs are scattered throughout s i g n i f i c a n t areas of the limb and trunk muscles (e.g. Burke and T s a i r i s , 1973; Edstrom and Kugelberg, 1968; Stalberg et a l , 1976) . There are however, some muscles which appear outwardly to be single structures whereas i n t e r n a l l y they incorporate a number of putative subvolumes (English and Letbetter, 1982; Letbetter, 1974; Richmond et a l , 1985). In these cases, MU t e r r i t o r i e s seem to be confined within one of the subvolumes. This finding has raised speculation about the nature of motor control i n a compartmentalized muscle because the presence of l o c a l i z e d MU t e r r i t o r i e s could conceivably permit d i f f e r e n t i a l control of separate regions of the muscle (Belser and Hannam, 1986; Herring et a l , 1979; 1989; English, 1985; Stalberg and Eriksson, 1987). Further evidence of focal MU d i s t r i b u t i o n has been given by the descriptions of d i f f e r e n t i a l a c t i v i t y in d i f f e r e n t regions of the anatomical complex jaw muscles during the performance of routine functional tasks (Herring et a l , 1979, 1989; Herring and Wineski, 1986; Weijs and Dantuma, 1981). There i s a dearth of information on MU t e r r i t o r y i n human muscles, and the jaw muscles i n p a r t i c u l a r . Nonetheless, the d i s t r i b u t i o n of MUs within the human masseter muscle i s reputed to be f o c a l , and more akin to the t e r r i t o r i e s of animal jaw muscles than those found i n human limb and trunk muscles (Buchthal and Schmalbruch, 1980; Herring et a l , 1979; 1989; Stalberg and Eriksson, 1987). Because of the implications of MU t e r r i t o r y for motor control and functional behaviour of muscles, MU mapping studies i n both animal and human muscles w i l l be reviewed. a. Motor Unit D i s t r i b u t i o n In animal muscles, the d i s t r i b u t i o n of MUs i s normally demonstrated by histochemical staining of muscle tissu e a f t e r glycogen deletion of constituent f i b r e s (Burke, 1981). Because the muscle tissu e i s studied h i s t o l o g i c a l l y , MU t e r r i t o r y i s usually viewed as a 2-dimensional map, but i n fact i t i s a 3-dimensional volume. The t o t a l number of f i b r e s i n a MU may not be represented i n only one muscle cross-section, p a r t i c u l a r l y i f there i s angulation of the fi b r e s r e l a t i v e to the plane of section (Burke and T s a i r i s , 1973). Multiple sections at d i f f e r e n t positions along the length of the muscle are often required to reconstruct the MU t e r r i t o r y . In addition, glycogen depletion i s not uniform throughout MUs of d i f f e r e n t types, with depletion being more d i f f i c u l t i n those units which r e l y mainly on oxidative metabolism (Burke, 1981; Burke et a l , 1973). Given the l i m i t a t i o n s of the technique, the volume occupied by a MU within a muscle may be quite substantial, i n some instances up to 2 0% of the muscle volume i n the case of the long-fibred rat 36 soleus muscle, or 14% of the volume for the cat medial gastrocnemius muscle (Burke, 1981; Burke et a l , 1974). Throughout these r e l a t i v e l y extensive t e r r i t o r i e s , the MU f i b r e s i n t e r d i g i t a t e with f i b r e s of many other MUs. When studied i n muscle cross-section however, most MU f i b r e s are d i s t r i b u t e d over only a part of the muscle cross-section, for example i n the rat anterior t i b i a l i s muscle SMU f i b r e s are found i n 12-2 6% of the t o t a l muscle cross-section, which suggests a possible segregation of populations of MUs (Edstrom and Kugelberg, 1968) . b. Compartmentalization of Muscles 1. Neuromuscular A primary muscle nerve branch has been defined as one of the branches of a muscle nerve as i t enters the muscle at i t s h i l u s (English and Weeks, 1984). The compartmentalization of muscles about primary muscle nerve branches has been postulated as the anatomical substrate for motor control (English and Weeks, 1984; English and Letbetter, 1982; Letbetter, 1974). Glycogen depletion methods involving prolonged neural stimulation have demonstrated that the primary branches of muscle nerves supply discrete subvolumes of a muscle. These t e r r i t o r i e s have been coined "neuromuscular compartments" (English, 1985). In the cat l a t e r a l gastrocnemius muscle, primary nerve branches appear to supply four d i s t i n c t regions of the muscle (English, 1985; English and Weeks, 1984) . Other cat ankle extensor muscles (medial gastrocnemius and plantaris) seem to be organized s i m i l a r l y although they exhibit 37 considerable differences i n complexity of t h e i r f i b r e architecture (English and Letbetter, 1982). In the rat extensor digitorum longus muscle, two primary nerve branches innervate separate "compartments" although no d i s t i n c t anatomical features appear to separate the muscle into two subvolumes (Balice-Gordon and Thompson, 1988). Three primary nerve branches, which are organized around in t e r n a l connective tissue septa of the muscle, have been described for the pig masseter muscle (Herring et a l , 1989; 1979). Prolonged stimulation of the two smaller anterior branches causes glycogen depletion i n anterior parts of the muscle whereas in the larger posterior branch glycogen depletion occurs throughout almost the whole muscle i n a f a s c i c l e - s p e c i f i c pattern (Herring et a l , 1989). Segmentotopic organization of projections from motoneurons to neuromuscular compartments has been documented (Balice-Gordon and Thompson, 1988; English, 1985; Herring et a l , 1989) . Although the motoneuron arrangement i s not p a r t i c u l a r l y strong i n some muscles, i t may to some extent explain the presence of neuromuscular compartments (Balice-Gordon and Thompson, 1988). English (1985) has postulated that the d i v i s i o n of muscles into neuromuscular compartments may explain the regional aggregation of muscle f i b r e s of the same histochemical type i n certa i n muscles. Within the neuromuscular compartments of the cat l a t e r a l gastrocnemius muscle, the v a r i a b i l i t y i n f i b r e type composition i s i n s i g n i f i c a n t , whereas marked differences i n f i b r e type composition are present between compartments (English and Letbetter, 1982). However, t h i s explanation for "histochemical 38 compartmentalization" i s contingent on SMUs being r e s t r i c t e d within i n d i v i d u a l neuromuscular compartments. Glycogen depletion studies involving stimulation of ind i v i d u a l axons of the cat l a t e r a l and medial gastrocnemius, and the pig masseter muscles have disclosed that SMUs are indeed confined to only one neuromuscular compartment within these muscles (English and Weeks, 1984; Herring et a l , 1989; 1979). In the l a t e r a l and medial gastrocnemius muscles the depleted SMU f i b r e s are usually d i s t r i b u t e d throughout a large volume of the compartment, whereas in the pig masseter muscle a number of the SMUs appear to be located within only one or two f a s c i c l e s and within these f a s c i c l e s only 5-20% of the t o t a l number of f i b r e s are normally depleted. In the pig masseter muscle in d i v i d u a l MU t e r r i t o r i e s are reputed to occupy approximately 5% of the t o t a l muscle volume (Herring et a l , 1989). However, the small t e r r i t o r i e s i n the pig masseter muscle may be due to s i g n i f i c a n t l y smaller f i b r e lengths, v i z . 15.6mm i n the pig masseter compared with up to 37.7mm i n the cat soleus (Herring et a l , 1989). It has been reported that the human masseter muscle has three primary nerve branches (Li Xiguang et a l , 198 6), therefore i t i s possible that a small number of neuromuscular compartments may be present. 11. S e r i a l S e r i a l l y - l i n k e d compartments of MUs have been i d e n t i f i e d i n a number of cat, neck and limb muscles (Armstrong et a l , 1988; 39 Bodine et a l , 1982; Richmond et a l , 1985). Prolonged stimulation of nerve bundles supplying the cat biventer c e r v i c i s muscle has produced glycogen depletion i n compartments which do not span the entire length of the muscle (Armstrong et a l , 1988). These compartments appear to be arranged i n - s e r i e s and are separated anatomically by tendinous i n s c r i p t i o n s (Armstrong et a l , 1988; Richmond et a l , 1985). A si m i l a r compartmentalization i s seen i n the rectus abdominis muscle (Duchen and Gale, 1985) . As i n the neuromuscular compartments of other muscles, SMUs are located within only one compartment (Armstrong et a l , 1988; Bodine et a l , 1982; Richmond et a l , 1985). This unusual organization leads to functional behaviour in which forces generated by one compartment are transmitted " i n - s e r i e s " through the other compartments, rather than d i r e c t l y on to ske l e t a l attachments. c. El e c t r o p h y s i o l o g i c a l Studies It i s not possible to determine the t e r r i t o r y of ind i v i d u a l MUs i n humans by using h i s t o l o g i c a l methods based on glycogen depletion (Buchthal and Schmalbruch, 1980). In humans, the size of SMUs are usually investigated by ele c t r o p h y s i o l o g i c a l methods (Buchthal and Schmalbruch, 1980; Stalberg and Antoni, 1980; Stalberg et a l , 1976). Electrodes incorporating multiple recording s i t e s , s i n g l e - f i b r e , and concentric electrodes have been employed to scan the cross-section of muscles to determine the area over which f i b r e s of a MU are di s t r i b u t e d (Buchthal and Schmalbruch, 1980; Stalberg and Eriksson, 1987; Stalberg et a l , 1976). Mean 40 scan lengths i n the brachial biceps and anterior t i b i a l i s muscles have been determined as 7mm (max. 15.1mm) and 7.3mm (max. 14.6mm) respectively, with MU areas covering only part of the muscle cross-section (Buchthal and Schmalbruch, 1980; Stalberg and Antoni, 1980; Stalberg et a l , 1976), and the f i b r e s are admixed with those of other MUs (Buchthal and Schmalbruch, 1980) . Based on these studies i t has been surmised that the MU t e r r i t o r i e s are oval or c i r c u l a r i n shape (Buchthal and Schmalbruch, 198 0; Stalberg and Eriksson, 1987). The t e r r i t o r i a l d i s t r i b u t i o n of MU fib r e s i n cross-sectional scans of the extensor digitorum communis muscle i s normally 6-9mm with no evidence of f i b r e grouping (Stalberg et a l , 1976), whereas i n instances of motoneuron disease f i b r e grouping occurs due to reinnervation of denervated f i b r e s (Schwartz et a l , 1976). In some of the larger limb muscles, MU fib r e s may be dis t r i b u t e d over a distance of up to 22mm (Buchthal et a l , 1959) . A s i m i l a r electrode scanning technique has been used to study the topography of MUs i n the human masseter muscle (Stalberg and Eriksson, 1987). With electrode penetrations r e s t r i c t e d to the lower part of the muscle, the mean scan length was determined to be 3.7mm, although a small number of units had scans of 9.1-12.5mm which covered the whole of the muscle cross-section. Despite some long cross-sectional scans, focal MU t e r r i t o r i e s of less than 5mm diameter were considered to be the norm (Stalberg and Eriksson, 1987). However, f i b r e density studies have yielded s i m i l a r results in both jaw and limb muscles, therefore although presumptive 41 masseter muscle MU t e r r i t o r i e s appear to be small, i t i s un l i k e l y that they are homogenous clumps (Stalberg et a l , 1986). This i n d i r e c t electrophysiological technique of establishing MU area i n a muscle cross-section has a number of pote n t i a l weaknesses. The technique was o r i g i n a l l y used to study MU area i n p a r a l l e l - f i b r e d muscles (Buchthal et a l , 1957). However, in a complex muscle such as the human masseter muscle findings should be interpreted with caution. Due to muscle f i b r e angulation, the t o t a l number of f i b r e s belonging to a unit may not be r e f l e c t e d in the muscle cross-section. The technique also does not detect MU t e r r i t o r y i n the long-axis of the muscle, therefore i t does not accurately address the d i s t r i b u t i o n of muscle f i b r e s within the muscle volume. In addition, the electrode technique i s e s s e n t i a l l y a random probe which gives l i t t l e i n d i c a t i o n of the r e l a t i v e positions of MU f i b r e s within the muscle volume, and t h e i r possible r e l a t i o n s h i p to putative muscle compartments. d. Functional Implications The extensive intermingling of MUs and t h e i r r e l a t i v e l y wide d i s t r i b u t i o n i n some animal and human muscles appears to present the optimum conditions for the controlled summation of asynchronously discharging MUs during the generation of muscle force. Nonetheless, any attempt to specify a sing l e function to a limb or jaw muscle i s , perhaps, s i m p l i s t i c when i t i s considered that many MUs are not necessarily active during the function of the parent muscle (English, 1985). The concept of SMUs l o c a l i z e d 42 within one of a small number of neuromuscular compartments within a muscle suggests a l e v e l of motor control which i s more s p e c i f i c than "whole muscle" control. This type of control would permit the ac t i v a t i o n of muscle compartments r e l a t i v e l y independently of one another, with the l e v e l of in t e r a c t i o n dependent on the p a r t i c u l a r task and muscle movement strategy at hand (English, 1985). The se l e c t i v e a c t i v a t i o n of muscle compartments during certain functional tasks appears to support t h i s concept (English, 1985; Herring et a l , 1979). The t e r r i t o r y of a SMU within a neuromuscular compartment varies between animals, and appears to be most fo c a l i n the pig masseter muscle where i t may represent as l i t t l e as 5% of the muscle volume. The presence of small MU t e r r i t o r i e s overlaid on the complex pig muscle architecture would p o t e n t i a l l y permit numerous l i n e s of muscle action by means of regional contraction. Recently, Herring et a l (1989) have described seven to eight "functional compartments" which appear to exhibit differences i n EMG a c t i v i t y patterns. However, these "compartments" do not l i e within s t r i c t anatomical boundaries, nor do they have any p a r t i c u l a r f i b r e orientation (Herring et a l , 1989). In addition, the EMG method was e s s e n t i a l l y q u a l i t a t i v e , i n which no SMU recordings were made, therefore there i s the d i s t i n c t p o s s i b i l i t y that there may be an overlap of MU populations i n the putative compartments. I f there are seven or eight semi-autonomous regions within the muscle, the motor control of such a system would have to be highly sophisticated to ensure the smooth integration of a 43 large number of compartments. Within a selected part of the human masseter muscle, Stalberg and Eriksson (1987) have suggested that there are many MUs with r e s t r i c t e d t e r r i t o r i e s but also some with very wide domains. Small MU t e r r i t o r i e s confer a pote n t i a l for s e l e c t i v e recruitment of muscle regions whereas more widespread units o f f e r the functional capacity for widespread contraction. This combination i s fea s i b l e , but i t i s possible that MUs with small cross-sections may have long scans i n the longitudinal d i r e c t i o n of the muscle. The small number of long scans suggests that putative compartments may be unusually large and occupy the whole muscle cross-section. e. Summary Si g n i f i c a n t differences i n MU f i b r e d i s t r i b u t i o n within muscles have been described between species, and between limb and jaw muscles. A feature of a number of animal limb and jaw muscles, i s t h e i r organization into subvolumes based on the d i s t r i b u t i o n of primary branches of muscle nerves. Single motor unit d i s t r i b u t i o n s appear to be r e s t r i c t e d to one of these "neuromuscular compartments", with jaw muscles exhibiting the most focal MU t e r r i t o r i e s . A small number of neuromuscular compartments are reputed to provide a l e v e l of motor control which i s more f i n e l y tuned than that of the whole muscle. There i s some evidence to suggest that the pig masseter muscle may be segregated into putative functional compartments which are 44 smaller than neuromuscular compartments, and based on d i f f e r e n t i a l EMG a c t i v i t y recorded i n many d i f f e r e n t regions of the muscle. However, i f t h i s i s so, a highly complex pattern of neural control would be required. Motor unit t e r r i t o r i e s i n human jaw muscles appear i n general to be smaller than those of limb muscles. However, instances of t e r r i t o r i e s extending the f u l l cross-section of the muscle have been noted. Such a finding has not been observed i n the limb muscles. The precise t e r r i t o r y of SMU f i b r e s i s not readi l y determinable because of inherent weaknesses i n the presently available i n v e s t i g a t i v e techniques. I t i s thus d i f f i c u l t to speculate on motor control of the complex jaw muscles when the nature of MU d i s t r i b u t i o n i s uncertain. 45 i l l . Motor Unit A c t i v i t y The muscle MU has been described as the basic unit of motor a c t i v i t y (Liddel & Sherrington, 1935), the f i n a l common pathway u t i l i z e d i n the neural control of movement. Each of these functional units of motor a c t i v i t y consists of an a-motoneuron located i n the brainstem or spinal cord, a motor axon and the ske l e t a l muscle f i b r e s supplied by t h i s axon. In a normal adult muscle, each MU consists of only one histochemical f i b r e type. When a sk e l e t a l muscle contracts i t s MUs are normally activated in a predictable sequence which i s related intimately to the size of t h e i r parent motoneurons (reviewed i n Burke, 1981). At the commencement of a graded muscle contraction, motoneurons with small somata are activated while larger motoneurons are recruited progressively as the force increases. The r e l a t i v e force contribution of the individual MU may be modulated by v a r i a t i o n in i t s frequency of discharge (Burke, 1981). The progressive recruitment of MUs and the modulation of t h e i r f i r i n g rates or "rate coding" are intimately involved i n the gradation of muscle force (Adrian & Bronk, 1929; Burke, 1981; Desmedt & Godaux, 1978; Milner-Brown et a l 1973b). Within the muscle as a whole, the inte r a c t i o n of MU recruitment and f i r i n g rate modulation permits the t r a n s i t i o n from a series of muscle twitches to a tetanic contraction as a smooth continuum during increasing force development (Buchthal & Schmalbruch, 198 0; Clamann, 1971; De Luca, 1985) . 46 Much evidence on the patterns of motor control has been gleaned from animal studies although the behaviour patterns are highly stereotyped, and the e f f e c t of decerebration on the functional organization of the motoneuron pool i s uncertain (Burke, 1981). Motor control of jaw muscle a c t i v i t y cannot be studied d i r e c t l y i n humans, but i t may be in f e r r e d from studies of MU behaviour. The use of human subjects for such studies confers many advantages including the a b i l i t y to perform highly-s k i l l e d , voluntary motor tasks (Freund, 1983; Miles et a l , 1986). Human MU behaviour may vary s i g n i f i c a n t l y during the performance of a given task (Petajan, 1981). Although the observed movement may appear to be the same, the task may be accomplished by varying degrees of muscle contraction depending on the l e v e l of coactivation of antagonistic muscles (Carew & Ghez, 1985; Petajan, 1981) , and muscle strategies also vary when a task i s repeated (Freund, 1983) . In addition to highly p l a s t i c human MU behaviour, there are apparent fundamental differences between spinal and o r o - f a c i a l motor mechanisms (Derfler & Goldberg, 1977; Dubner et a l , 1978). For example, the spinal and supra-spinal control of limb muscles during locomotion often requires d i f f e r e n t muscle a c t i v a t i o n strategies i n each leg, whereas i n the o r o - f a c i a l region a c t i v i t i e s such as mastication and suckling require b i l a t e r a l l y s y n e r g i s t i c patterns of muscle behaviour (Dubner et a l , 1978). Variations i n behaviour have also been observed between limb and jaw muscle SMUs which are capable of performing multiple tasks 47 (Eriksson et a l , 1984; Thomas et a l , 1987; 1986). As MUs of s k e l e t a l muscles i n the o r o - f a c i a l region are under neural control v i a t h e i r motor innervation, the major mechanisms involved i n the control of MU a c t i v i t y w i l l be reviewed. a. Motor Unit Recruitment The concept of motoneuron recruitment, which was introduced by Sherrington (1925), i s one of the p r i n c i p a l determinants involved i n the control of muscle force (Burke, 1981). The motoneurons which innervate one muscle are integrated to form a motoneuron pool (Burke et a l , 1977) . The basis of the c o r t i c a l control of such a motoneuron pool are multiple excitatory inputs from c o r t i c o s p i n a l neurons to the motoneurons of the pool (Henneman & Mandell, 1981; Miles, 1987). As the muscle begins to contract c o r t i c o s p i n a l c e l l s d e l i v e r progressively more current u n t i l i n d i v i d u a l motoneurons of the pool reach t h e i r a c t i v a t i o n threshold and commence f i r i n g (Miles, 1987). The e x c i t a b i l i t y of each motoneuron i s c l o s e l y related to i t s input resistance, which varies inversely with the c e l l size (Henneman & Mandell, 1981; Henneman et a l , 1974) . Thus for most voluntary and r e f l e x contractions motoneurons r e c r u i t i n an orderly manner with small motoneurons incorporating small slowly conducting axons being recruited f i r s t , while progressively larger ones with bigger axons are recruited as the l e v e l of muscle a c t i v i t y increases (Henneman, 1981; Henneman et a l , 1965a) . The s i z e of each muscle MU i s c l o s e l y related to the size of 48 i t s parent motoneuron, therefore i n a graded muscle contraction MU recruitment based on the "size p r i n c i p l e " p r e v a i l s (Henneman & Mandell, 1981; Henneman et a l , 1965a). The axonal conduction of a MU i s related to a number of c h a r a c t e r i s t i c s of i t s muscle f i b r e s such that an early recruited MU with a low conduction v e l o c i t y has a low twitch tension, a long contraction time, and a high resistance to fatigue whereas larger, faster twitch units are recruited at higher muscle forces (Edstrom & Grimby, 1986) . Direct evidence for a consistent pattern of MU recruitment has been gleaned predominantly from animal studies (Clark et a l , 1978; Miles, 1987) . In the hindlimb muscles of the cat and the temporalis muscles of non-human primates the e a r l i e s t recruited units have lower twitch tensions and a c t i v a t i o n thresholds than units recruited l a t e r (Burke, 1967; Clark et a l , 1978; Henneman et a l , 1965a; McPhedran et a l , 1965). Many studies i n human limb and jaw muscles have also demonstrated an orderly recruitment of MUs according to the siz e p r i n c i p l e during slowly developing muscle contractions (Desmedt & Godaux, 1975; Freund et a l , 1975; Goldberg & Derfler, 1977; Milner-Brown et a l , 1973a; Yemm, 1977). Because the process of MU recruitment i s b a s i c a l l y orderly under many conditions, i t permits consistency of force coding (Desmedt, 1983). I f recruitment order within the motoneuron pool was v e r s a t i l e i t would be almost impossible to perform s k i l l e d motor behaviour (Henneman, 1981). There i s a general c o r r e l a t i o n between the functional threshold and the mechanical force output among limb and jaw 49 muscle MUs throughout the recruitment range (Burke, 1981; Goldberg & Derfler, 1977; Milner-Brown et a l , 1973a; Monster & Chan, 1977; Yemm, 1977). However functional thresholds are not pr e c i s e l y fixed in the motoneuron pool and may vary within c e r t a i n l i m i t s as a function of the input source, and they are not e n t i r e l y stable with time (Burke, 1981) . I t i s perhaps not sur p r i s i n g that MUs with s i m i l a r recruitment thresholds w i l l f i r e occasionally i n reverse order due to inherent "noise" i n the system. In non-human primate temporalis muscle MUs, Clark et a l (1978) observed consistent recruitment thresholds during tonic contractions. However, Miles et a l (1986) observed length-related changes i n the ac t i v a t i o n thresholds of human masseter MUs, with recruitment thresholds increasing dramatically as jaw gape was increased which tends to confound previous theories of recruitment order within the muscle (Desmedt & Godaux, 1975; Yemm, 1977). S i g n i f i c a n t task-dependent differences i n unit a c t i v a t i o n thresholds have also been observed i n biceps MUs activated by fle x i o n versus supination, although the extent to which recruitment reversals occurred i s uncertain (ter Haar Romeny, 1982) . It i s generally recognized that voluntary control of MU recruitment patterns i s not possible (Burke, 1981). However, unit functional thresholds may decrease dramatically during b a l l i s t i c muscle contractions to the extent that there may be apparent changes i n the MU recruitment order as powerful motor commands to the motoneuron pool activate many MUs almost simultaneously 50 (Burke, 1 9 8 1 ; Desmedt, 1 9 8 3 ; Freund, 1 9 8 3 ) . There are also instances when an interchange i n units of very d i f f e r e n t thresholds occur during tonic contractions (Burke, 1 9 8 1 ; Grimby St Hannerz, 1 9 6 8 ) . Modulation of cutaneous afferents may d i f f e r e n t i a l l y a f f e c t d i f f e r e n t sized units i n both animals and humans (Kanda et a l , 1 9 7 7 ; Grimby & Hannerz, 1 9 6 8 ; Stephens et a l , 1 9 7 8 ) . Consistent deordering of MU recruitment during slow ramp contractions has been observed i n multifunctional human muscles that can be used as prime movers or as synergists (Desmedt, 1 9 8 0 ; Schmidt & Thomas, 1 9 8 1 ; Thomas et a l , 1 9 8 7 ; 1 9 7 8 ) . Changes i n recruitment order have been observed i n the f i r s t dorsal interosseous and abductor p o l l i c i s muscles, both of which have two degrees of freedom (Desmedt & Godaux, 1 9 8 1 ; Thomas et a l , 1 9 8 7 ) . Although apparent varia t i o n s i n recruitment patterns may occur i n the f i r s t dorsal interosseous muscle for abduction, or f l e x i o n of the index finger, or abduction of the thumb together with f l e x i o n of the index finger, separate groups of MUs do not appear to be recruited for each task (Thomas et a l , 1 9 8 6 ) . Units capable of performing more than one. task have also been observed in the human masseter muscle although no apparent differences i n recruitment thresholds were discerned between tasks (Eriksson et a l , 1 9 8 4 ) . Any reordering of MU recruitment suggests differences i n the organization of synaptic inputs to the motoneuron pool (Burke, 1 9 8 1 ) . Changes i n synaptic e f f i c i e n c y as a r e s u l t of post-tetanic potentiation, and the a c t i v a t i o n of sensory afferents with an uneven d i s t r i b u t i o n of the terminals to the motoneuron pool have 5 1 been implicated i n MU recruitment a l t e r a t i o n s (Kanda et a l , 1977; Luescher et a l , 1979). In the human biceps muscle, some units may perform multiple tasks whereas other units are apparently activated s e l e c t i v e l y when d i f f e r e n t tasks are performed (ter Haar Romeny et a l , 1982). A combination of multi-task and t a s k - s p e c i f i c units have apparently also been observed i n the human masseter muscle (Eriksson et a l , 1984), although the extent to which t h i s occurs i s uncertain. These findings suggest possible differences i n input a c t i v i t y to the motoneuron pool for d i f f e r e n t SMU tasks. b. Interaction of Motor Unit F i r i n g Rate and Recruitment The role of the motoneuron pool i s to synthesize a multitude of c e n t r a l l y and peripherally generated signals so that the motor output e l i c i t s controlled tensions i n the muscle (Henneman & Mandell, 1981). Motoneuron pool output may be measured by MU recruitment patterns but also i n terms of the f i r i n g frequency c h a r a c t e r i s t i c s of SMUs because both mechanisms are important i n the control of motor performance (Burke, 1981; Henneman & Mandell; 1981). During voluntary muscle contractions i n humans, the f i r i n g frequency of MUs appears to vary rnonotonically with the force produced by the muscle as a whole (De Luca et a l , 1982; Edstrom & Grimby, 1986) . Based on t h i s finding, De Luca et a l (1982) suggested that increased excitatory inputs to the motoneuron pool increase the f i r i n g rate of a l l active MUs c o l l e c t i v e l y . The 52 strong c o r r e l a t i o n between the f i r i n g rates of MUs within a muscle in r e l a t i o n to changes i n muscle force, and as a function of time, has been coined the "common drive" (De Luca, 1985) . As a motor control concept, i t indicates that the motoneuron pool i s modulated as a whole rather than at the l e v e l of i n d i v i d u a l motoneurons (De Luca, 1985). There are, however, c o n f l i c t i n g findings concerning the role of frequency modulation i n the gradation of muscle contractions. Bigland & Lippold (1954) have suggested that muscle force i n MUs of hand muscles may be varied s i g n i f i c a n t l y with very l i t t l e frequency modulation. I t has also been proposed that MU f i r i n g frequency i s s t a b i l i z e d at r e l a t i v e l y low muscle forces, and further graded contraction occurs by means of MU recruitment (Bracchi et a l , 1966). However, the present consensus i s that both MU recruitment and f i r i n g frequency are i n t e r - r e l a t e d but that t h e i r r e l a t i v e contribution to motor control of muscle force may vary between muscles (Burke, 1981; Desmedt & Godaux, 1978; G r i l l n e r & Udo, 1971; Milner-Brown et a l , 1973b). In general, the small muscles of the hand r e c r u i t t h e i r MUs below 50% of maximum voluntary contraction (MVC) and then r e l y on rate modulation for further force increments (De Luca, 1985; Milner-Brown et a l , 1973b). In c o n t r a d i s t i n c t i o n , larger more powerful limb muscles r e l y predominantly on MU recruitment up to 90% of MVC (De Luca, 1985) . In the non-human primate, MU recruitment appears to occur over the entire range of muscle contraction whereas the human masseter muscle i s much more r e l i a n t 53 on MU rate-coding, and thus appears to behave d i f f e r e n t l y from powerful limb muscles (Clark et a l , 1978; Derfler & Goldberg, 1977) . Two types of MU f i r i n g patterns have been described i n humans: a tonic "T" pattern with a low o v e r a l l f i r i n g frequency and regular inter-spike i n t e r v a l s , and a k i n e t i c "K" pattern with high frequency i r r e g u l a r discharges (Tokizane & Shimazu, 1964). Most studies, however, have been unable to confirm the existence of "T" or "K" groups (for example Clamann, 1970; Freund et a l , 1973; Hannerz, 1974). I t appears that almost a l l units may discharge t o n i c a l l y or p h a s i c a l l y depending on the p r e v a i l i n g behavioural set and that "T" and "K" f i r i n g patterns are not i n d i c a t i v e of two d i f f e r e n t types of MUs (Freund, 1983). Human and animal a-motoneurons tend to f i r e r e l a t i v e l y consistently at slow rates when they are activated t o n i c a l l y near t h e i r functional threshold (reviewed i n Burke, 1981). However, unlike the sensory system, a-motoneurons do not normally commence f i r i n g from zero. The lowest sustainable f i r i n g frequency appears to be 6-8Hz, without s i g n i f i c a n t differences between units of d i f f e r e n t force thresholds (Clamann, 1970; Freund et a l , 1975; Milner-Brown et a l , 1973b). Even highly-trained subjects using audiovisual feedback cannot consistently drive units below 6Hz (Freund, 1983; Petajan, 1981). Only i n instances of rapidly alternating movements, and i n patients with basal ganglia or cerebellar diseases may the f i r i n g frequency be maintained below 6Hz (Freund et a l , 1973). 54 The lowest sustainable rate of steady MU f i r i n g appears to be equivalent to the f i r i n g rate at which SMU twitches begin to fuse (Kernell, 1974). The lowest sustainable f i r i n g freqency (LSFF) appears to vary between muscles (Person & Kudina, 1972; Petajan, 1981; Stalberg and T r o n t e l j , 1978; Tanji & Kato, 1981). Petajan (1981) has shown that the LSFF i s greater for f a c i a l muscles (10Hz) than limb muscles (6Hz) . Nordstrom et a l (1989) have indicated that i t i s d i f f i c u l t to drive masseter MUs consistently below 8-lOHz, although Eriksson et a l (1984) have suggested that they may be driven without pauses between 5 and 8Hz. Given that jaw muscles appear to f i r e f aster than limb muscles, there i s also a difference i n the inter-spike i n t e r v a l v a r i a b i l i t y between limb and jaw muscles (Derfler & Goldberg, 1978). Limb muscles appear to maintain more controlled inter-spike i n t e r v a l s for a greater range of f i r i n g frequencies than the jaw muscles (Derfler & Goldberg, 1978; Tokizane & Shimazu, 1964). Renshaw c e l l s have been implicated i n f i r i n g frequency modulation of spinal motoneurons (Henneman & Mandell, 1981). As trigeminal motoneurons do not appear to have such recurrent c o l l a t e r a l s , recurrent i n h i b i t i o n cannot aid i n the maintenance of consistent discharge patterns of jaw muscle MUs (Derfler & Goldberg, 1978). In addition to differences i n LSFFs between limb and jaw muscle MUs, there are also apparent differences i n the LSFF for d i f f e r e n t tasks i n masseter MUs (Eriksson et a l , 1984). In multi-task human limb muscle MUs there are seemingly no differences between the LSFFs for d i f f e r e n t tasks (Thomas et a l , 1987; 1986), 55 whereas i n masseter units capable of performing both intercuspal clenching and jaw retrusion there are s i g n i f i c a n t differences i n measurable LSFFs, and hence excitatory drive, although the extent to which t h i s occurs for d i f f e r e n t tasks i n units throughout the muscle i s uncertain. (Eriksson et a l , 1984). c. Summary Muscle force i s expressed through the action of MUs and can be produced by varying the combinations and l e v e l s of MU a c t i v i t y , which indicates subtle, complex motor control of MU behaviour. Motor unit recruitment and f i r i n g rate modulation are important determinants of any muscle action. In general, the recruitment process i s orderly for tonic muscle contractions although i t s r e l a t i v e importance throughout the spectrum of muscle contraction appears to vary between some limb and jaw muscles. F i r i n g rate modulation seems to play a more dominant role i n the masseter than s i m i l a r l y powerful limb muscles. Some MUs of both limb and jaw muscles are capable of performing multiple tasks. The excitatory drive to the jaw muscle MUs however seems to vary according to the task performed, but whether t h i s occurs i n units throughout the masseter muscle i s presently unknown. 56 IV. Reflex Behaviour Peripheral sensory inputs from o r o - f a c i a l s i t e s such as the teeth, tongue, muscles, and temporomandibular j o i n t s are capable of e l i c i t i n g brainstem reflexes which are important to normal and pathological functioning of the jaws. The l i t e r a t u r e on brainstem reflexes a f f e c t i n g the jaws i s copious and has been reviewed extensively by Matthews (1975), Dubner et a l (1978), Luschei and Goldberg (1981) , and Lund et a l (1983) . A number of the simple reflexes serve protective functions, whereas others are rarely used i n function and yet i n combination they provide a neural basis for complex jaw a c t i v i t i e s such as mastication, swallowing, and suckling (Dubner et a l , 1978; Sessle, 1981). Brainstem jaw reflexes most commonly involve muscles which e f f e c t jaw movement in an opening, cl o s i n g or l a t e r a l d i r e c t i o n . The prime movers in such movements are the masseter, temporalis, d i g a s t r i c and pterygoid muscles. Under experimental conditions, the jaw jerk or myotatic ref l e x i s evoked by stretching the jaw elevator muscles (conventionally by downwards displacement of the mandible). Transient jaw closure i s caused by stimulation of muscle spindles i n the jaw closing muscles which e l i c i t monosynaptic r e f l e x e x c i t a t i o n (reviewed in Dubner et a l , 1978). In man, voluntary contraction of other muscles can increase the amplitude of the r e f l e x response (Bratzlavsky, 1976; Hannam, 1972). The r e f l e x i s mediated v i a the trigeminal mesencephalic and motor nuclei, and i s the trigeminal 57 analogue of the spinal myotatic reflexes, although i t d i f f e r s from the spinal reflexes due to the absence of i n h i b i t i o n of antagonistic jaw muscles (Lund et a l , 1983; Luschei and Goldberg, 1981). A myotatic r e f l e x has not been evoked i n the jaw opening muscles, most l i k e l y due to the lack of i n t r i n s i c muscle spindles (Lund et a l , 1983). The jaw opening r e f l e x was f i r s t described by Sherrington (1917), who evoked the response i n the cat by i n t r a o r a l stimulation. When mechanical or e l e c t r i c a l s t i m u l i are applied to o r o - f a c i a l tissues of animals, short latency e x c i t a t i o n occurs in jaw opening muscles v i a a disynaptic pathway, whereas two periods of i n h i b i t i o n occur i n the jaw elevator motoneurons (Kidokoro et a l , 1968; Sumino, 1971). In humans the i n h i b i t o r y response i n jaw elevator muscles appears to be comparable, but no early e x c i t a t i o n i s c l e a r l y demonstrable i n the l a t e r a l pterygoid or d i g a s t r i c muscles, although a l a t e response has been reported in the d i g a s t r i c muscle (Desmedt and Godaux, 197 6; Matthews, 197 6; Yemm, 1972). The degree of i n h i b i t i o n of jaw elevator muscle a c t i v i t y appears to be related to the in t e n s i t y of the applied stimulus, with the early i n h i b i t o r y response reported to have the lowest threshold i n animal and most human studies (Godaux and Desmedt, 1975; McGrath et a l , 1981). Because the d i g a s t r i c muscle i s much more e a s i l y accessible than the l a t e r a l pterygoid muscle, most studies on the jaw opening r e f l e x use the d i g a s t r i c muscle as a model. Nonetheless, Sessle and Gurza (1982) have demonstrated presumptive r e f l e x e x c i t a t i o n and i n h i b i t i o n , i n response to 58 i n t r a o r a l stimulation, i n both heads of the l a t e r a l pterygoid muscle i n non-human primates. A d i f f e r e n t sequence of excitatory and i n h i b i t o r y r e f l e x events has been reported by Widmer (1987) for the i n f e r i o r head of the human l a t e r a l pterygoid muscle. Evidence for a l a t e r a l jaw r e f l e x has been obtained in the rabbit by Lund et a l (1971). This r e f l e x has been described as a v a r i a t i o n of the jaw opening r e f l e x (Hannam, 1979) , and may be protective i n nature. Lateral jaw movements have been observed i n response to tooth interferences or temporomandibular j o i n t inflammation (Dubner et a l , 1978), although the r e f l e x has not yet been unequivocally demonstrated i n man (Hannam, 1979) . Another p o t e n t i a l l y protective r e f l e x has been described by Hannam et a l (1968) and Miles and Wilkinson (1982) , as the response of the jaw elevator muscles to unloading during voluntary clenching. Transient i n h i b i t i o n of jaw elevator muscles and a c t i v a t i o n of the jaw opening d i g a s t r i c muscles appears to prevent rapid approximation of the teeth. Apart from the p o s s i b i l i t y of species differences, comparisons of studies are often complicated by differences i n the methods used to measure r e f l e x responses. Both multiunit and SMU EMG recordings have been used for analysis i n many human studies, and averaging techniques are employed for quantitation (for example Cadden and Newton, 1989; Widmer, 1987; Yemm, 1972). The v a l i d i t y of multiunit EMG as a r e l i a b l e , quantitative measure of r e f l e x muscle a c t i v i t y i s , however, disputed (Lavigne et a l , 1983; Miles et a l , 1987; Turker and Miles, 1987). Turker et a l (1989) have 59 recently demonstrated that the r e l a t i v e p o s i t i o n of the stimulus during r e p e t i t i v e f i r i n g , and MU f i r i n g frequency, are important determinants of r e f l e x i n h i b i t i o n i n the human masseter muscle. It has also been observed that MU synchronization can lead to apparent excitatory responses when averaging techniques are used (Matthews et a l , 1988; Widmer and Lund, 1988). Because r e f l e x behaviour of jaw muscles may be studied more e f f e c t i v e l y at a motoneuron or SMU l e v e l , the e f f e c t s of brainstem reflexes on human SMUs and animal motoneurons w i l l be reviewed. a. Animal Studies Synaptic events associated with jaw reflexes are usually studied using microelectrode techniques applied to jaw muscle motoneurons (Goldberg, 1976; Kidokoro et a l , 1968). Trigeminal i n t r a c e l l u l a r studies have been performed mainly i n cats, although motoneuron studies i n the guinea-pig have been reported (Goldberg and Tal , 1978). The e f f e c t of i n t r a o r a l neural stimulation on the e x c i t a b i l i t y of jaw muscle motoneurons has been studied most extensively i n the masseter and d i g a s t r i c motoneurons of the cat (Goldberg, 1976), with the most common overt e f f e c t being r e f l e x opening of the jaw. When a low threshold e l e c t r i c a l shock i s applied to an i n t r a o r a l nerve, i n h i b i t o r y and excitatory responses may be detected in masseteric motoneurons (Goldberg and Nakamura, 1968; Goldberg, 1976; Kidokoro et a l , 1968). The i n h i b i t o r y e f f e c t appears to be biphasic, with hyperpolarization occurring with a latency of 2-3ms and a duration of 5-7ms. As the motoneuron membrane then begins to depolarize, a reversal i n p o l a r i t y occurs and a second hyperpolarizing p o t e n t i a l i s evident at a latency of 40ms. The second period of membrane hyperpolarization i s followed by a return to prestimulus l e v e l s approximately 2 00ms a f t e r the i n i t i a l d e livery of the stimulus (Goldberg and Nakamura, 1968; Kidokoro et a l , 1968). The f i r s t phase of hyperpolarization i s considered to be the r e s u l t of an i n h i b i t o r y post-synaptic p o t e n t i a l (IPSP), however in co n t r a d i s t i n c t i o n , a presynaptic i n h i b i t o r y mechanism in addition to a post-synaptic event i s implicated i n the second hyperpolarizing period (Goldberg, 1976; Goldberg and Nakamura, 1968; Kidokoro et a l , 1968). Excitatory responses have also been recorded i n response to low-threshold afferent stimulation i n some masseter muscle motoneurons. Short latency (2.5ms) depolarization a f t e r stimulation of the maxillary nerve has been observed, although membrane depolarization beyond res t i n g p o t e n t i a l occurs more commonly at latency of 18ms, between the f i r s t and second hyperpolarization phases (Kidokoro et a l , 1968; Goldberg, 1976). When a low-threshold stimulus i s applied to the l i n g u a l or i n f e r i o r dental nerve of the cat, a short latency (2-4ms) excitatory post-synaptic p o t e n t i a l (EPSP) i s generated which evokes e x c i t a t i o n i n d i g a s t r i c muscle motoneurons (Kidokoro et a l , 1968; Nakamura et a l , 1973). The cat does not perform s i g n i f i c a n t l a t e r a l jaw movements due to the anatomy of i t s temporomandibular j o i n t s , therefore the e f f e c t of afferent stimulation of l a t e r a l pterygoid motoneurons 61 i s uncertain. Sessle and Gurza (1982) have studied reflexes i n the l a t e r a l pterygoid muscles of non-human primates using multiunit and SMU EMG recording techniques. Intraoral afferent stimulation appears to be most e f f e c t i v e i n evoking r e f l e x a c t i v i t y i n both heads of the muscle. Reflex e x c i t a t i o n with a latency of 12ms followed by i n h i b i t i o n l a s t i n g 10-3Oms was reported. A small number of SMU recordings appeared to confirm multiunit r e f l e x records. The r e f l e x l y induced suppression of l a t e r a l pterygoid muscle a c t i v i t y i s at least q u a l i t a t i v e l y s i m i l a r to i n h i b i t o r y periods occurring i n primate and non-primate jaw muscles (Dubner et a l , 1978; Goldberg, 1976; Yemm, 1972). b. Human Studies The r e f l e x e x c i t a b i l i t y of motoneurons i s normally inferred from SMU responses i n the human jaw muscles because i n t r a c e l l u l a r recordings are not possible i n human trigeminal motoneurons. Single motor unit r e f l e x studies have been performed predominantly i n the masseter muscle, with l i t t l e or no information currently available on the behaviour of other jaw muscles such as the d i g a s t r i c or pterygoid muscles. Using mildly-noxious s t i m u l i applied to the l i p , Miles and Turker (1986) have demonstrated that masseteric MUs are in h i b i t e d to a s i m i l a r degree provided the units have the same prestimulus f i r i n g frequency, and therefore the same l e v e l of prestimulus e x c i t a b i l i t y . Miles and Turker (1986) infer r e d that the f i r i n g frequency of the masseteric motoneurons, rather than the siz e of 62 the MU, was the major determinant of the i n h i b i t o r y response. The demonstration that i n h i b i t i o n i s more profound i n a unit with a prestimulus f i r i n g frequency of 10 than 15Hz has important implications for i n h i b i t o r y reflexes, nociceptive and otherwise, because a stimulus w i l l p r e f e r e n t i a l l y i n h i b i t the most slowly f i r i n g units i n a population i r r e s p e c t i v e of MU s i z e (Miles et a l , 1987; Miles and Turker, 1986). The importance of t h i s finding i s apparent when attempts are made to quantitate multiunit r e f l e x responses within and between subjects. The uncontrolled nature of the prestimulus f i r i n g patterns renders the multiunit r e f l e x response e s s e n t i a l l y q u a l i t a t i v e (Miles and Turker, 1986). Further studies on the e f f e c t of l i p stimulation on masseteric MUs have demonstrated the apparent d i v i s i o n of the i n h i b i t o r y period into two phases, separated by a small number of MU action potentials (Miles et a l , 1987). These i n h i b i t o r y phases appear to mirror those recorded i n animal jaw c l o s i n g motoneurons (Kidokoro et a l , 1968), and also the periods of exteroceptive suppression recorded i n human jaw closing muscles using multiunit EMG techniques (reviewed by Lund et a l , 1983), although the latencies are more variable i n the multiunit studies. There are, however, apparent differences i n thresholds of the two i n h i b i t o r y phases. Miles et a l (1987) noted that the second phase could be e l i c i t e d by stimulus i n t e n s i t i e s close to threshold, whereas higher i n t e n s i t i e s were required to e l i c i t the f i r s t phase, i n contrast to the s i t u a t i o n observed i n masseteric motoneurons (Kidokoro et 63 a l , 1968) and i n other human jaw clos i n g muscle r e f l e x studies (reviewed by Lund et a l , 1983)• The d i s p a r i t y i n stimulus thresholds for the second phase of i n h i b i t i o n may be due, in part, to the p e r i o r a l location of the stimulus. Cadden and Newton (1989) have noted that the early phase of exteroceptive suppression may be recorded e a s i l y using i n t r a o r a l s t i m u l i , whereas only very intense s t i m u l i to the skin of the l i p evoked a r e l i a b l e response. The degree of jaw opening may also have a modulatory e f f e c t on an i n h i b i t o r y r e f l e x i n the masseter muscle MUs (Turker and Miles, 1989). With the prestimulus f i r i n g frequency of the units controlled, there are apparent differences i n some MU responses with the i n h i b i t o r y e f f e c t diminishing as the teeth are approximated. Turker and Miles (1988) have also highlighted the importance of the stimulus p o s i t i o n r e l a t i v e to the preceding SMU spikes (whose f i r i n g frequencies are constrained). The sequential i n j e c t i o n of st i m u l i permits the c a l c u l a t i o n and interpretation of the motoneuron membrane recovery curve during an i n h i b i t o r y r e f l e x i n masseteric MUs. c. Summary The mechanisms mediating the integration of jaw muscles are dramatically d i f f e r e n t from those occurring i n the limbs. Oro-f a c i a l sensory afferents appear to play a s i g n i f i c a n t role i n the i n i t i a t i o n and modulation of jaw reflexes, i n contrast to the rec i p r o c a l innervation favoured by the limb muscles. 64 The e f f e c t s of peripheral sensory afferents can be studied at a motoneuron l e v e l i n animals. However, jaw anatomy often d i f f e r s s i g n i f i c a n t l y from man therefore r e f l e x events cannot be studied e f f e c t i v e l y i n a l l jaw muscles, and comparisons with human studies are often problematic. In addition, animals are anaesthetized and sometimes decerebrate during recording procedures, therefore t h e i r behaviour patterns are highly stereotyped, unlike the conditions p r e v a i l i n g during human studies. Multiunit EMG r e f l e x studies i n humans are at best q u a l i t a t i v e . Parameters such as MU f i r i n g frequency and stimulus p o s i t i o n appear to have s i g n i f i c a n t e f f e c t s on the appearance of jaw r e f l e x responses. In order to study r e f l e x behaviour quantitatively, s t r i c t experimental paradigms incorporating SMU recordings seem to be necessary. At present, the r e f l e x behaviour of SMUs i n human jaw muscles has only been studied i n the masseter muscle. 65 V . Motor Unit Twitch Tensions Recruitment of motoneurons normally progresses from small to large motoneurons in a stereotyped manner, r e s u l t i n g i n the early recruitment of small motor units (MUs) followed by larger MUs as the force of muscle contraction increases (Henneman, 1981). The physiological properties of MUs are c l o s e l y linked to the size of the parent motoneurons. Larger motoneurons innervate MUs with larger numbers of f i b r e s . These larger MUs develop higher mechanical forces or "twitch tensions" and faster twitch contraction times than smaller MUs (Burke et a l , 1973; Henneman et a l , 1965a). The r e l a t i v e sizes and numbers of MUs d i f f e r i n d i f f e r e n t muscles according to the precision of movement control performed by the muscle (Buchthal and Schmalbruch, 1980). Goldberg and Derfler (1977) have attempted to quantify single motor unit (SMU) amplitude as a method of estimating SMU s i z e . However, t h i s method i s unreliable because the configuration of an SMU amplitude i s highly dependent on the geometrical r e l a t i o n s h i p of the electrode recording t i p and the f i b r e s of the SMU under study (Buchthal and Schmalbruch, 1980; Desmedt, 1981; Miles et a l , 1986). A method of estimating SMU s i z e based on twitch tension c h a r a c t e r i s t i c s has been c i t e d as a more predictable and r e l i a b l e method (e.g. Brooks, 1986; Carew and Ghez, 1985). However, SMU twitch tensions i n human muscles are highly dependent on the r e l i a b i l i t y of an i n d i r e c t method of measurement. A description of t h i s method of twitch tension measurement i n human muscles w i l l be presented together with an assessment of factors which may p o t e n t i a l l y • modify such measurements. a. Spike-Triggered Averaging O r i g i n a l l y Buchthal and Schmalbruch (1970) u t i l i z e d an intramuscular stimulation technique which incorporated a transducer system i n the muscle tendon, to produce contraction of small numbers of MUs i n various human muscles. The mechanical contributions of these small groups of units were calculated by averaging the resultant force recorded at the muscle tendon. This novel spike-triggered averaging (STA) technique was modified by Stein et a l (1972) so that the mechanical response or "twitch tension" of an SMU could be extracted from the t o t a l muscle force in both human and animal muscles. This was accomplished by recording a compound action potential (CAP) from a SMU, during voluntary a c t i v a t i o n i n humans or by cont r o l l e d e l e c t r i c a l stimulation i n animals, then averaging the force recorded for a s p e c i f i c epoch a f t e r consecutive SMU CAPs. I t was infer r e d that the averaged force response was an accurate measure of the SMU twitch tension because of the asynchronous f i r i n g pattern of motor units within the muscle at low force l e v e l s (Stein et a l , 1972). Spike-triggered averaging has since been widely used in human and animal studies to extract the twitch p r o f i l e s of ind i v i d u a l motor units (Burke, 1981; Calanchie and Bawa, 1986; Goldberg and Derfler, 1977; Milner-Brown et a l , 1973a; Yemm, 1977) but the 67 method i s susceptible to several influences which may bias i t s re s u l t s . Central to the STA technique i s the concept of a l i n e a r mechanical response from an SMU following a single neural impulse, but t h i s premise has been questioned (Partridge and Benton, 1981). The e f f e c t of an impulse from the parent motoneuron may have a variable e f f e c t on the ind i v i d u a l f i b r e s of the SMU, depending on previous a c t i v i t y of the SMU and on the a c t i v i t y patterns of other motor units within the muscle as a whole (Burke et a l , 1976; Demieville and Partridge, 1980). In addition, muscle length and v e l o c i t y , and motor unit f i r i n g frequency may have non-linear e f f e c t s on consecutive mechanical responses of an SMU (Calanchie and Bawa, 1986; Nordstrom et a l , 1989; Partridge and Benton, 1981). In animal experiments, the twitch c h a r a c t e r i s t i c s of SMUs are usually determined by e l e c t r i c a l stimulation of the parent motoneurons at rates of less than 1 pulse per second (pps) (Burke, 1967; McPhedran et a l , 1965). With increasing stimulation rates, consecutive SMU twitches fuse to a degree dependent on the SMU contraction time (Calanchie and Bawa, 1986). Twitch p r o f i l e s of SMUs i n the medial gastrocnemius and soleus muscles of the cat have been shown to vary with the rate of e l e c t r i c a l stimulation of the motor units (Calanchie and Bawa, 1980). With an increasing stimulus rate of 0.2-15pps, the twitch amplitude, contraction time, and ha l f - r e l a x a t i o n time decreased. The decrease i n twitch amplitude was most apparent with increase i n the stimulus rate. The slowly contracting units (mainly soleus muscle) were affected 68 to the greatest degree by an increase i n stimulus rate. Within the human f i r s t dorsal interosseous muscle, however, twitch p r o f i l e s may remain constant when units are stimulated at a slow f i r i n g rate or when they are v o l u n t a r i l y driven, provided the f i r i n g rate i s constrained (Milner-Brown et a l , 1973a). In the f i r s t dorsal interosseous muscle, SMU f i r i n g rates up to 10Hz did not a f f e c t twitch amplitudes and contraction times. Calanchie and Bawa (1986) contended that these findings were due to analysis of a population of fast contracting units because s i m i l a r results were observed i n fast twitch units of cat medial gastrocnemius muscle at comparable rates of stimulation. Nonetheless, i n human muscles SMU twitch tensions may be underestimated due to p a r t i a l fusion of voluntary driven units (Calanchie and Bawa, 1986; Milner-Brown et a l , 1973a; Nordstrom et a l , 1989; Stein et a l , 1972; Yemm, 1977). The twitch tension of human SMUs are usually recorded with the SMU f i r i n g as slowly as possible. In the upper limbs, minimal f i r i n g rates are 8-12Hz (Milner-Brown et a l , 1973b; Monster and Chan, 1977), and by v i r t u e of the slow, continuous nature of t h i s f i r i n g pattern, p a r t i a l fusion of consecutive SMU twitches i s probable. Monster and Chan (1977) demonstrated the ef f e c t of fusion on averaged unit responses i n the human extensor digitorum communis muscle by averaging SMU twitches with the f i r i n g rates of 8, 12, and 16Hz. The largest twitch was recorded with a minimum f i r i n g frequency of 8Hz and was smallest when the background f i r i n g frequency was 16Hz. A s i m i l a r decrease i n twitch amplitude with increasing unit f i r i n g frequency was demonstrated i n human f i r s t dorsal interosseous muscles by Milner-Brown et a l (1973a). Nevertheless, Nordstrom et a l (1989) have shown that i n a fast contracting muscle such as the human masseter muscle, a r e l a t i v e l y unfused twitch may be obtained provided that the i n t e r -spike i n t e r v a l s before and a f t e r the SMU spikes used to tr i g g e r the averaging procedure are c a r e f u l l y selected. At voluntary f i r i n g frequency of 7.5-lOHz during STA the twitch p r o f i l e does not seem to be affected appreciably (Miles et a l , 1987; Nordstrom et a l , 1989; Stein et a l , 1972; Yemm, 1977). b. Biomechanical Linkage of the Jaw During the development of t h e o r e t i c a l models, anatomical j o i n t s are often considered to be hinge-like, and, i n subsequent analyses the loads are treated as r i g i d bodies with a constant centre of mass and a constant moment of i n e r t i a around the j o i n t axes (Partridge and Benton, 1981). Such models of human j o i n t s are s i m p l i s t i c , e s p e c i a l l y when complex j o i n t s such as the paired temporomandibular a r t i c u l a t i o n s are considered. In the non-human primate jaw, muscle forces are re s i s t e d both at the b i t e point and the temporomandibular a r t i c u l a t i o n during mastication and tooth-clenching (Hylander, 1975). Likewise, the human jaw has been described as a loaded beam with multiple supports, i n which the summed forces of the jaw elevator muscles are opposed by reaction forces at the temporomandibular a r t i c u l a t i o n and the tooth b i t e point (Smith, 1978). Therefore, when biomechanical analyses of the human or primate jaw are contemplated, calculations of 70 reaction forces and moments i n the l a t e r a l jaw projection are necessary (Hylander, 1975). Midline i n c i s a l b i t i n g and b i l a t e r a l molar b i t i n g acts may be analysed adequately i n the l a t e r a l jaw dimension, however, u n i l a t e r a l b i t i n g acts should also be analysed in the f r o n t a l jaw projection because j o i n t reaction forces frequently d i f f e r between working and non-working side temporomandibular a r t i c u l a t i o n s (Hylander, 1975). Goldberg and Derfler (1977) and Yemm (1977) employed a b i t e force transducer located u n i l a t e r a l l y i n the premolar region of the dental arch to record twitches i n the human masseter (Yemm, 1977; Goldberg and Derfler, 1977) and temporalis muscles (Yemm, 1977) , and assumed that the twitch tensions generated by SMUs i n the muscles were r e f l e c t e d i n the force recorded at the b i t e point by STA. In monkeys, Clark et a l (1978) employed a midline b i t e force transducer i n the i n c i s o r region to measure the SMU twitch tensions i n the temporalis muscle again without considering the biomechanical implications. Since tension generated by a l l or part of the masseter muscle, which i s located midway between these s i t e s of resistance v i z . b i t e point and temporomandibular a r t i c u l a t i o n , i s only p a r t l y r e s i s t e d at the s i t e of the recording device, twitch tensions reported previously for SMUs i n t h i s muscle are l i k e l y to have been too low. A s i m i l a r biomechanical s i t u a t i o n applies for the primate and human temporalis muscles and has resulted i n apparently low twitch tension values for SMUs i n these muscles (Luschei and Goldberg, 1981). Because the jaw behaves as a loaded beam under conditions of isometric 71 contraction, jaw reaction forces and moments should be considered, and putative twitch tensions measured at a b i t e point recalculated to r e f l e c t jaw torque. Biomechanical constraints are further complicated by the fact that i n humans the jaw muscle lever arms vary between subjects (Hannam and Wood, 1989), therefore twitch tension recordings i n jaw muscles may also be biased by variations i n the c r a n i o f a c i a l morphology of the t e s t sample. c. E f f e c t s of Muscle Architecture The concepts of muscle s t i f f n e s s and compliance are important to the understanding of the e f f e c t of muscle architecture on muscle twitch tensions, therefore d e f i n i t i o n s of these terms from Houk and Zev Rymer (1981) and Carew and Ghez (1985) w i l l be described. Muscle s t i f f n e s s may be defined i n terms of a force to length r a t i o . This r a t i o may be measured under both s t a t i c and dynamic conditions: s t a t i c s t i f f n e s s - The r a t i o of change i n force and the length change causing the force, measured under steady state conditions. dynamic s t i f f n e s s - Incremental dynamic s t i f f n e s s (Sf/Sx). The r a t i o of increase i n force to increased length, measured under transient conditions. Instantaneous dynamic s t i f f n e s s (df/dx). This i s equivalent to the measured slope of a p l o t of time against force during the constant v e l o c i t y phase of a ramp change i n muscle length. compliance - The r a t i o of change i n length to change i n force i . e . the inverse of s t i f f n e s s . The a r c h i t e c t u r a l design of a muscle has a s i g n i f i c a n t e f f e c t on i t s motor performance (Armstrong et a l , 1988). The precise mechanical action, the contraction behaviour, and the length-tension properties of a muscle are linked to the complexity of the f i b r e arrangement within muscles (Loeb et a l , 1987). The anatomical arrangement of connective tiss u e within muscle may also influence muscle function (Borg and C a u l f i e l d , 1980). Differences i n compliance between muscles have been related to the d i f f e r i n g quantities and arrangements of connective t i s s u e within muscles (Borg and C a u l f i e l d , 1980). However, the e f f e c t of connective tissue arrangement on the mechanical properties of muscle i s unclear (Borg and C a u l f i e l d , 1980). Connective tiss u e i s present i n the muscle sarcolemma, between muscle f i b r e s , connected to the ends of f i b r e s , and i n aponeuroses and tendons which connect muscles to s k e l e t a l structures (Partridge and Benton, 1981; Proske and Morgan, 198 4). These connective t i s s u e elements have inherent e l a s t i c properties, and appear to operate both " i n s e r i e s " and " i n p a r a l l e l " with the c o n t r a c t i l e components of muscles (Close, 1973; H i l l , 1950; Partridge and Benton, 1981). The d i s t r i b u t i o n of connective tissue 73 varies considerably between muscles (Loeb and Gans, 1986; Proske and Morgan, 1984). In simple muscles such as the fusiform biceps b r a c h i i , connective tissue (epimysium) envelops the muscle mass to form a discrete bundle. Within more complex muscles such as the multipinnate d e l t o i d or masseter muscles, muscle f i b r e s attach to connective tiss u e tendons which form aponeurotic sheets that spread out l a t e r a l l y within the muscles (Loeb and Gans, 1986; Proske and Morgan, 1984) . This connective tiss u e architecture i s important i n the generation of muscle s t i f f n e s s . When stretch i s applied to a muscle, i t i s d i s t r i b u t e d between muscle f i b r e s and tendons according to t h e i r r e l a t i v e s t i f f n e s s e s . However, muscle f i b r e s t i f f n e s s i s non-linear, even at r e l a t i v e l y constant levels of a c t i v a t i o n (Proske and Morgan, 1987). In addition, although muscle compliance decreases with increases i n load, i t i s subject to s i g n i f i c a n t v a r i a t i o n depending on the damping c h a r a c t e r i s t i c s of muscle tissues (Proske and Morgan, 1987). The extent to which muscle architecture and connective tissue arrangement i n p a r t i c u l a r a f f e c t SMU twitch tensions i s uncertain. I t i s conceivable that increasingly complex connective tissue septa, i n addition to complex f i b r e arrangements could a f f e c t twitch tension measurements. Certainly, reports of SMU twitch p r o f i l e s i n p a r a l l e l - f i b r e d muscles with simple connective tissue architecture such as the human f i r s t dorsal interosseous muscle are f a i r l y consistent (Desmedt, 1983; Milner-Brown et a l , 1973a; Thomas et a l , 1986). They are not, however, for the multipinnate masseter muscle. Yemm (1977) measured SMU twitch tensions of 9.8-297.OmN, while Goldberg and Derfler (1977) reported twitches as high as 2N. Although t h i s inconsistency may have been due to the d i f f e r e n t types of units sampled the complex in t e r n a l anatomy of the masseter muscle complicates the int e r p r e t a t i o n of twitch tension measurements. Motor unit tension per se depends on the innervation r a t i o , the cross-sectional area of the muscle f i b r e s and t h e i r s p e c i f i c tension output, but these factors may be modified by muscle architecture (Burke and T s a i r i s , 1973; Loeb et a l , 1987). Monster and Chan (1977) demonstrated the presence of a mechanical s t i f f n e s s gradient for SMUs with d i f f e r e n t force thresholds i n which the r e l a t i v e s t i f f n e s s of the muscle-tendon-force transducer interface was greater for larger units. Therefore, any mechanical s t i f f n e s s gradient due to the sel e c t i v e a c t i v a t i o n of d i f f e r e n t sizes of SMUs with d i f f e r e n t force thresholds would have a non-linear e f f e c t on measured twitch tensions. To compound t h i s e f f e c t , the compliance of tissues such as skin and tendon l y i n g between the active unit and the force transducer can also cause non-linear e f f e c t s as muscle forces vary according to the recruitment threshold of d i f f e r e n t SMUs (Burke, 1981). By convention, the l i n e of action of a muscle force i s determined by connecting the centroids of the regions of o r i g i n and i n s e r t i o n of the muscle (Weijs, 1980) . This method i s ef f e c t i v e i n simple, p a r a l l e l - f i b r e d muscles with discrete regions of o r i g i n and ins e r t i o n . In a pinnate muscle however, the ca l c u l a t i o n of i t s l i n e of action i s more complex because 75 connective t i s s u e septa a l t e r the d i s t r i b u t i o n of force within the muscle (Weijs, 1980). Nonetheless, provided the configuration of the muscle architecture i s known, a putative l i n e of action may be calculated. However, to confer a single l i n e of action on a pinnate muscle presumes uniform behaviour of MUs within the muscle. English (1985) has demonstrated that a l l MUs are not necessarily active during the function of the parent muscle. In the multipinnate pig masseter muscle, Herring et a l (1979) demonstrated multiple l i n e s of action based on contraction of discrete regions of the muscle. A s i m i l a r heterogenous behaviour has been demonstrated i n the rabbit masseter muscle (Weijs and Dantuma, 1981). In human muscles, d i f f e r e n t contraction strategies can be used to perform the same task, as demonstrated i n the masseter muscle (Eriksson et a l , 1984; Sasaki et a l , 1989) and i n the adductor p o l l i c i s brevis muscle (Thomas et a l , 1987), suggesting non-uniform behaviour within the muscles. The observed pote n t i a l for d i f f e r e n t i a l a c t i v a t i o n i n a multipinnate muscle could conceivably a l t e r the environment in which SMU twitches are generated i n an uncontrolled manner. Such unpredictable changes i n muscle s t i f f n e s s as a r e s u l t of heterogenous contraction patterns within a complex muscle may a l t e r the amplitude of twitches recorded remotely from units. d. E f f e c t s of Muscle Coactivation Anatomical j o i n t s are moved by agonistic and antagonistic muscles (Carew and Ghez, 1985; DeLuca and Mambrito, 1987). 76 Contraction of the agonist may occur i n concert with relaxation of the antagonist muscle by means of r e c i p r o c a l innervation (Sherrington, 1909) . The net r e s u l t i s a high torque and low s t i f f n e s s at the j o i n t . A l t e r n a t i v e l y coactivation of the agonist and antagonist may occur (De Luca and Mambrito, 1987; Smith, 1981). This leads to increased s t i f f n e s s at the j o i n t , as a consequence of increased contraction and s t i f f n e s s of both agonist and antagonist muscles. Muscle coactivation i s often used i n the maintenance of a steady limb p o s i t i o n (Loeb, 1985) and to control posture (Somjen, 1983) because i t provides a more stable muscle set against unexpected changes i n external forces. By increasing muscle s t i f f n e s s , coactivation a l t e r s the intern a l environment of both agonist and antagonist muscles. The mechanical s t i f f n e s s gradient has been shown to increase non-l i n e a r l y as SMU force thresholds increase (Monster and Chan, 1977) . Any further increment of s t i f f n e s s as a r e s u l t of coactivation of antagonistic muscles could have an additive e f f e c t on the amplitude of SMU twitches within muscles. Summary The interpretation of SMU twitch tension measurements i n human muscles may be complicated by a number of factors including the STA technique i t s e l f , j o i n t biomechanics, muscle architecture, and muscle co-activation. Precise control of SMU f i r i n g rate i s necessary during the STA procedure, and rigorous c r i t e r i a are essen t i a l for the sel e c t i o n of in d i v i d u a l force records p r i o r to 77 averaging. The implications of biomechanical linkage should also be addressed as i t may be erroneous to assume that the twitch tensions measured at a s i t e remote from the muscle SMU accurately r e f l e c t the t o t a l mechanical contribution of SMUs. This i s p a r t i c u l a r l y so when the temporomandibular a r t i c u l a t i o n s are considered. Muscle architecture, and s t i f f n e s s and compliance i n pa r t i c u l a r , may a f f e c t twitch tension measurements e s p e c i a l l y i n multipinnate muscle such as the masseter, where the internal architecture may vary considerably. The e f f e c t s of coactivation of antagonistic muscles also p o t e n t i a l l y complicates the interp r e t a t i o n of twitch tensions, i n a l l human muscles. 78 11. STATEMENT OF THE PROBLEM Regional differences i n muscle a c t i v i t y patterns have been recorded electromyographically i n anatomically complex muscles, and i n a number of animal studies such functional heterogeneity has been related to d i s t i n c t muscle subvolumes or "compartments". The functional quantum of muscle contraction i s the motor unit, but there i s very limited information on single motor unit behaviour i n the human jaw muscles. The complex architecture of the human masseter muscle i n p a r t i c u l a r confers the potential for multiple l i n e s of action during function, and i s r e f l e c t e d i n motor units which appear to behave d i f f e r e n t l y according to the task performed, and whose t e r r i t o r i e s may be d i s t r i b u t e d i n discrete regions of the muscle. The extent of t h i s relationship between peripheral functional and anatomical organization i s however unclear. The degree to which motor unit r e f l e x behaviour i s modified by d i f f e r e n t a c t i v a t i o n strategies i s also uncertain. In addition, there i s doubt regarding the v a l i d i t y of motor unit twitch tensions previously measured i n the masseter muscle because these estimations may be biased by muscle contraction strategies and jaw biomechanics, neither of which seem to have been previously considered as c o n t r o l l i n g variables. 79 In the present study, the following s p e c i f i c questions were posed : 1. What are the sizes and three-dimensional locations of single motor unit t e r r i t o r i e s within the human masseter muscle, and how do they r e l a t e to putative muscle compartments? 2 . How t a s k - s p e c i f i c are masseter motor units during voluntary muscle contraction? Are units r e s t r i c t e d to s p e c i f i c muscle regions or compartments based on t h e i r task s p e c i f i c i t y ? Is motor unit control, as manifested by lowest sustainable f i r i n g frequency, modulated according to the task performed? 3. , How i s motor unit r e f l e x behaviour i n the masseter muscle modified by exteroceptive stimuli? Do d i f f e r e n t a c t i v a t i o n strategies a f f e c t unit r e f l e x behaviour? 4. How does unit r e f l e x behaviour i n the l a t e r a l pterygoid muscle, both a synergist and antagonist of the masseter muscle, compare with that i n the masseter muscle? 5. How do changes i n muscle contraction strategies and jaw biomechanical linkage a f f e c t twitch tension measurements i n the masseter muscle? It was reasoned that answers to these questions would enhance the current understanding of normal motor unit topography and behaviour within the human masseter muscle s p e c i f i c a l l y , and provide further insight into the organizing p r i n c i p l e s of complex human muscles generally. 80 In order to answer the proposed questions, several general technical methods had to be developed, v i z . a r e l i a b l e method for SMU EMG recording i n the masseter muscle, a method of imaging jaw muscles, and a ste r e o t a c t i c method for locating needle electrode recording s i t e s within muscles. The development of these methods w i l l be described i n the following chapter devoted to general experimental methods. 81 GENERAL METHODS The methods employed i n the following studies contain elements common to a l l the experiments and elements which are related to pa r t i c u l a r experimental paradigms. For c l a r i t y , methods common to the experiments w i l l be presented i n a section e n t i t l e d "General Methods". In subsequent sections, s p e c i f i c methods w i l l be combined with the re s u l t s for each experimental paradigm due to th e i r inherent interdependency. A. SINGLE MOTOR UNIT RECORDING 1. Technique Single motor unit (SMU) electromyographic (EMG) recordings were performed using a monopolar needle electrode (MF37, TECA Corp., P l e a s a n t v i l l e , NY) . The needle was fabricated of sta i n l e s s s t e e l , and insulated with Teflon (PTFE) except at i t s c o n i c a l l y -sharpened t i p . The needle was s l i g h t l y f l e x i b l e , with a length of 37mm, a diameter of 0.35mm (28 gauge), and a recording area of 0.12mm2. A surface electrode (SynCor, Lec Tec Corp., Minnetonka, MN) placed over the main b e l l y of the masseter muscle acted as a reference electrode. A second surface electrode fixed to the nape of the neck served as a ground. The needle was introduced percutaneously at ri g h t angles to the skin surface and i t s po s i t i o n s t a b i l i z e d by means of a l i g h t metal platform taped to the skin of the cheek (Figure 1). Single 82 Figure l . Single motor unit recording technique i n the masseter muscle. A surface reference electrode with a hole i n i t s centre i s located over the body of the r i g h t masseter muscle. A metal g r i d i s positioned over the reference electrode and secured to the skin of the face by adhesive tape. A monopolar needle electrode i s inserted into the muscle through the hole i n the reference electrode, then s t a b i l i z e d i n s i t u by the metal g r i d . 83 motor unit a c t i v i t y was amplified (Model AL 2010, Axon Instruments Inc., Burlingame, CA), and bandpass-filtered. With the masseter muscle l i g h t l y activated, the needle was moved i n and out of the muscle along a medio-lateral t r a j e c t o r y u n t i l a compound action po t e n t i a l (CAP) was obtained from a group of SMU f i b r e s located at the needle t i p . 11. E f f e c t of Bandpass-Filtering The signal obtained by a monopolar needle represents the temporal and s p a t i a l summation of action potentials (AP) from a variable number of muscle f i b r e s located around the needle t i p . The shape of the SMU CAP may be affected by the distance between the recording t i p and the muscle f i b r e group, and the volume conduction properties of the muscle. Distant f i b r e s are responsible for the slower components of the CAP due to the low-pass f i l t e r e f f e c t of muscle tissu e on volume conduction (Stalberg and T r o n t e l j , 1979). When the f i l t e r i n g c h a r a c t e r i s t i c s of the EMG amplifier are altered the shape of the SMU CAP may vary s i g n i f i c a n t l y , p a r t i c u l a r l y when the high-pass f i l t e r i s changed. Figure 2 shows changes i n the waveform shape of a CAP recorded from a continuously f i r i n g masseter SMU i n a normal subject when the high-pass f i l t e r was progressively changed from 100Hz to 1kHz, with the low-pass f i l t e r remaining constant at 10kHz. When the high-pass f i l t e r s e t t i n g was altered from 100 to 3 00Hz, the amplitude of the signal did not decrement s u b s t a n t i a l l y , although slower frequency components from more distant f i b r e s were reduced. 84 Figure 2 . E f f e c t s of bandpass-filtering a single motor unit EMG si g n a l . Waveform configuration of an averaged (n=16) SMU compound action p o t e n t i a l with changes i n amplifier bandpass-filters. In a. the bandwidth i s lOOHz-lokHz, i n b. 300Hz-10kHz, and i n c. lkHz-lOkHz. 85 When a high-pass f i l t e r of 1kHz was used, the amplitude of the waveform was diminished quite dramatically as the signal tended towards the f i r s t derivative of the o r i g i n a l s i g n a l . The duration of the CAP and, correspondingly, the in t e g r a l of the waveform shape, were also considerably reduced, i n f e r r i n g that the needle was recording from a s i g n i f i c a n t l y smaller population of SMU fi b r e s and thus appeared to be more focal i n terms of i t s volume of pick-up. The increase i n s e l e c t i v i t y of EMG recordings achieved by hi g h p a s s - f i l t e r i n g has been described previously (Gath and Stalberg, 1976; Stalberg and T r o n t e l j , 1979), and i s a useful technique when se l e c t i v e preservation of high frequency components of a signal i s desired. The higher s p a t i a l resolution achieved by bandpass-filtering i s useful i n single f i b r e EMG studies, and also in SMU t e r r i t o r i a l mapping. When multiple needles are used simultaneously, i t minimizes the p o s s i b i l i t y of recording from the same group of SMU f i b r e s . The recording s e l e c t i v i t y of the monopolar electrode obtained by f i l t e r i n g the low frequency components from the amplified signal was investigated i n four masseter MUs of two normal male subjects aged 34 years. A small adhesive tape, graduated i n mms was attached to the Teflon coating of the needle. The needle was inserted percutaneously at ri g h t angles to the skin surface and supported by a metal framework (as described previously), so that the scale on the g r i d could be v i s u a l i z e d r e l a t i v e to the metal framework 86 Figure 3 . Measurement of needle electrode i n s e r t i o n along a medio-l a t e r a l t r a j e c t o r y within the masseter muscle. A small adhesive tape graduated i n nuns attached to a needle electrode whose t i p i s located within the muscle. Needle penetration i n the medio-lateral t r a j e c t o r y i s measured r e l a t i v e to an o r i f i c e i n the metal framework. 8 7 Figure 4 . Changes i n a single motor unit EMG signal according to the p o s i t i o n of the electrode recording t i p . Changes i n waveform shape of an averaged (n=16) SMU compound action p o t e n t i a l as the needle t i p i s moved ±2mm along a medio-lateral axis from the location of the signal of maximum amplitude. 88 (Figure 3) . The amplifier bandpass-filters were adjusted to eliminate signal frequency components beyond the range lkHz-lOkHz throughout the recording session. The needle was moved within the muscle u n t i l a SMU CAP was observed on a d i g i t a l oscilloscope screen (Model 2221, Tektronix Canada Inc., Vancouver, B.C.) during the performance of a gentle tooth clenching task. While the SMU f i r e d continuously, the needle was moved manually along i t s medio-l a t e r a l t r a j e c t o r y u n t i l a CAP of maximum amplitude was obtained i . e . the needle t i p was closest to the SMU. I t was considered too cumbersome to use a micromanipulator (Stalberg and Eriksson, 1987) to move the needle i n the muscle cross-section, because the needle was repeatedly inserted and retracted i n order to locate a CAP of maximum amplitude. The CAP was used to t r i g g e r the oscilloscope sweep. An averaged SMU CAP was recorded and plotted (Model HC1000, Tektronix Canada Inc., B.C.). The needle was moved along a medio-l a t e r a l trajectory, f i r s t l y deep, then s u p e r f i c i a l to the primary recording s i t e i n mm increments by means of the graduated scale on the needle referenced to the supporting metal framework. Averaged CAPs from the SMU were obtained at positions +2, +1, -1, -2mm r e l a t i v e to the primary recording s i t e (Figure 4). There was a s i g n i f i c a n t reduction i n the amplitude of the SMU CAP as the needle was moved further from the s i t e of the SMU within the muscle v i z . a 55-85% decrease i n the sig n a l amplitude at a distance of 2mm. Four masseter SMUs were examined systematically i n t h i s manner. The voltage r a t i o s (dB) for each unit are depicted i n Figure 5. 89 Figure 5. Variations i n voltage r a t i o s of four single motor unit EMG signals with changes i n the po s i t i o n of the electrode recording t i p . The voltage r a t i o (dB) i s plotted against distance from the location of the signal of maximum amplitude compound action potentials for four SMUs. The error bars represent one standard deviation. 2 ~5 3 g < cc U J 1 § - 1 0 \ \ • / \ / / 1 i T" -2 -1 0 +1 DISTANCE (mm) +2 90 Based on these findings, the volume of pick-up of the monopolar needle electrode was estimated to be approximately ±2mm, when an amplifier bandwidth of lkHz-lOkHz was used. This i s comparable with the value for monopolar needles reported by Stalberg (1980). An amplifier bandwidth of lkHz-lOkHz was therefore considered p o t e n t i a l l y useful for current experiments requiring a high degree of s p a t i a l resolution. Nonetheless the EMG signal i s considerably truncated and not suitable for waveform quantitation, nor i s i t an appropriate bandwidth for an i n i t i a l survey of SMU a c t i v i t y in a s p e c i f i c muscle location. In the following experiments, SMU EMG signals were i n i t i a l l y bandpass-filtered lOOHz-lOkHz when viewed on the d i g i t a l oscilloscope screen, even though t h i s amplifier bandwidth did not always provide an optimum signal to noise r a t i o . Since a bandwidth of 300Hz-10kHz yielded an acceptable amplitude r a t i o between fas t and slow components of the s i g n a l , an adequate d e f i n i t i o n of waveform s i n g u l a r i t i e s , and an acceptable signal to noise r a t i o , bandpass-filtering of 300Hz-10kHz was used i n the majority of ensuing SMU recordings. An exception was made in the MU mapping study where a bandwidth of lkHz-lOkHz was employed. B. STEREOTACTIC LOCATION OF NEEDLE ELECTRODE RECORDING SITES 1. Morphological Application Magnetic Resonance (MR) imaging has become a useful technique for the three-dimensional (3-D) reconstruction of human anatomical structures, including the o r o - f a c i a l complex. Three-dimensional 91 reconstructions of o r o - f a c i a l tissues, including muscles, have permitted quantitation of jaw muscle cross-sectional areas (Hannam and Wood, 1989) . Recently MR imaging has been employed as an imaging modality for stereotactic neurosurgery (Leksell et a l , 1985; Schad et a l , 1987). Clear v i s u a l i z a t i o n of target anatomical structures has been achieved using MR images generated i n three orthogonal planes (Leksell et a l , 1985). Good tissue contrast and the a b i l i t y to obtain t i s s u e sections at multiple angles has offered d i s t i n c t advantages over such invasive procedures as X-ray Computerized Tomography (CT), ventriculography and angiography. Computerized tomography i s also a sensitive method for imaging human tissues; however, i t s use i s limi t e d because of the pot e n t i a l radiation hazard, e s p e c i a l l y when normal, healthy tissues are to be imaged. MR imaging has been reported to be superior to CT i n the characterization of normal o r o - f a c i a l t i s s u e architecture (Seltzer and Wang, 1987). Although the resolution of MR imaging does not as yet permit v i s u a l i z a t i o n of jaw muscle f i b r e s , muscle compartments separated by connective t i s s u e septa, and discrete f a s c i c l e s can be demonstrated within jaw muscles (Lam et a l , 1989) . Because of the potential u t i l i t y of MR imaging as an adjunct in the st e r e o t a c t i c location of s p e c i f i c anatomical e n t i t i e s within human o r o - f a c i a l tissues, and jaw muscles i n p a r t i c u l a r , a b r i e f review of the MR imaging technique w i l l be presented. 92 11. Magnetic Resonance Imaging Technique Proton MR imaging i s a non-invasive imaging technique, which u t i l i z e s a high strength s t a t i c magnetic f i e l d , pulsed radiowaves, and switched gradient magnetic f i e l d s to generate images of anatomical features (Lam et a l , 1989). These imaging systems exploit the magnetic properties of the hydrogen nucleus (proton) to generate the MR signal (Nixon, 1987). Hydrogen protons, which possess an inherent spin, can ex i s t i n two spin states and the magnetic dipole moments which are produced are oriented randomly within a tis s u e . The (superconducting) magnets used i n MR imaging possess stationary magnetic f i e l d s from 0.15-1.5 Tesla. When a tissue containing protons i s placed into a strong stationary magnetic f i e l d (SMF) a net magnet dipole moment (the magnetizing vector, MV) i s produced within the tiss u e , as the in d i v i d u a l magnetic dipole moments a l i g n p a r a l l e l with the SMF. Under these conditions, each proton precesses at the same rate, although the precession i s not synchronized i . e . not i n phase. Energy derived from the pulsed radiowaves causes the protons to precess i n phase. When the radiofrequency (RF) pulse i s switched o f f , the protons dephase and high energy protons return to the low-energy state. The time for dephasing i s c a l l e d "T2", or the spin-spin relaxation time. The time taken for protons to ree s t a b l i s h t h e i r equilibrium condition i n the two d i f f e r e n t energy states i s c a l l e d "TI", or the s p i n - l a t t i c e relaxation time. RF pulse sequences such as "inversion recovery" and "spin-echo" which ex p l o i t t i s s u e TI and T2 differences respectively, permit improved d i f f e r e n t i a t i o n of anatomical features (Stewart, 1987). Radiofrequency receiver c o i l s fabricated from copper are placed close to the subject during the imaging procedure. Throughout the RF pulse sequence, the MV cuts across these copper c o i l s . The RF c o i l picks up multiple signals, each with s p e c i f i c features of phase and frequency which describe each signal's location within the tissue being imaged. Both hard and soft tissues may be imaged, but soft tissue d e f i n i t i o n i s better p a r t i c u l a r l y when appropriate RF pulse sequences are employed. The RF signal i n t e n s i t i e s of musculoskeletal tissues vary considerably, being of highest i n t e n s i t y for f a t and medullary bone, intermediate i n t e n s i t y for muscle and of low in t e n s i t y for blood vessels, ligaments, tendons and c o r t i c a l bone (Berquist, 1987). Materials which contain s i g n i f i c a n t amounts of iron, cobalt, n i c k e l , and chromium may exhibit ferromagnetic properties due t h e i r high magnetic permeabilities (Lam et a l , 1989; Seltzer and Wang, 1987). M e t a l l i c dental f i l l i n g s and appliances produce low signal a r t i f a c t s on MR images; however, these are less severe than the degradation e f f e c t s observed on CT images (Fache et a l , 1987; Seltzer and Wang, 1987). 111. Development of a Stereotactic Method Needle electrodes are often employed to determine the d i s t r i b u t i o n of groups of f i b r e s or single f i b r e s of a motor unit (MU) within a muscle (Stalberg et a l , 1976; Stalberg and Eriksson, 94 1987; Buchthal and Schmalbruch, 1980). The technique, based upon uni a x i a l cross-sectional scanning, was o r i g i n a l l y used to study MU t e r r i t o r y i n p a r a l l e l - f i b r e d limb and trunk muscles (Buchthal et a l , 1957). However many muscles, such as those i n the human jaws, are multipinnate, incorporating numerous connective tissue septa which form compartments (Schumacher, 1982). The re s u l t s of needle electrode scans through such muscles are necessarily biased because the t o t a l number of fi b r e s belonging to a MU may not be re f l e c t e d i n the p a r t i c u l a r cross-section sampled due to the various f i b r e angulations. A preferred approach would be to sample several regions of the muscle simultaneously, and to record the locations of the recording s i t e s i n three dimensions of space. The a v a i l a b i l i t y of MR imaging and i t s apparent safety prompted the development of a st e r e o t a c t i c method for locating needle electrode recording s i t e s within the human masseter muscle as a precursor to mapping i t s MU t e r r i t o r i e s . IV. The Position of the Needle Electrode Tip Relative to Anatomical Landmarks. I n i t i a l l y , a reference plane was defined by a r i g i d extraoral square g r i d made of non-ferrous materials (Figure 6) . The g r i d incorporated four copper sulphate (0.001M) f i l l e d glass tubes with four 90 degree line-angles. I t was attached r i g i d l y to a p l a s t i c b i t e - f o r k which was indented and held firmly between the teeth. The contour of each b i t e - f o r k was customized by means of a c r y l i c r e s i n , at a tooth separation of about 1mm from f u l l tooth 95 F i g u r e 6. A r e f e r e n c e g r i d a t t a c h e d t o a b i t e - f o r k . The four glass tubes form a square. Four point angles are v i s i b l e , with a distance of 42mm between each point. 96 intercuspation. At the end of each recording session, while the needle was s t i l l placed intramuscularly, an o p t i c a l system (Reflex Metrograph, HF Ross, Salisbury, Wilts., England) with a resolution of 0.1mm (Takada et a l , 1983) was used to measure the three-dimensional coordinates of the grid's point angles. Planar regression was used to calculate a plane of reference from the positions of the four point angles. The length of the needle electrode was a known variable. Therefore, by determining the three-dimensional coordinates of the needle hub and a second v i s i b l e point on the needle, the p o s i t i o n of the needle t i p r e l a t i v e to the coordinates of the reference plane was calculated (Figure 7). A Picker V i s t a MR1100 system with a 0.15T magnet was used to obtain images of selected anatomical regions. During imaging sessions, each subject again held the reference g r i d between the teeth. A surface r e c e i v i n g - c o i l (copper) was placed over the gr i d , the masseter muscle and the temporomandibular j o i n t (TMJ). Spin-echo sequences with a fixed r e p e t i t i o n time (TR:833ms) and echo time (TE:60ms) were used to obtain a series of contiguous, 5mm sections i n the a x i a l plane (Figure 8). The t o t a l imaging time was 30min. The images included the o r o - f a c i a l region ( s p e c i f i c a l l y the r i g h t masseter muscle, the i p s i l a t e r a l temporomandibular j o i n t , and the maxillary r i g h t canine tooth) and the copper sulphate within the glass tubes attached to the g r i d (Figure 7) . The copper sulphate appeared as ovoid markers (el l i p s e s ) i n contiguous sections. From each a x i a l section, selected anatomical features 97 Figure 7. Relationship of the reference g r i d to o r o - f a c i a l landmarks and Magnetic Resonance imaging plane of section. The pos i t i o n of the g r i d r e l a t i v e to the r i g h t masseter muscle and a needle electrode within the muscle i s shown on the r i g h t . On the l e f t , the r e l a t i o n s h i p of 16 contiguous a x i a l Magnetic Resonance sections to the right masseter muscle, the r i g h t a r t i c u l a r condyle, the teeth, and the g r i d are depicted. 98 Figure 8 . A x i a l spin echo Magnetic Resonance image. The masseter muscle (M), the mandibular ramus (R), the maxillary teeth including the canine (Ca), and ovoid markers (arrows) are depicted. The image was obtained with a surface c o i l (TR:833ms, TE:60ms). The scale bar represents 2cm. and the centroid of each e l l i p s e were traced then d i g i t i z e d by-means of a d i g i t i z e r and computer, (Models HP9874 and HP350, Hewlett Packard Canada, Vancouver, B.C.)- Each set of d i g i t i z e d sections was configured to form a three-dimensional matrix and displayed graphically. The location of the reference plane r e l a t i v e to the anatomical landmarks' was calculated according to trigonometric p r i n c i p l e s . The positions of the centroids of the copper sulphate e l l i p s e s and the regression l i n e s through these points were displayed on the computer terminal i n order to reconstruct the square shape of the g r i d (Figure 9). The plane defined by the g r i d was also calculated. The reference square previously obtained from the Metrograph data was also displayed and made co-planar with the MR-generated reference square (Figure 9) . I t was superimposed f i r s t by rotating the square around a selected point angle, then by t r a n s l a t i n g the Metrograph-generated square u n t i l a p o s i t i o n of best f i t was achieved. This computer merging of two reference squares, plus the data sets associated with them, permitted the location of the needle electrode t i p to be displayed graphically within the reconstructed masseter muscle. V. Error of Measurement The accuracy with which the needle t i p p o s i t i o n could be determined r e l a t i v e to the Metrograph-generated reference plane was tested i n the following way. A needle electrode was inserted through a simulated masseter muscle i n a dry s k u l l so that the needle t i p was v i s i b l e . This permitted the location of the needle 100 Figure 9 . Metrograph and Magnetic Resonance-generated reference squares. On the l e f t , the centroids of the imaged copper sulphate e l l i p s e s are plotted and regression l i n e s through the points form the Magnetic Resonance-generated reference square. Superimposition of the Magnetic Resonance and metrograph-generated reference squares are demonstrated on the r i g h t . The scale bar represents 2 cm. 101 t i p to be calculated as before, and a comparison to be made with i t s actual d i g i t i z e d p o s i t i o n r e l a t i v e to the reference points. This error was ±0.8mm, but did not take into account any movement a r t i f a c t which may have occurred with a l i v i n g subject. The Picker V i s t a MR1100 system used i n t h i s study had been examined previously for inherent n o n - l i n e a r i t i e s caused by inhomogeneities i n the s t a t i c magnetic and radiofrequency f i e l d s . A volume of 12100cm3 at the centre of the magnet c o i l was determined to be homogenous i n terms of d i s t o r t i o n (Stewart, 1987). The subjects' heads were positioned within t h i s l i n e a r environment. Errors were also possible during the reconstruction of the MR image-generated reference plane. Occasionally i t was d i f f i c u l t to locate the centroid of an e l l i p s e when an air-bubble was present i n one of the glass tubes. In these instances any such spurious data were eliminated before the c a l c u l a t i o n of regression l i n e s . At l e a s t three centroid points on each limb of the g r i d were used to calculate the regression l i n e s . The accuracy of t h i s procedure was always determinable because the reconstruction of the g r i d t h e o r e t i c a l l y formed a square with sides of known dimension, i n t h i s case 42mm. In order to f i n a l i z e the shape and s p a t i a l l o c ation of the MR-generated gri d , the section i n t e r v a l was adjusted. For an i n t e r v a l of 5mm the error was ±10%, due to the nature of the tuning curves for each section i n t e r v a l used with the Picker system. Section i n t e r v a l s adjusted within these l i m i t s (5.0±0.5mm) produced a square-shaped reconstruction with sides of 42.Oil.5mm. 102 Rotation and t r a n s l a t i o n of the metrograph-generated square on to the MR-generated one were achieved mathematically, and v i s u a l comparison of the goodness of f i t of the two coplanar squares was considered adequate before proceeding to display the p o s i t i o n of the needle t i p within the muscle. Through-calibration of the st e r e o t a c t i c system was c a r r i e d out with a male subject aged 3 2 years. The subject was imaged according to the protocol described previously. The MR images were d i g i t i z e d and a three-dimensional reconstruction was c a r r i e d out which included the reference square. A needle electrode was supported at the hub with i t s t i p fixed to the r i g h t canine tooth midcoronally at the mesiobuccal l i n e angle. The point angles of the g r i d , and the two normally-used points on the needle were measured with the Metrograph. The Metrograph procedure was then repeated. This t e s t incorporated a l l errors including those related to use of the Metrograph, MR imaging, the d i g i t i z i n g procedure, subject movement a r t i f a c t , and intraoperator error. The po s i t i o n of the needle t i p r e l a t i v e to the tooth i s demonstrated in Figure 10. The r e p r o d u c i b i l i t y of i t s location over both t r i a l s was estimated to be ±1.4mm, which was considered acceptable for p r a c t i c a l use. VI. Application i n vivo The same subject who p a r t i c i p a t e d i n the methodological study was used to demonstrate the f e a s i b i l i t y of using the st e r e o t a c t i c system i n vivo. A monopolar needle electrode with a surface 103 Figure 10. Val i d a t i o n of ste r e o t a c t i c needle electrode placement. A three-dimensional s l i c e reconstruction of the r i g h t masseter muscle (M) , ri g h t a r t i c u l a r condyle (C) , r i g h t maxillary canine tooth (Ca), viewed i n three planes. The r e l a t i o n s h i p of the needle electrode t i p to the canine tooth i s shown. C INFERIOR m \ca LATERAL CORONAL 5 0 m m 104 reference electrode overlying the masseter muscle were used to record a c t i v i t y from a SMU i n the r i g h t masseter muscle. The electrode was introduced percutaneously at r i g h t angles to the skin surface, and i t s p o s i t i o n fixed to the skin of the cheek (q.v.). SMU a c t i v i t y was amplified (Model AL 2010, Axon Instruments Inc., Burlingame, CA), and bandpass-filtered (300Hz-10kHz) . The muscle was activated (5-10% of maximum) by b i t i n g on the i n c i s o r teeth, and a compound action p o t e n t i a l (CAP) was obtained from a group of SMU f i b r e s located at the needle electrode t i p . The locating g r i d was then introduced between the teeth and the three-dimensional coordinates of the needle hub, an additional point on the needle close to the skin surface, and the four point angles on the g r i d were recorded and stored for o f f - l i n e c a l c u l a t i o n of the three-dimensional coordinates of the electrode t i p , and to display the three-dimensional location of the recording s i t e within the computer reconstruction of the muscle, (Figure 11). During the placement of the reference g r i d and the recording of known points on the needle and the reference g r i d using the Metrograph, there was the p o s s i b i l i t y that needle movement could occur within the muscle. In order to investigate t h i s , the g r i d was removed and the electrode leads reconnected to the EMG amplifier. The subject then reactivated the muscle using the same task as before ( l i g h t i n c i s a l clenching). The SMU CAP recorded by the needle was sampled and averaged, then plotted (Models 2221 and 105 Figure 11. Location of a needle electrode recording s i t e within the r i g h t masseter muscle. A three-dimensional reconstruction of the r i g h t masseter muscle (M) , r i g h t a r t i c u l a r condyle (C), r i g h t maxillary canine (Ca), viewed i n three planes. The l o c a t i o n of the needle electrode within the muscle i s shown. 1 §Ca [ INFERIOR M -Ca LATERAL CORONAL 50mm 106 Figure 12. Changes i n single motor unit waveform shape due to needle electrode movement within the masseter muscle, a. Averaged (n=16) SMU compound action p o t e n t i a l obtained during EMG recording session, b. Averaged (n=16) compound action p o t e n t i a l of the same unit a f t e r metrograph data c o l l e c t i o n . 1 I 0.2mV 5ms 1 i i i I I 107 HC1000, Tektronix Canada Inc., Vancouver, B.C.). Figure 12 shows the SMU CAP waveforms recorded before (a) and a f t e r (b) metrograph data c o l l e c t i o n . The s i n g u l a r i t i e s of each waveform shape were remarkably s i m i l a r which indicated that both CAPs were most l i k e l y recorded from the same SMU. The amplitude of the signal was s l i g h t l y reduced i n "b" which suggested that t h e i r had been minimal needle displacement, less than 1mm, during the insertion/removal of the g r i d or during the short time taken to record the metrograph data points. 108 SPECIFIC STUDIES (Methods and Results) A. TERRITORIAL MAPPING OF MOTOR UNITS IN THE MASSETER MUSCLE 1. Single Motor Unit Recording Technique Single motor unit EMG experiments were ca r r i e d out on two male and one female subjects aged 31-3 6 years. Each subject had a complete natural dentition and no his t o r y of jaw dysfunction. The subjects were selected because of t h e i r knowledge of basic neurophysiological p r i n c i p l e s of SMU ac t i v a t i o n which i s an advantage i n t h i s type of experiment. Two needle electrodes were inserted into the rig h t masseter muscle, and t h e i r p osition s t a b i l i z e d using a l i g h t metal platform (Figure 13). Reference electrodes were attached to the skin over the muscle. At the beginning of each recording session, the needles were normally situated approximately 15-2Omm apart (as measured on the skin surface). Single motor unit a c t i v i t y was amplified (Model AL 2010, Axon Instruments Inc., Burlingame, CA) and bandpass-filtered (lkHz-lOkHz). Units were activated by strategies which permitted continuous f i r i n g at a low frequency (approximately 10-12Hz) by means of auditory and v i s u a l feedback. When a c l e a r l y defined CAP was obtained from a SMU located at one of the needle t i p s i t was used to t r i g g e r the oscilloscope sweep (Model 2221, Tektronix Canada Inc., Vancouver, B.C.). Any synchronous a c t i v i t y recorded from the second needle was displayed on the second channel of the oscilloscope as a time-locked event, 109 Figure 13. Technique for single motor unit t e r r i t o r i a l mapping i n the masseter muscle. Paired monopolar needles inserted through a metal g r i d fixed over the right masseter muscle. Two surface reference electrodes placed d i s t a l to the metal g r i d . 110 and therefore was considered to originate from the same SMU. I f no synchronous a c t i v i t y was recorded, the second needle was repositioned progressively closer to the t r i g g e r i n g s i t e u n t i l a synchronous CAP was recorded. At each location the needle electrodes were moved in and out of the muscle along the conventional t r a j e c t o r y used i n u n i a x i a l cross-sectional scanning u n t i l CAPs of maximum amplitude (>0.1mV) were obtained at both recording s i t e s . The SMU responses from the two recording s i t e s were sampled and averaged, then plotted (Model HC1000, Tektronix Canada Inc., Vancouver, B.C.). Paired SMU recordings were obtained in t h i s way for a t o t a l of 32 units. 11. Selection of Muscle Recording Sites In order to probe d i f f e r e n t parts of the muscle adequately, each muscle was divided into eight regions by d i v i d i n g the muscle into major s u p e r f i c i a l and deep parts, which i n turn were divided into quadrants (Figure 14). i l l . Location of Electrode Recording Sites At the end of each EMG recording session, a customized reference g r i d (Figure 6) was placed between the teeth of the subj ect. The four point angles of the gr i d , and two known points on each needle were d i g i t i z e d three-dimensionally (3-D) i n s i t u , by means of a Reflex Metrograph, and the data stored d i g i t a l l y for o f f - l i n e analysis. Because the length of each needle was known, 111 Figure 14. Masseter muscle recording regions. The r i g h t masseter muscle i s divided into s u p e r f i c i a l and deep parts which i n turn are divided into quadrants, numbered 1-8. 112 the p o s i t i o n of the needle t i p s r e l a t i v e to the Metrograph-based reference plane could be calculated (q.v.). P r i o r to the f i r s t EMG recording session, the pertinent oro-f a c i a l anatomy of each of the three subjects had been imaged by Magnetic Resonance (MR), as described previously, and an MR-generated plane of reference calculated for each subject. This permitted the reference plane from the metrograph data recorded a f t e r each EMG session to be made coplanar with the MR-generated plane for that subject. A l l relevant reference points were then superimposed to permit the location of the paired needle t i p s to be displayed within the reconstructed masseter muscle (see Figure 15) . IV. Data Display and Analysis The paired recording s i t e s for each subject were displayed within a graphic reconstruction of the r i g h t masseter muscle viewed i n three planes. Since the 3-D coordinates of these needle t i p locations were known, the l i n e a r distance between each p a i r could be calculated (see Figure 15). The distance between each p a i r of recording s i t e s was also calculated along antero-posterior, s u p e r i o r - i n f e r i o r , and medio-l a t e r a l axes, and the mean distances computed for the 3 2 paired recording locations. S t a t i s t i c a l comparisons between the mean distances i n each axis were performed using paired t - t e s t s . 113 Figure 15. The location of two paired needle electrode recording t i p s within the masseter muscle. A three-dimensional s l i c e reconstruction of the ri g h t masseter muscle (M) , the ri g h t a r t i c u l a r condyle (C) , r i g h t maxillary canine (Ca), viewed i n three planes. The location of the two needle electrode t i p s , a and b, within the masseter muscle are depicted. Time-locked averaged (n=16) SMU compound action potentials recorded by the t r i g g e r i n g electrode at recording s i t e "a", and by the second electrode at recording s i t e "b" are also shown, (arrowed) . In t h i s instance the l i n e a r distance between the paired recording s i t e s was 11mm. 114 V. RESULTS a. D i s t r i b u t i o n of Triggering Electrode Sites within the Muscle Single motor units were activated by tasks such as l i g h t intercuspal clenching and jaw retrusion during which the units f i r e d f r e e l y without muscle discomfort. When a c l e a r l y defined compound action potential (CAP) was obtained from a SMU located at one of the needle t i p s , the unit was f i r e d slowly and continuously at low le v e l s of e f f o r t (5-10% of maximum). Unit a c t i v a t i o n strategies which permitted slow, regular f i r i n g appeared to vary throughout the muscle. In almost a l l instances the second electrode, which was i n i t i a l l y located at a distance of 15-22mm, had to be reinserted within the muscle at a pos i t i o n nearer to the t r i g g e r i n g electrode (as measured on the skin surface). In many instances the second needle had to be repositioned 2-3 times progressively closer to the t r i g g e r i n g s i t e , u n t i l time-locked CAPs were recorded at both needle t i p s . On no occasion were synchronous CAPs not obtained from the unit investigated at each experimental session. A t o t a l of 32 paired ( i . e . time-locked) SMU recordings were made i n various parts of the masseter muscles of three subjects. The d i s t r i b u t i o n by region of the 32 t r i g g e r i n g s i t e s i s depicted i n Figure 16. 115 Figure 16. D i s t r i b u t i o n by region of 32 single motor unit t r i g g e r i n g s i t e s i n the ri g h t masseter muscle. Each number represents the t o t a l number of units sampled i n that region. 116 Figure 17. Distance between paired recording s i t e s i n the ri g h t masseter muscle. A histogram of the distance between 3 2 paired recording s i t e s . CO b or O 10 UJ _i z CO UL o 5 5.5 7.5 9.5 11.5 13.5 15.5 17.5 19.5 4.5 6.5 8.5 10.5 12.5 14.5 16.5 18.5 DISTANCE BETWEEN PAIRED RECORDINGS (mm) 117 Figure 18. Axes of orientation of single motor unit t e r r i t o r i e s i n the masseter muscle. A diagrammatic i l l u s t r a t i o n of the mean distance between 32 pairs of recording s i t e s measured along antero-posterior, s u p e r i o r - i n f e r i o r , and medio-lateral axes. The distance along an antero-posterior axis i s approximately double that measured along s u p e r i o r - i n f e r i o r or medio-lateral axes (p<0.01). I, L, P, S, M, and A represent i n f e r i o r , l a t e r a l , posterior, superior, medial, and anterior d i r e c t i o n s respectively. I 118 b. Distances between Paired Needle Electrode Recording Sites The mean l i n e a r distance measured three dimensionally between the 3 2 paired recording s i t e s from which synchronous SMU a c t i v i t y could be obtained was 8.8±3.4mm, and ranged from 5-20mm. A d i s t r i b u t i o n histogram for those data i s shown i n Figure 17. c. Orientation of Putative Motor Unit T e r r i t o r i e s The mean distances measured along antero-posterior, superior-i n f e r i o r , and medio-lateral axes for the 32 paired SMU recording s i t e s were 6.1±4.Omm (range 0.3-19.0), 3.8±2.5mm (0.1-11.5), and 3.2±2.3mm (0.1-10.0) respectively. The unit t e r r i t o r i e s therefore appeared to have a preferred axis of orientation, (shown diagrammatically i n Figure 18) because the distances between the 32 pa i r s of recording s i t e s measured along an antero-posterior axis were approximately double those measured along a superior-i n f e r i o r or medio-lateral axis (p < 0.01) . The MU t e r r i t o r i e s thus appeared to be "cigar-shaped", and arranged i n layers throughout the muscle i r r e s p e c t i v e of t h e i r locations. B. MOTOR UNIT TASK PROFILES IN DIFFERENT REGIONS OF THE MASSETER MUSCLE 1. Single Motor Unit Recording Method a. Technique Single motor unit a c t i v i t y was recorded from 50 masseter SMUs 119 i n three male and two females aged 26-32 years. A l l subjects had complete natural dentitions and no history of jaw dysfunction. Each subject had a knowledge of the neurophysiological p r i n c i p l e s of SMU ac t i v a t i o n . The masseter muscle was divided into eight regions (q.v.) to permit thorough investigation of SMUs i n d i f f e r e n t parts of the muscle (see Figure 14). A single needle electrode was placed percutaneously into a preselected region of the r i g h t masseter, and i t s pos i t i o n s t a b i l i z e d (q.v.) [Figure 1]. Single motor unit a c t i v i t y was amplified and band-pass f i l t e r e d (300Hz-10kHz), then pulse-discriminated. During minimal a c t i v a t i o n of the muscle, the needle was moved within the muscle u n t i l a SMU CAP of maximum amplitude was obtained. The f i r i n g rate of the unit was controlled by means of auditory and v i s u a l feedback. b. Task P r o f i l e s A protocol was established to determine the task s p e c i f i c i t y of each of the 50 units examined. I n i t i a l l y , the SMU under study was activated by any i n t r a o r a l strategy which permitted the unit to f i r e f r e e l y without s i g n i f i c a n t pauses. The subject then attempted to maintain unit a c t i v a t i o n while a number of d i f f e r e n t i n t r a o r a l strategies were performed consecutively. The 13 tasks attempted are defined i n Table 1. I f the unit ceased f i r i n g or did not f i r e during a p a r t i c u l a r task, the task which activated the unit i n i t i a l l y was used i n order to recover unit f i r i n g . The 120 Table 1. Motor unit a c t i v a t i o n strategies. A description of the methods of motor unit a c t i v a t i o n together with t h e i r respective codes. MOTOR UNIT ACTIVATION STRATEGIES: m -a -b -j ~ k -1 -d -e -c -coactivation directed v e r t i c a l l y * coactivation with jaw protrusion* r i g h t premolar clench directed v e r t i c a l l y l e f t premolar clench directed v e r t i c a l l y i n c i s a l clenching directed v e r t i c a l l y i n c i s a l clenching directed a n t e r i o r l y r i g h t molar clench directed v e r t i c a l l y l e f t molar clench directed v e r t i c a l l y jaw retrusion with molar contact jaw retrusion with no tooth contact r i g h t canine clench directed v e r t i c a l l y l e f t canine clench directed v e r t i c a l l y intercuspal clench directed v e r t i c a l l y * Simultaneous contraction of jaw elevator and antagonistic jaw depressor muscles which i n the case of a. permitted the maintenance of a jaw p o s i t i o n approximately 6mm from f u l l intercuspation on a habitual path of jaw closure, and i n b. maintained the jaw i n a fixed, protrusive p o s i t i o n with the teeth approximately 6mm apart. 121 number and types of tasks which a unit performed during continuous f i r i n g were then noted p r i o r to d e f i n i t i v e EMG recordings. Once the tasks which could be performed by the unit were known, a SMU CAP was sampled, averaged, then plotted (Models HP 2221 and HP1000, Tektronix Canada Inc., B.C.) during the performance of each task. The unit f i r e d without s i g n i f i c a n t pauses during t h i s recording session. On a few occasions the needle was gently repositioned to r e t a i n a CAP of maximum amplitude during the performance of d i f f e r e n t tasks. To ensure unequivocally that d i f f e r e n t tasks were always performed by the same unit, each task was repeated i n a random order, and addi t i o n a l , averaged CAPs plotted for v i s u a l comparison of the waveform shape. c. Inter-Spike Interval Measurement When the task s p e c i f i c i t y of the unit was established, the subject controlled the f i r i n g frequency of the unit during the performance of each task (after recording a representative SMU CAP), by means of v i s u a l and auditory feedback. The subject was instructed to increase the f i r i n g frequency of the unit r e l a t i v e l y slowly, then to decrease unit f i r i n g to i t s lowest sustainable f i r i n g frequency (LSFF) [Petajan, 1981] three times i n succession, and f i n a l l y to maintain unit f i r i n g at the LSFF. When the unit reached the LSFF on the t h i r d occasion (Figure 19), 201 consecutive CAPs were pulse-discriminated and t h e i r i n t e r v a l s measured i n r e a l time with a data a c q u i s i t i o n unit and a d i s c -based computer (Models HP 3852a and HP 350, Hewlett Packard 122 Figure 19. Single motor unit inter-spike i n t e r v a l measurement at the lowest sustainable f i r i n g frequency. A p l o t of inter-spike i n t e r v a l duration against inter-spike i n t e r v a l number for a continuously f i r i n g single masseter motor unit. The lowest sustainable f i r i n g frequency was reached by slow increases and decreases i n voluntary f i r i n g rate. The period during which consecutive inter-spike i n t e r v a l s were measured (at the lowest sustainable f i r i n g frequency) i s marked by a horizontal bar, (see t e x t ) . 1 0 0 2 0 0 3 0 0 4 0 0 Inter-spike Interval Number 123 Canada, Vancouver, B.C.). 11. Location of Electrode Recording Sites Prior to EMG experiments a customized reference g r i d was fabricated for each subject. A l l subjects were then imaged by Magnetic Resonance (MR) , with the g r i d i n s i t u as described previously, and MR-reference planes calculated. At the end of each EMG recording session, the point angles of the reference g r i d , and two known points on the needle were d i g i t i z e d three-dimensionally i n s i t u (q.v.). The superimposition of metrograph-based and MR reference planes permitted the location of the needle electrode t i p s with the masseter muscle of each subject as described previously. i l l . Data Analysis and Display The SMU recording s i t e s for each of the subjects were displayed within a three-dimensional reconstruction of the right masseter muscle. The d i s t r i b u t i o n of units according to the eight s p e c i f i e d regions was then coll a t e d , together with the number and types of tasks performed by each unit. Comparisons were made between muscle regions according to the s p e c i f i c tasks performed and the number and proportion of times they occurred i n each region, to determine whether p a r t i c u l a r tasks were performed more frequently i n d i f f e r e n t regions. For the purpose of these analyses the eight muscle regions were also merged into larger d i v i s i o n s . 124 For each SMU, the mean inter-spike i n t e r v a l (ISI) of 200 in t e r v a l s obtained from 201 consecutively occurring CAPs at the LSFF was calculated for the d i f f e r e n t tasks performed. Inter-spike i n t e r v a l s were plotted i n a histogram format to determine the nature of the ISI d i s t r i b u t i o n for each task. Mean ISIs for d i f f e r e n t tasks performed by in d i v i d u a l units were then compared s t a t i s t i c a l l y by means of ANOVA. I V . RESULTS a . D i s t r i b u t i o n o f R e c o r d i n g S i t e s w i t h i n t h e M u s c l e The d i s t r i b u t i o n by subject of units sampled can be found i n Table 4 and the d i s t r i b u t i o n by region of the 50 s i t e s from which SMU a c t i v i t y was recorded from a l l subjects i s shown i n Figure 20. Recordings were made throughout a l l regions of the muscle. b . R e l a t i o n s h i p o f S i n g l e M o t o r U n i t T a s k P r o f i l e s t o S p e c i f i c R e g i o n s o f t h e M u s c l e 1. S i n g l e M o t o r U n i t W a v e f o r m C h a r a c t e r A l l 50 units were activated by more than one task. The number of tasks performed by each unit ranged from 2 - 6 , with a t o t a l of 155 tasks recorded. Single motor unit CAPs plotted for a t y p i c a l unit are shown in Figure 21. While the unit f i r e d continuously, three d i f f e r e n t tasks were performed v i z . l e f t canine clenching "a", i n c i s a l clenching "b", and intercuspal clenching "c". The shape of the waveforms were remarkably consistent for d i f f e r e n t tasks, and also 125 Figure 20. D i s t r i b u t i o n by region of 50 single motor units recorded i n the r i g h t masseter muscle. 126 F i g u r e 21. C o n s i s t e n c y o f a s i n g l e m o t o r u n i t waveform f o r d i f f e r e n t a c t i v a t i o n s t r a t e g i e s . Averaged (n=16) compound action potentials for a unit activated by three d i f f e r e n t strategies are represented by "a", "b", and "c". "a r" and "br" represent repeat recordings of "a" and "b". "a m" represents "a" a f t e r Metrograph data had been recorded. 127 during repeat recordings, "a r" and "hr". In t h i s instance the unit was also reactivated, sampled, then plotted ("am") a f t e r two known points on the needle electrode and the four point angles of the reference g r i d had been recorded i n s i t u by a Metrograph (q.v.) to permit s t e r e o t a c t i c location of the needle recording s i t e within the masseter muscle. 11. Unit A c t i v a t i o n Strategies with and without Tooth Contact Units recorded i n each of the eight designated muscle regions were divided into those which performed tasks involving both tooth contact and non-contact (dental/non-dental [DND] units) and those which f i r e d only during tooth-contact tasks (dental u n i t s ) , (see Figure 22). In muscle regions 5 to 8, the deep part of the muscle, only s i x out of 2 6 were purely dental units. In regions 1 to 4, corresponding to the s u p e r f i c i a l part of the muscle, however, DND and dental units were present i n equivalent numbers. The anterior deep part of the muscle (areas 6 and 7) had only one purely dental unit out of a possible 12 units, whereas the s u p e r f i c i a l posterior part (areas 1 and 4) comprised predominantly dental units. The posterior deep part (areas 5 and 8) and the anterior s u p e r f i c i a l part (areas 2 and 3) had more DND than dental u n i t s . In the superior deep (areas 5 and 6) and i n f e r i o r deep parts (areas 7 and 8) DND units were dominant, whereas i n the superior s u p e r f i c i a l (areas 1 and 2) and i n f e r i o r s u p e r f i c i a l parts (areas 3 and 4) DND 128 Figure 22. D i s t r i b u t i o n by muscle region of dental and dental/non-dental single masseter motor units. Dental/non-dental units are in parentheses. 129 units were present i n si m i l a r numbers. There were no dental units present i n the anterior i n f e r i o r part (areas 3 and 7) of the muscle. 111. S p e c i f i c Unit A c t i v a t i o n Strategies In the deep part of the masseter muscle (areas 5-8) each of the a c t i v a t i o n strategies defined i n Table 1, except r i g h t molar clenching directed v e r t i c a l l y , were associated with at least one of the SMUs. I n c i s a l clenching directed v e r t i c a l l y , jaw retrusion with no tooth contact, l e f t canine and intercuspal clenching were the tasks which activated units most consistently. A l l tasks were carri e d out by units i n the s u p e r f i c i a l part of the muscle (areas 1-4). Left canine, intercuspal and i n c i s a l clenching directed v e r t i c a l l y were most frequently associated with units i n t h i s part of the masseter. In the posterior s u p e r f i c i a l part of the muscle (areas 1 and 4) i n c i s a l and intercuspal clenching directed v e r t i c a l l y were most often e f f e c t i v e i n ac t i v a t i n g units whereas r i g h t and l e f t molar clenching and jaw retrusion with tooth contact were not. Jaw muscle coactivation directed v e r t i c a l l y and i n c i s a l clenching were commonly associated with units i n the s u p e r f i c i a l anterior part of the muscle (areas 2 and 3) whereas ri g h t and l e f t pre-molar clenching, i n c i s a l clenching directed a n t e r i o r l y , and jaw retrusion without tooth contact did not activate them. In the posterior deep part of the muscle (areas 5 and 8) intercuspal clenching was the most common ac t i v a t i n g task whereas ri g h t and 130 Figure 23. Motor unit task p r o f i l e s i n d i f f e r e n t regions of the masseter muscle. Figure 23, Legend. The tasks most commonly associated with unitary a c t i v i t y i n s p e c i f i c muscle regions are preceded by a f i l l e d star and those tasks during which a c t i v i t y was not recorded are preceded by a cross. The muscle i s subdivided i n three d i f f e r e n t ways. 131 MOTOR UNIT ACTIVATION STRATEGIES: •11'"?%, a 6 c d e f 9 h i i k I m coactivation directed vertically coactivation with jaw protrusion right premolar clench directed vertically left premolar clench directed vertically incisal clench directed vertically incisal clench directed anteriorly right molar clench directed vertically left molar clench directed vertically Jaw retruslon with molar contact jaw retruslon with no tooth contact right canine clench directed vertically left canine clench directed vertically intercuspal clench directed vertically l e f t molar clenching were not e f f e c t i v e a c t i v a t o r s . Jaw muscle coactivation directed v e r t i c a l l y was most consistently associated with unit a c t i v i t y i n the anterior deep part (areas 6 and 7) while jaw muscle coactivation with jaw protrusion and i n c i s a l clenching directed a n t e r i o r l y , and ri g h t molar clenching were not. Only l e f t premolar clenching was not associated with units in the superior s u p e r f i c i a l part of the muscle (areas 1 and 2) , while jaw muscle coactivation directed v e r t i c a l l y , and intercuspal and i n c i s a l clenching directed v e r t i c a l l y were the most e f f e c t i v e tasks. In the i n f e r i o r s u p e r f i c i a l part (areas 3 and 4) intercuspal and i n c i s a l clenching directed v e r t i c a l l y were common ef f e c t i v e a c t i v a t i n g tasks, while r i g h t and l e f t molar clenching and jaw retrusion with tooth contact were the le a s t . A l l tasks except jaw muscle coactivation with protrusion and ri g h t molar clenching were associated with a c t i v i t y i n the superior deep part (areas 5 and 6) . Jaw muscle coactivation directed v e r t i c a l l y was the most common. In the i n f e r i o r deep part (areas 7 and 8) , intercuspal and i n c i s a l clenching directed v e r t i c a l l y were most common ac t i v a t i n g tasks while i n c i s a l clenching directed a n t e r i o r l y and ri g h t and l e f t molar clenching was l e a s t . These data are summarized i n Figure 23. c. Task-related Single Motor Unit Inter-Spike Intervals An array of contiguous ISIs were recorded at the LSFF for d i f f e r e n t tasks i n 49 out of 50 units. In the other unit f i r i n g ceased before ISIs could be recorded. 133 Although the subjects were highly s k i l l e d i n the performance of motor unit f i r i n g using auditory and visual-feedback, there were readily-apparent differences i n each subject's a b i l i t y to sustain the LSFF for d i f f e r e n t tasks, and obvious v a r i a t i o n s i n the a c t i v a t i o n threshold for d i f f e r e n t tasks. Within units, the f i r i n g frequency could be controlled e a s i l y for some tasks whereas for others there were a small number of random pauses (corresponding to long ISIs) i n unit f i r i n g as the subject attempted to maintain the LSFF. The range of mean ISIs for 153 tasks i n 49 units varied from 41±10ms-169±71ms. Within units, the difference between minimum and maximum mean ISIs for d i f f e r e n t tasks ranged from 7-79ms (mean 37ms). In Table 2, the mean ISIs are tabulated for the tasks performed by a l l units sampled. There was a consistent asymmetrical d i s t r i b u t i o n of ISIs within the d i f f e r e n t tasks i n each unit. A l l d i s t r i b u t i o n s were unimodal and p o s i t i v e l y skewed. When ISI arrays for each task were transformed to natural logarithms, however, the ISIs for each task were normally d i s t r i b u t e d . This permitted comparisons of mean ISIs for d i f f e r e n t tasks i n ind i v i d u a l units to be compared using the parametric ANOVA. Inter-spike i n t e r v a l s were recorded for at least two tasks i n 47 out of 50 units. There were s i g n i f i c a n t differences between mean ISIs for d i f f e r e n t tasks performed i n 45 out of 47 units at the 99% confidence l i m i t (p<0.01) . In the other two units there were s i g n i f i c a n t differences at the 95% confidence l i m i t (p<0.05). 134 Table 11. Single motor unit behavioural p r o f i l e s i n d i f f e r e n t regions of the masseter muscle. The types of tasks and the mean inter-spike i n t e r v a l s are c o l l a t e d by muscle region for 50 units. MUSCLE REGION UNIT TASK ISI mean±lSD (ms) K3 i 139±51 e 94±43 c 82±47 K4 e 156±39 m 144142 a 124±39 SI a 145±47 k 80131 K7 m 107138 f 76±30 T7 e 169171 m 140165 T9 e 159164 m 129154 c 96139 K2 e 125166 h 88136 a 65124 M3 j 70137 m 60115 a 45±4 b 43110 M5 a 70130 1 57±9 e 52±9 S2 k 161149 e 135130 1 129126 TI g 166156 k 87131 135 MUSCLE REGION UNIT TASK ISI Mean+ISD (ms) 2 T3 1 122±35 e 95±30 3 E3 e 118±39 m 89±8 M6 e 123±45 b 99±36 m 98±46 a 89±30 1 61±12 S6 m 143±51 k 131±44 e 111±30 1 106±32 a 105±28 j 96±42 T6 a 136±58 1 131±31 m 120±43 T13 m 163±61 e 142±55 a 130±57 1 112±34 4 E7 1 136±26 f 130±22 Itl 118±17 K5 a 100±40 Itl 94±42 1 65±21 M2 c 98±29 k 94±31 e 93±30 r 79±23 d 62±17 136 MUSCLE REGION UNIT TASK ISI meanilSD (ms) 4 S4 k 89±23 1 75±12 e 66±10 T2 m 161±49 k 146±53 e 143±41 b 107±37 T5 e 164±65 1 142±55 m 136±48 T8 e 160±43 m 151±40 C 119+35 5 E l d 164±32 e 151±40 m 137±44 k 126±17 E2 m 136±28 k 128±19 a 114±16 E4 k 149±35 a 105±11 m 103±13 E8* f 151±52 m 139±34 E12 m 147±39 j 113±43 M4 j 95±28 1 64±12 S5 m 131±42-a 94±28 j 89±37 T4 c 156±45 i 126±38 137 MUSCLE REGION UNIT TASK ISI meant1SD (ms) 5 T12 j 114142 1 100132 a 90127 m 76±9 e 67115 6 E5 a 117122 m # E9 m 137145 a 122127 E l l a 106135 m 88122 h 87122 j 41110 S3 k 164147 e 147133 1 144131 T i l a 142156 j 120145 i 110139 e 84131 7 E10 m 139125 1 118121 e 109119 E13 k 134130 e 116116 a 110125 1 101114 E14 m # j # Kl a 114154 d 82140 i 76136 c 54115 K6 m 139155 a # 138 MUSCLE REGION UNIT TASK ISI meanilSD (ms) 7 Ml a 67±44 k 57±23 1 # S7 a 138±54 k 129±52 e 126±48 1 102±34 8 E6 1 138129 e 135±43 m 102±22 K8* a 131±41 m 124±51 K9 e 141±39 1 127±42 m 118±37 k 112±36 M7 m 119±49 j 70±13 e 69±19 T10 m 147±62 b 117±58 1 113±45 e 109138 * p<0.05 # ISIs not recorded 139 In d i f f e r e n t regions of the muscle, the tasks performed by each unit were ranked i n order of decreasing mean ISI i . e . i n increasing order of LSFF (Table 2) . In the deep part of the muscle (areas 5-8) the LSFF occurred with intercuspal clenching directed v e r t i c a l l y i n seven out of 23 units, and with coactivation directed v e r t i c a l l y i n f i v e out of 23 units. I n c i s a l clenching directed v e r t i c a l l y e l i c i t e d the LSFF i n s i x out of 24 units i n the s u p e r f i c i a l part of the muscle (areas 1-4). Three out of six units i n muscle area 1 exhibited t h e i r LSFF when the units were activated by i n c i s a l clenching directed v e r t i c a l l y . In area 3, the LSFF for two out of six units occurred with i n c i s a l clenching directed v e r t i c a l l y , and an additional two units by intercuspal clenching directed v e r t i c a l l y . Three out of nine units i n area 5, three out of f i v e units i n area 7, and two out of f i v e i n area 8 performed the LSFF with intercuspal clenching directed v e r t i c a l l y , whereas two out of four units i n region area 6 and three out of f i v e i n area 7 had t h e i r LSFF during muscle coactivation directed v e r t i c a l l y . C o l l e c t i v e l y these r e s u l t s indicate that there are regional differences i n SMU behaviour although there was no obvious systematic pattern. 140 C. MOTOR UNIT INHIBITORY REFLEX BEHAVIOUR IN THE MASSETER AND LATERAL PTERYGOID MUSCLES 1. Single Motor Unit Recording Technique a. Masseter Single motor unit EMG recordings were obtained from 22 units i n three male and one female subjects aged 2 6-50 years. The subjects had complete natural dentitions and reported no history of jaw dysfunction. A needle electrode was inserted percutaneously into the middle part of the body of the masseter muscle, and i t s p o s i t i o n secured by means of a metal framework, as described previously (Figure 1) . Ten units were activated by single tasks such as i n c i s a l and intercuspal clenching. An additional 12 units were activated by means of at least two d i f f e r e n t strategies. Single motor unit a c t i v i t y was amplified, bandpass-filtered (300Hz-10kHz), pulse-discriminated, and sampled with a data a c q u i s i t i o n unit and d i s c -based computer (Models HP 3852a and HP350, Hewlett-Packard Canada, Vancouver, B.C.). Ongoing SMU a c t i v i t y was sampled every ms and stored as a series of events occurring within a maximum sampling period of 5min. Single motor unit recordings involving d i f f e r e n t tasks were repeated to determine consistency of r e f l e x responses. b. Lateral Pterygoid The responses of 30 SMUs were recorded from f i v e males aged 2 2-36 years. A l l the subjects had complete natural dentitions and 141 Figure 24. Single motor unit recording technique i n the l a t e r a l pterygoid muscle. A precurved needle was inserted transmucosally in the buccal sulcus d i s t a l to the r i g h t maxillary t h i r d molar aiming for the lower part of the l a t e r a l aspect of the l a t e r a l pterygoid plate, then secured to the buccal surfaces of the molar teeth. 142 no h i s t o r y of jaw dysfunction. In each case, a modified monopolar technique was used to record SMU a c t i v i t y i n the i n f e r i o r head of the l a t e r a l pterygoid muscle. A precurved needle electrode was introduced transmucosally in the buccal sulcus d i s t a l to the ri g h t maxillary t h i r d molar, then secured to the buccal surfaces of the molar teeth (Figure 24). A surface reference electrode was attached to the ri g h t earlobe. The location of the needle within the l a t e r a l pterygoid muscle was established by observing increased EMG a c t i v i t y during jaw protrusion and con t r a l a t e r a l abduction, and minimal a c t i v i t y during jaw closure and intercuspal clenching. SMUs were activated by minimal jaw protrusion, c o n t r a l a t e r a l abduction or jaw opening against resistance. Unit a c t i v i t y was amplified and sampled as for masseter muscle SMUs. 11. Intraoral Stimulation For both muscles, bipolar stimulating electrodes were fixed to the buccal gingiva adjacent to the ri g h t canine eminence and iso l a t e d single pulses of 1ms duration were delivered v i a a constant current unit (Stimulator model S44 and Is o l a t i o n unit model SIU 5, Grass, Quincy, Mass). For masseter muscle recordings, the i n t e n s i t y of the stimulus was adjusted by the subject u n t i l i t was perceived as a mild tapping, non-noxious sensation; the range varied between 3-4T, where T was the minimum current perceived by the subject. In the case of l a t e r a l pterygoid recordings, each subject adjusted the i n t e n s i t y of the stimulus 143 u n t i l i t was perceived as approximately 80% of noxious. The i n t e n s i t y of the stimulus was described as a firm tapping or pr i c k i n g sensation which on occasions was mildly uncomfortable. 111. Reflex Paradigm The following r e f l e x paradigm, o r i g i n a l l y described by Turker and Miles (1988), was used to examine r e f l e x responses i n both masseter and l a t e r a l pterygoid SMUs. When a clearly-defined motor unit was activated, the f i r i n g frequency was constrained by the subject to either 10±2Hz or 15±2Hz using v i s u a l and auditory feedback. Inter-spike i n t e r v a l (ISI) analysis was performed on-line so that a computer-generated signal would t r i g g e r a stimulus whenever two ISIs occurred within the predetermined f i r i n g range. Successive s t i m u l i were then delayed by increasing i n t e r v a l s of 1ms from the occurrence of the prestimulus spike (Figure 25) . A minimum of 12 ISIs occurred between consecutive s t i m u l i . Thus the maximum stimulus frequency was 0.8Hz, and i n most instances less than t h i s . When the prestimulus f i r i n g frequency was 10Hz, a t o t a l of 78 s t i m u l i were delivered, whereas at 15Hz, a t o t a l of 54 s t i m u l i were used. An additional experiment was performed i n the l a t e r a l pterygoid muscle. In two subjects, a t o t a l of 50 e l e c t r i c a l pulses of 1ms duration were delivered at Is i n t e r v a l s when the l a t e r a l pterygoid muscle was inactive, and the responses were displayed on a storage oscilloscope screen. Here, both sub-noxious (6T) and noxious (8T) l e v e l s of i n t e n s i t y were used. 144 Figure 25. EMG response from a single l a t e r a l pterygoid motor unit when a controlled i n t r a o r a l e l e c t r i c a l stimulus i s delivered, "a" and "b" represent consecutive inter-spike i n t e r v a l s which occur within a predetermined f i r i n g range, i n t h i s instance 10±2Hz. The 65th stimulus i s shown and occurs a f t e r a delay of 65ms measured from the spike at the end of i n t e r v a l "b". The same technique was used for masseter motor units. 145 IV. Data Analysis and Display Data for each MU were displayed i n the form of raster plots in which each spike immediately preceding the stimulus was aligned at time zero along with spikes occurring 500ms before and af t e r t h i s spike (see Figures 26, 27). Data were also displayed as peristimulus time histograms (Figure 28) i n which MU data were reformatted so that the s t i m u l i were aligned at time zero and in d i v i d u a l spikes were placed i n 5ms bins 250ms before and af t e r the stimulus. The Wilcoxon matched-pair sign rank t e s t was used to i d e n t i f y s i g n i f i c a n t differences between prestimulus control and peristimulus ISIs for masseter and l a t e r a l pterygoid SMUs, and between peristimulus ISIs for d i f f e r e n t tasks i n ind i v i d u a l masseter units . VI. RESULTS a. E f f e c t of F i r i n g Frequency 1. Masseter In general, SMUs could be f i r e d at 10 or 15Hz by means of auditory and v i s u a l feedback, although some units f i r e d more consistently at 15Hz. Units could be activated by multiple strategies which included intercuspal or molar clenching, and jaw muscle coactivation. Reflex responses were recorded i n 22 units whose prestimulus f i r i n g frequency was 10±2Hz and 10 units whose prestimulus frequency was 15±2Hz. A l l units behaved i n a s i m i l a r manner when tested at the same prestimulus f i r i n g frequency. When the 146 Figure 26. Raster display of spikes and inter-spike i n t e r v a l s for a single masseter motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 1 0 ± 2 H z . Each spike immediately preceeding the stimulus i s aligned at time zero. Spikes occurring during a period of 500ms before and af t e r these spikes are shown. Seventy eight increasingly delayed s t i m u l i are depicted by a diagonal l i n e , and are arranged sequentially from above down. -500 0 +500 (ms) 147 Figure 27. Raster display of spikes and inter-spike i n t e r v a l s for a single masseter motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 15±2Hz. Each spike immediately preceeding the stimulus i s aligned at time zero. Spikes occurring during a period of 500ms before and a f t e r these spikes are shown. F i f t y four increasingly delayed s t i m u l i are depicted by a diagonal l i n e , and are arranged sequentially from above down. -500 0 +500 (ms) 148 Figure 28. Peristimulus time histograms for three masseter single motor units. Data have been reformatted so that s t i m u l i are aligned at time zero and ind i v i d u a l spikes are placed i n 5ms bins 250ms before and a f t e r the stimulus p o s i t i o n . The v e r t i c a l scale represents 5 spikes. -140 , 0 x +140 (ms) 149 prestimulus f i r i n g frequency was 10Hz, a l l 22 units displayed an in h i b i t o r y e f f e c t when peristimulus i n t e r v a l s were compared with prestimulus control i n t e r v a l s (p<0.01), which seemed to be related to the po s i t i o n of the stimulus r e l a t i v e to the prestimulus spike. In each unit the duration of the peristimulus i n t e r v a l was increased when the stimulus was injected up to approximately 1-50ms a f t e r the prestimulus spike, and i n many instances the peristimulus i n t e r v a l was lengthened u n t i l the stimulus was delivered 70ms af t e r the prestimulus spike. A t y p i c a l example of an i n h i b i t o r y response i n a masseter MU with a prestimulus f i r i n g frequency of 10Hz i s shown i n Figure 26. When the prestimulus f i r i n g frequency was constrained to 15Hz, a l l ten units were in h i b i t e d (p<0.05) although the degree of i n h i b i t i o n was less marked than that occurring i n units f i r i n g with a prestimulus frequency of 10Hz. In general, there was an increase i n the duration of peristimulus i n t e r v a l s when the stimulus was injected approximately 0-20ms af t e r the prestimulus spike. Thereafter there was no dramatic e f f e c t , (Figure 27). Peristimulus histograms constructed for each of the 22 units f i r i n g at 10Hz, revealed periods of apparent i n h i b i t i o n whose duration varied between units (Figure 28). 11. Lateral pterygoid Here the most consistent method of unit a c t i v a t i o n was l i g h t i n c i s a l clenching, and jaw opening against minimal resistance applied to the menton of the chin. In one subject, however, unit 150 a c t i v i t y was so l a b i l e that jaw protrusion of approximately 4mm e l i c i t e d unitary a c t i v i t y . In no instance was a c t i v i t y detected during i p s i l a t e r a l abduction or jaw closure. In a l l subjects a number of in d i v i d u a l motor units could be activated by more than one strategy, v i z . horizontal protrusion (with and without i n c i s a l tooth contact) and c o n t r a l a t e r a l abduction, and t h e i r f i r i n g could be sustained at both 10 and 15Hz peristimulus f i r i n g frequencies. A number of these motor units had d i f f e r i n g preferred lowest sustainable f i r i n g frequencies. In four subjects, unit f i r i n g frequency could be constrained r e l a t i v e l y e a s i l y to 10Hz, whereas i n one subject discrete single motor units f i r i n g occurred consistently at 15-20HZ or greater. Reflex responses were recorded i n 15 units whose prestimulus f i r i n g frequency was 1012Hz and 15 units whose prestimulus frequency was 15±2Hz. For each subject, l a t e r a l pterygoid units behaved i n a s i m i l a r manner when tested at the same prestimulus f i r i n g frequency. At a constrained f i r i n g frequency of 10Hz, 14 out of 15 units displayed an i n h i b i t o r y e f f e c t when peristimulus i n t e r v a l s were compared with prestimulus control i n t e r v a l s (p<0.01), which appeared to be dependent on the p o s i t i o n of the stimulus r e l a t i v e to the prestimulus spike (Figure 29). There was an increase i n the duration of the peristimulus ISI when the stimulus was injected approximately 1-35ms af t e r the prestimulus spike, but thereafter there was no obvious e f f e c t . An example of unit a c t i v i t y which demonstrates an increased peristimulus i n t e r v a l r e l a t i v e to the prestimulus control i n t e r v a l i s depicted 151 Figure 29. Raster display of spikes and inter-spike i n t e r v a l s for a single l a t e r a l pterygoid motor unit/ f i r i n g at a controlled prestimulus f i r i n g frequency of 10±2Hz. Each spike immediately preceeding the stimulus i s aligned at time zero. Spikes occurring during a period of 500ms before and af t e r these spikes are shown. Seventy eight increasingly delayed s t i m u l i are depicted by a diagonal l i n e , and are arranged sequentially from above down. 152 Figure 30. Raster display of spikes and inter-spike i n t e r v a l s for a single l a t e r a l pterygoid motor unit, f i r i n g at a controlled prestimulus f i r i n g frequency of 15±2Hz. Each spike immediately preceeding the stimulus i s aligned at time zero. Spikes occurring during a period of 500ms before and a f t e r these spikes are shown. F i f t y four increasingly delayed s t i m u l i are depicted by a diagonal l i n e , and are arranged sequentially from above down. i -500 0 +500 (ms) 153 Figure 31. Peristimulus time histograms for three l a t e r a l pterygoid single motor units. Data have been reformatted so that s t i m u l i are aligned at time zero and i n d i v i d u a l spikes are placed in 5ms bins 250ms before and a f t e r the stimulus p o s i t i o n . The v e r t i c a l scale represents 5 spikes. (ms) j i 154 i n Figure 29. In some instances there was an increased duration of the f i r s t post-stimulus ISI when the stimulus p o s i t i o n was delayed more than 35ms af t e r the prestimulus spike. At a constrained f i r i n g frequency of 15Hz only f i v e out of 15 units displayed an i n h i b i t o r y e f f e c t (p<0.01). In most instances there was no s i g n i f i c a n t e f f e c t on the duration of the peristimulus i n t e r v a l when the stimulus p o s i t i o n was delayed more than 4ms. A raster p l o t for a t y p i c a l unit f i r i n g with a prestimulus frequency of 15Hz i s shown i n Figure 30. Peristimulus time histograms constructed for each of the 15 units f i r i n g at 10Hz, revealed periods of apparent i n h i b i t i o n surrounding a period of apparent exci t a t i o n , even though i n h i b i t i o n alone had previously been demonstrated i n the same raw data for each unit. The timing of patterns of apparent i n h i b i t i o n and e x c i t a t i o n varied between units, (Figure 31). Intraoral e l e c t r i c a l stimulation when the l a t e r a l pterygoid muscle was quiescent did not produce any di s c e r n i b l e excitatory response even at noxious l e v e l s of stimulation. b. E f f e c t s of Different Motor Unit A c t i v a t i o n Strategies on Reflex I n h i b i t i o n Additional r e f l e x responses were recorded from 12 of the 2 2 masseter units i n a l l four subjects. The prestimulus f i r i n g frequency was 10±2Hz. Continuous f i r i n g of each unit was maintained while 2-3 tasks were performed consecutively. Unit a c t i v a t i o n strategies included i n c i s a l , canine, intercuspal and 155 Figure 32. Raster displays of spikes and inter-spike i n t e r v a l s for a single masseter motor unit, f i r i n g at a c o n t r o l l e d prestimulus f i r i n g frequency of lO±2Hz, activated by three d i f f e r e n t tasks, and subject to constant i n t r a o r a l e l e c t r i c a l s t i m u l i (see t e x t ) . Figure 32, legend. Each spike immediately preceding the stimulus i s aligned at time zero. Spikes occurring during a period of 500ms before and a f t e r these spikes are shown. Seventy eight increasingly delayed s t i m u l i are depicted by a diagonal l i n e , and are arranged sequentially from above down. Data are shown for three tasks, intercuspal clenching (a) , i n c i s a l clenching (b) , and l e f t canine clenching (c). The prestimulus f i r i n g rates for t h i s unit and the stimulus strength were held constant. 156 158 Figure 3 3 . Peristimulus time histograms for a single masseter motor unit activated by three d i f f e r e n t tasks. Data have been reformatted so that s t i m u l i are aligned at time zero and ind i v i d u a l spikes are placed i n 5ms bins before and a f t e r the stimulus p o s i t i o n . The v e r t i c a l scale represents 5 spikes. The three tasks were l e f t canine clenching, i n c i s a l clenching, and intercuspal clenching. In a l l three cases, the stimulus strength was constant, consisting of single pulses delivered to the gin g i v a l tissues (see t e x t ) . s -140 0 +140 (ms) 159 u n i l a t e r a l molar clenching. A t o t a l of s i x d i f f e r e n t tasks were performed by a l l subjects i n the sample. The delivery of an i n t r a o r a l e l e c t r i c a l stimulus e l i c i t e d r e f l e x i n h i b i t i o n i n a l l 12 units when peristimulus i n t e r v a l s and prestimulus control i n t e r v a l s were compared (p<0.01). The i n h i b i t o r y e f f e c t always took the form of an increased duration of peristimulus i n t e r v a l s . However, the degree of i n h i b i t i o n of each unit appeared to vary according to the task performed (Figure 32). There were s i g n i f i c a n t differences between peristimulus i n t e r v a l s for at le a s t two d i f f e r e n t tasks (p<0.01) i n 10 out of 12 units, although there were no s i g n i f i c a n t differences between peristimulus i n h i b i t o r y responses obtained for the same task when the r e f l e x paradigm was repeated i n four units. When peristimulus time histograms were constructed for d i f f e r e n t tasks, the duration of periods of i n h i b i t i o n varied even although the tasks were performed by the same unit f i r i n g at a constant frequency and subjected to a constant stimulus paradigm (Figure 33). D. TWITCH TENSION MEASUREMENTS OF MOTOR UNITS IN THE MASSETER MUSCLE l . Force Transducer C a l i b r a t i o n A prefabricated transducer (Model PS10A, Kyowa, Tokyo, Japan) was used to measure i n c i s a l b i t e - f o r c e . I t incorporated a 160 Figure 34. Method of applying loads to the force transducer. A planar view of the transducer i s depicted i n "a". A , P , M , and L represent the antero-posterior and medio-lateral axes along which the t e s t load was moved. In "b" the tes t load i s shown applied perpendicular to the force transducer surface v i a a small metal sphere. This could be moved s t e r e o t a c t i c a l l y along antero-posterior and medio-lateral axes. 161 Figure 3 5 . C a l i b r a t i o n of the plotted against voltage (V) for perpendicular to the centre of represents the l i n e of best f i t biteforce-transducer. Force (N) a series of known loads applied the force transducer. The slope for a l l values plotted. 162 Figure 36. Asymmetrical loading of the transducer. A p l o t of voltage (V) against distance from the centre of the force transducer (mm) for a constant load applied v i a a metal sphere. Voltages measured i n an antero-posterior d i r e c t i o n ( » A " - " P " ) are represented by "+", and i n a medio-lateral d i r e c t i o n ("M"-"L") by open squares. 2.20 <D CD CO 2.04 1.88 1.72 ED 5 • • + • • + • • + • • • + • + 1.56 • 1.40 -1.00 -0.60 -0.20 0.20 Distance (mm) 0.60 1.00 / i M 163 unidimensional strain-gauge transducer which was 6mm i n diameter and 1.5mm i n height. To maximize s e n s i t i v i t y and to reduce e l e c t r i c a l noise, the transducer was powered by a customized 5V D.C. power source and D.C. amplification was used. A l l forces were sampled using a 12 b i t data a c q u i s i t i o n unit (DAU) [Model HP 3852a, Hewlett-Packard Canada, Vancouver, B.C.], which had a resolution of ±10V. C a l i b r a t i o n studies were performed to determine the r e p r o d u c i b i l i t y and l i n e a r i t y of the transducer. A bench-mounted c a l i b r a t i o n system incorporating a s t e r e o t a c t i c locating device permitted various loads to be applied to the transducer. The transducer was rated by the manufacturer to record forces up to 98N, although the working range of i n c i s a l b i t e forces s u f f i c i e n t to activate SMUs was consistently less than 9.8N. Each load was applied perpendicular to the centre of the transducer v i a a small s t a i n l e s s s t e e l sphere (Figure 13). Loads were applied throughout the working range of the transducer (0-9.8N) for the present experiment and the resultant signals amplified at high-gain (9.8N = +10V), sampled on a d i g i t a l oscilloscope, then measured (Model 2221, Tektronix Canada Inc., Vancouver, B.C.). In p a r t i c u l a r , a series of loads were applied i n small increments up to 3N i n order to t e s t the s e n s i t i v i t y of the transducer at very low force l e v e l s , because the i n c i s a l bite-forces required to activate masseter MUs were often i n t h i s range (see Figure 35) . Signals from the transducer, amplified at high-gain, proved to be reproducible and highly l i n e a r even at very low force l e v e l s . The 164 maximum resolution of the DAU over the working range of the transducer (0-9.8N) for the present experiment was estimated to be 2.4mN, which was equivalent to one d i g i t a l count on the DAU display. The e f f e c t of asymmetrically loading the transducer was also examined. The metal sphere through which the tes t load was applied, could be manipulated s t e r e o t a c t i c a l l y so that the device was loaded at d i f f e r e n t positions on i t s f l a t surface. A 1.96N load was applied and the sphere moved by 0.1mm increments in medio-lateral then antero-posterior d i r e c t i o n s (Figure 34). Amplified signals were sampled and measured at each position, then plotted (Figure 36). The sphere could be moved 0.5mm l a t e r a l l y or antero-posteriorly from the centre of the transducer with less than a 10% decrement i n the signal amplitude. The inherent noise recorded from the transducer was approximately 8mV, with an amplifier bandwidth of 0 - 3 0 k H z . The noise was predominantly i n the high frequency range and could be reduced to a n e g l i g i b l e amount by f i l t e r i n g high frequency signals above 1kHz. Therefore a bandwidth of 0-lkHz was used for a l l b i t e -force recordings. There was minimal signal d r i f t i n g with time v i z . approximately 12mV over a 60s epoch. 11. Bite-Force and Single Motor Unit Recordings Subjects were adult volunteers aged 31-4 9 years, with complete dentitions and no history of jaw dysfunction. A modified monopolar technique was used to record SMU a c t i v i t y 165 Figure 3 7 . I n c i s a l biteforce transducer. Cobalt-chrome metal castings cemented over the maxillary and mandibular i n c i s a l teeth. The lower casting incorporates a small metal sphere which contacts the centre of the force transducer located i n the upper casting. from 32 units i n the r i g h t masseter muscles of four subjects. In each case, a needle electrode was introduced percutaneously at r i g h t angles to the skin surface, then s t a b i l i z e d as described previously. A surface reference electrode was attached to the skin over the body of the muscle. Single motor unit a c t i v i t y was amplified, bandpass-filtered (300Hz-10KHz), then pulse discriminated by adjusting the amplitude of the SMU compound action p o t e n t i a l (CAP) u n t i l i t lay within a predetermined voltage-gated window. Each SMU action p o t e n t i a l and i t s associated discriminated pulse were viewed on a d i g i t a l o scilloscope. A p a i r of Co-Cr metal castings were cemented over the maxillary and mandibular i n c i s o r teeth used to customize the transducer for each subject. The lower casting contained a small s t a i n l e s s s t e e l sphere which contacted the centre of a f l a t force transducer i n the upper casting (Figure 37) . The transducer alignment was preset on mounted dental casts, and could be adjusted so that b i t e forces were only delivered perpendicular (P) to the dental occlusal plane, or 30 degrees anterior to t h i s d i r e c t i o n (A) , without a l t e r i n g the jaw p o s i t i o n (Figure 38) . The precise p o s i t i o n of contact of the metal sphere with the transducer was inspected v i s u a l l y to determine contact with the centre of the device, and adjusted when necessary by manipulation of the subject's jaw position. Jaw p o s i t i o n was constant throughout the experiment. 167 Figure 38. A mid-sagittal p r o f i l e of the s k u l l showing the projection of the ri g h t a r t i c u l a r condyle, the r i g h t masseter muscle, the f i r s t molar and i n c i s o r teeth, the functional occlusal plane, and the two possible orientations, P and A, of the force transducer. STMT (P) and (A) represent the mechanical contribution of a SMU to the o v e r a l l force recorded at transducer orientations P and A. L(P) and L(A) represent the b i t e point moment arms. Fm represents the twitch tension of a SMU, and Lm i t s moment arm. With a fulcrum positioned at the condyle, the torque produced by the unit (Fm x Lm) i s i n the jaw closing d i r e c t i o n . This torque would be r e s i s t e d by an equal and opposite torque produced at the bi t e point. At load c e l l orientation P the torque would equal STMT(P) x L(P) , and at transducer p o s i t i o n A i t would equal STMT(A) X L(A). 168 111. Data Sampling When a clearly-defined motor unit was activated at transducer orientation P, the f i r i n g frequency was kept within 7.5-10Hz by v i s u a l and auditory feedback. Discriminated SMU pulses were sampled every 0.5ms. The spike-triggered force record was sampled every ms with a data a c q u i s i t i o n unit and a disc-based computer (HP3852a and HP350, Hewlett-Packard Canada, Vancouver, B.C.), then stored as a series of events occurring within a sampling period of 30s. A STA technique (Stein et a l , 1972) was used to extract the mechanical contribution of i n d i v i d u a l SMUs to the o v e r a l l force recorded at load c e l l orientations P and A, t h i s contribution being defined as the spike-triggered "measured tension" (STMT). Each subject then maintained SMU f i r i n g while the transducer was realigned to orientation A, and the sampling protocol was repeated. In four units, the same protocol was repeated i n order to t e s t the r e p r o d u c i b i l i t y of the STMT measurements. In some instances, additional samples were recorded at both transducer orientations with the motor unit f i r i n g slowly as before, but with the subject d e l i b e r a t e l y co-activating antagonistic jaw muscles so as to " s t i f f e n " the mandible against p o t e n t i a l displacement i n any d i r e c t i o n . During each sampling session, the threshold force of SMU a c t i v a t i o n was measured from a cal i b r a t e d d i g i t a l voltmeter connected to the output of the transducer. 169 IV. Analysis of force Rigorous constraints were used i n the sel e c t i o n of ind i v i d u a l spike-triggered records before averaging. Individual SMU spikes were accepted only i f they were preceded and followed by an i n t e r v a l of at lea s t 100ms. These pre- and post-spike i n t e r v a l s were selected to minimize twitch summation and were s i m i l a r to c r i t e r i a s p e c i f i e d by Miles et a l (1987) and Nordstrom et a l (1989). Thus only force records obtained when the motor unit was f i r i n g at 10Hz or less were averaged i . e . the pre- and post-spike i n t e r v a l s were greater than 100ms. In order to remove the e f f e c t s of fluctuations i n the steady-state, background forces p r i o r to the averaging process, the mean value of the f i r s t 10ms epoch of the force record occurring a f t e r each preselected SMU was calculated and the force record reset to t h i s baseline. Individual force records, each of 100ms duration, were then averaged (n>8 0) and the peak STMT and time to peak contraction calculated for each unit at transducer orientations P and A (Figure 39). To confirm that the res u l t s were not affected by t h i s method of baseline resetting, the mean value of the l a s t 10ms epoch of the force record (ie 90-100ms) occurring a f t e r a SMU spike was calculated for 10 units. Each record was reset to t h i s baseline before the ind i v i d u a l force records were again averaged. As the peak STMTs and times to peak contraction were calculated by the two methods were not s i g n i f i c a n t l y d i f f e r e n t , mean values set to the i n i t i a l 10ms epoch were used throughout the remainder of the experiment. 170 Figure 3 9 . An example of a spike-triggered force recording. Offsets i n the much larger background b i t i n g force were removed before averaging by subtracting the mean value of the f i r s t ten data points from the remaining points i n each i n d i v i d u a l record. The peak amplitude of each averaged record, the "STMT", was defined as the length of a perpendicular drawn from the highest point of the response to a l i n e j o i n i n g the f i r s t and l a s t points in the record. The horizontal l i n e represents zero counts i n the data array. STMT i n t h i s example corresponds to 2 7 counts or 67mN. 171 V. Biomechanical analysis The locations of the load c e l l a p p l i c a t i o n point and the centres of the mandibular condyles were measured by means of tracings of calibr a t e d l a t e r a l cephalometric radiographs taken for each subject. These measurements were used to calcu l a t e the moment arm of the b i t e point from the condylar centre, on the assumption that forces were always delivered perpendicular to the orientation of the load c e l l (Figure 38). A constant twitch tension produced by a given SMU was expected to produce a constant torque i n the jaw cl o s i n g d i r e c t i o n , provided the unit's lever arm (an unknown variable) remained the same. Normally, t h i s would be r e f l e c t e d by an increased peak STMT when the transducer was oriented so as to cause a decreased tooth-j o i n t moment arm, and by a decreased peak STMT when the transducer was oriented to produce an increased moment arm. For each SMU, the peak STMT produced at the b i t e point by each SMU was used to calculate the b i t e opening torque (STTOR) around the mandibular condyles. This was defined as peak STMT x tooth moment arm, and was determined for each of the two transducer orientations. In contrast to the STMT values, which were expected to vary systematically and i n d i r e c t proportion to the b i t e point for an i n d i v i d u a l subject, STTOR values were expected to remain constant as load c e l l orientation changed, since a constant closing torque generated by a SMU would be matched with an equal and opposite opening torque at the b i t e point. I t was assumed that the demonstrations of unequal STTORs would s i g n i f y e i t h e r a change 172 i n the e f f e c t i v e length of the unit's lever arm, an a l t e r a t i o n i n STMT due to events influencing the transfer of SMU torque to the b i t e point, or both. VI. RESULTS a. Spike-Triggered Measured Tensions STMT measurements were obtained from 3 2 SMUs. The range of contraction times was 25-67ms. Threshold forces of SMU a c t i v a t i o n ranged from 0.05-6.57N as subjects aimed to maintain the slow f i r i n g rates of 7.5-10 spikes/s. In 24 units, STMTs were recorded at both transducer orientations P and A, while the units f i r e d continuously. In eight units, p r o f i l e s were recorded only at orientations P (six units) and A (two units) only, because the units ceased f i r i n g during the recording session. The range of peak STMTs amplitudes at load c e l l o rientation P was 5.9-96.0mN (mean 36.2±19.6mN, n=3 0 units) , and at orientation A was 4.9-130.3mN (mean 38.2±28.4mN, n=26 u n i t s ) . Repeated STMT measurements at orientations P and A for four units were within 3% of each other. As expected, i n 24 units for which paired data were available, the amplitudes of the STMT varied according to the orientation of the transducer. Typical STMTs for one unit are depicted i n Figure 40, which shows a much greater amplitude at transducer orientation P than at A. The peak STMTs measured at transducer orientation P were greater than those at A for a t o t a l of 11 units, and were smaller for nine units. Peak STMTs were s i m i l a r (±10%) at both 173 Figure 40. Spike-triggered averages of the load c e l l output (STMTs) recorded at transducer orientations P and A (upper and lower traces r e s p e c t i v e l y ) . P represents 93 averaged events, and A 81 events. The horizontal scale bar i s 30ms, and the v e r t i c a l bar i s 50mN. 50mN 30ms 174 orientations of the transducer for f i v e u n i t s . According to our assumptions, the plots of the STMTs developed at transducer orientations P and A for a given number of units should l i e on a s t r a i g h t l i n e for each subject, with the slope of each l i n e determined by the r a t i o of t h e i r respective force transducer moment arms in that subject. As expected, the slopes of the regression l i n e s d i f f e r e d between subjects. However, the c o e f f i c i e n t s of c o r r e l a t i o n between data points and the f i t t e d l i n e s were not s i g n i f i c a n t i n any instance (Figure 41). b. Spike-Triggered Torque Measurements In a l l subjects, matched STTOR values calculated for the P and A orientations of the b i t e force were not constant. Plots of one variable against the other did not l i e on a s t r a i g h t l i n e , and paired t - t e s t s c a r r i e d out between P and A STTOR values proved to be s i g n i f i c a n t l y d i f f e r e n t (P<0.001) for the 24 units tested, (see Figure 42). c. E f f e c t s of Muscle Coactivation In 11 units, STMTs could be altered markedly by d i f f e r e n t , subjectively-reported degrees of antagonistic muscle co-contraction i r r e s p e c t i v e of the orientation of the load c e l l . The peak STMT increased by 1.3 to 2.5 times with increasing co-a c t i v a t i o n . Figure 43 shows STMTs obtained from one unit, with and without subjective attempts to coactivate other muscles. In addition, t h i s unit could be driven without tooth contact, 175 Figure 41. Peak STMTs measured at transducer o r i e n t a t i o n P plotted against peak STMTs measured at orientation A, for 2 4 single motor units. Values for four subjects are i d e n t i f i e d by open/closed squares and open/closed t r i a n g l e s . Regression l i n e s are shown for 3 subjects i d e n t i f i e d by open/closed squares and closed t r i a n g l e s for whom more than two data points were available. 176 Figure 42. STTORs calculated from P-STMTs plotted against STTORs calculated from A-STMTs# for 24 single motor un i t s . The unit slope l i n e represents the expected r e l a t i o n of paired STTORs when P-STTOR equals A-STTOR. <10 - 3 10 8 ^ 6 DC e </) 4 i CL 2 + / v / + X + + 6 9 A-STTOR (Nm) 12 15 xicr 177 Figure 43. STMTs for a single motor unit recorded at transducer orientations P and A with and without antagonistic muscle co-act i v a t i o n . The e l e c t r i c a l noise i n the force recording system i s shown when the unit continued to f i r e without tooth contact, n = number of averaged events contributing to each STMT. The horizontal bar represents 3 0ms, the v e r t i c a l bar represents 50mN. C o - a c t i v a t i o n No Co-ac t i va t ion STMT(P) / n=87 n=108 STMT(A) / n=80 ru82 No Tooth Contact n=83 178 i n d i c a t i n g that the appropriate degree of coactivation could reduce i t s measured STMT from 78.4mN to zero. During the recording sessions, the same unit, and four others, could be f i r e d consistently by co-activation without contact on the transducer, as shown i n Figure 43. An additional four SMUs which could also be activated by co-activation, but were not incorporated i n the study because they could not be v o l u n t a r i l y driven when the teeth contacted the transducer. 179 DISCUSSION 1. GENERAL METHODS A. Single Motor Unit Recording A monopolar needle electrode i n combination with a surface reference electrode were used i n a l l SMU EMG experiments. The needle was inserted e a s i l y into the masseter muscle with only minor discomfort as i t penetrated the outer f a s c i a of the muscle. Discomfort during the recording session appeared to occur only i f the needle t i p was located close to a nerve branch, i n which case the needle was moved u n t i l the discomfort ceased. Muscle a f t e r -pain was n e g l i g i b l e , probably as a consequence of the needle being i n s i t u for no longer than 45 minutes during any EMG recording session, and because no vigorous clenching or chewing tasks were performed which may cause tiss u e damage as the needle t i p moves within the muscle. Although monopolar electrodes are reported to be less e l e c t r i c a l l y stable than other needle electrodes types (Kimura, 1983), t h i s was not evident i n the present SMU EMG experiments. A consistently high signal to noise r a t i o during EMG recording sessions permitted easy discrimination of SMU CAPs i n both masseter and l a t e r a l pterygoid muscles. S i n g u l a r i t i e s of SMU CAP shape were retained with only minimal changes i n form as a function of time. 180 Electromyographic signals were routinely bandpass-filtered to eliminate low frequency components of the waveforms. For a l l experiments except mapping of MU t e r r i t o r y , the EMG amplifier bandwidth was 3 00Hz-10kHz which yielded d i s t i n c t i v e SMU CAPs with multiple high frequency components. The upper frequency response was maintained consistently at 10kHz i n order to r e t a i n the amplitude of the fast components of the signal (Stalberg and Antoni, 1983). A h i g h p a s s - f i l t e r of 1kHz was used i n the t e r r i t o r i a l mapping study i n order to achieve an increased s e l e c t i v i t y of SMU EMG recordings. With a bandwidth of lkHz-lOkHz, the volume of pick-up of the needle electrode was estimated to be of radius 2mm, at least for the high frequency components of the s i g n a l . This finding was comparable with that reported previously for monopolar needles (Stalberg, 1980). The increased s p a t i a l resolution achieved by bandpass-filtering from lkHz-lOkHz s i g n i f i c a n t l y reduced the l i k e l i h o o d of paired needle recordings occurring from the same group of SMU f i b r e s which was an advantage in the MU mapping study. The combined attributes of the monopolar needle electrode v i z . minimum discomfort during i n s e r t i o n into the muscle, i t s low noise c h a r a c t e r i s t i c s , and good s p a t i a l resolution made the needle u n i v e r s a l l y appropriate for multiple SMU recordings i n the masseter muscle. 181 B. Stereotactic Location of the Needle Electrode Recording Sites Although s t e r e o t a c t i c surgery has been performed on humans since 1947 (Spiegel et al) and the s t e r e o t a c t i c p r i n c i p l e s on which i t was based were developed early i n the 2 0th century, the invasiveness of ste r e o t a c t i c procedures have precluded t h e i r use in studies involving normal humans subjects. The present s t e r e o t a c t i c technique represents an i n d i r e c t method of lo c a t i n g needle electrode recording s i t e s within the human masseter muscle. The method has combined a SMU EMG recording technique, Magnetic Resonance (MR) imaging, the three-dimensional reconstruction of o r o - f a c i a l tissues, and a common reference system. The reference system took the form of a square-shaped g r i d with the four corners of the square defining the reference plane. A square shape was selected for ease of f a b r i c a t i o n , but only three known points were i n fact necessary to ca l c u l a t e a plane of reference. The g r i d was required to be constructed of non-ferrous materials because of the subsequent imaqing procedure. A combination of brass struts and a p l a s t i c b i t e f o r k implanted within a c r y l i c formed a suitable r i g i d , non-bulky design on which to support the copper s u l p h a t e - f i l l e d tubes. The decision to use the occlusal surfaces of the teeth as a method of f i x i n g the g r i d in a reproducible p o s i t i o n posed a po t e n t i a l problem of tissue movement a r t i f a c t . Minor separation of the teeth was necessary so that the b i t e f o r k segment of the grid could be placed between the 182 teeth a f t e r each EMG recording session. This was a concern because of possible movement of the needle located within the muscle during i n s e r t i o n and removal of the g r i d . In p r a c t i c e however, t h i s proved to be minimal as judged by the minor changes i n SMU waveform shape before and a f t e r the procedure (see Figure 12). Each needle electrode was inserted into the muscle at r i g h t angles to the skin and then supported to minimize any bending of the needle within the t i s s u e s ; however, the p o s s i b i l i t y of unforeseen needle bending within the muscle remained e s s e n t i a l l y uncontrolled. During d i g i t i z i n g of reference points on the needle and the g r i d by a Reflex Metrograph, there were po t e n t i a l errors due to subject movement and by the operator recording the reference points. A l l subjects practised head positioning with the aid of r i g i d nose and chin supports, customized with rubber impression material, so that head movement was n e g l i g i b l e during each short recording session (approximately 60s). One operator (ASM) who had previously practised the recording technique performed a l l Metrograph recording procedures. A previous study of the Metrograph's recording c h a r a c t e r i s t i c s has shown that d i g i t i z i n g of points i n three planes of space without d i r e c t contact with the object can be obtained with a consistent measurement accuracy, and that s p e c i f i c points can be recorded with an accuracy of 10.1mm even by inexperienced operators (Takada et a l , 1983). Anatomical features i n the o r o - f a c i a l region may be observed on CT and MR images. Computed Tomography was considered for 183 imaging the o r o - f a c i a l region because i t can depict both sof t and hard tissues with good resolution, but i t s use i s l i m i t e d due to the p o t e n t i a l radiation hazard (2-4rads/slice), e s p e c i a l l y when normal tissu e i s involved (Seltzer et a l , 1987). Computed Tomography images are also degraded s i g n i f i c a n t l y by m e t a l l i c dental restorations and appliances. MR imaging was preferable because i t i s non-invasive and can also provide hard and s o f t t i s s u e images from multiple a x i a l , coronal and s a g i t t a l scans. The spin-echo pulse sequence was selected for t h i s study as i t i s reported to be the optimum sequence for musculoskeletal MR imaging because i t provides a high degree of ti s s u e d i f f e r e n t i a t i o n (Lam et a l , 1989). With t h i s p a r t i c u l a r sequence adipose ti s s u e , medullary bone, and the aqueous copper sulphate solution within the reference g r i d yielded high i n t e n s i t y signals which appeared white on MR images. Aqueous copper sulphate solution (0.001M) was selected s p e c i f i c a l l y because of i t s high i n t e n s i t y signal (Stewart, 1987). The teeth and c o r t i c a l bone produce very low i n t e n s i t y signals and thus appeared black. Muscle tissu e produced a signal of intermediate i n t e n s i t y and appeared grey on imaged sections. The copper receiver c o i l which was placed over the r i g h t masseter muscle produced signals of even d i s t r i b u t i o n throughout each a x i a l MR section. Accurate s p a t i a l information i s e s s e n t i a l for e f f e c t i v e stereotaxy. I f eddy currents are produced during a pulse sequence, inhomogeneities and n o n - l i n e a r i t i e s are induced which may d i s t o r t 184 the image. Fortunately, the Picker V i s t a MR1100 system used i n t h i s study had been investigated previously for such problems (Stewart, 1987). Each subject's head was positioned e n t i r e l y within the volume of 12100cm3 at the centre of the magnetic c o i l which was deemed to be homogenous and l i n e a r during imaging. The magnet used i n the present series of MR images possessed a stationary magnetic f i e l d of 0.15 Tesla. This provided adequate resolution for the purposes of t h i s study because fine tissue d e t a i l within anatomical structures was not mandatory. However, more powerful magnets would permit improved tissue d i f f e r e n t i a t i o n . Although the resolution of MR imaging at present does not permit v i s u a l i z a t i o n of muscle f i b r e s , muscle compartments separated by connective tiss u e septa and discrete f a s c i c l e s can be seen (Lam et a l , 1989). This should be an advantage when the int e r n a l architecture of a muscle i s considered in future electrophysiological studies. For each subject involved i n s t e r e o t a c t i c EMG studies, the relevant anatomical features and the centroids of the copper sulphate spheres were traced then d i g i t i z e d as a two stage procedure p r i o r to configuration of the data into a three-dimensional matrix for graphic display. With advancing computer technology, i t should be possible to d i g i t i z e MR images d i r e c t l y from a computer monitor to minimize tr a c i n g errors. MR imaging i s compromised by materials which contain iron, cobalt, n i c k e l and chromium due to t h e i r inherent ferromagnetic properties (Stewart, 1987). Since conventional EMG recording 185 electrodes, such as the monopolar type used i n t h i s study, are usually made of s t a i n l e s s s t e e l , t h e i r d i r e c t v i s u a l i z a t i o n by MR i s impractical. Even with electrodes of low magnetic permeability (e.g. tungsten), there remains the s i g n i f i c a n t problem of tr a n s f e r r i n g the subject to an imaging f a c i l i t y without producing electrode movement. In addition, each subject was required to l i e supine and motionless during the 30 minute imaging period. There i s also the major l o g i s t i c a l problem of multiple SMU experiments performed on a number of subjects at multiple recording sessions. The present i n d i r e c t method of locating needle electrode t i p s within muscles avoided these problems. 11. SPECIFIC STUDIES A. MOTOR UNIT TERRITORIES IN THE MASSETER MUSCLE The SMU recording method used i n t h i s study was based on the technique of electrophysiological cross-sectional scanning of MUs in human muscles (Schwartz et a l , 1976; Stalberg & Eriksson, 1987; Stalberg & Antoni, 1980), but with a number of modifications. Stalberg and Antoni (1980) o r i g i n a l l y described the use of a s i n g l e - f i b r e electrode to record a single f i b r e action potential which acted as a t r i g g e r . A second concentric needle was used to probe for MU a c t i v i t y , synchronous with the s i n g l e - f i b r e action po t e n t i a l , i n the cross-section of the muscle, without any knowledge of the s p a t i a l r e l a t i o n s h i p of the needle recording 186 locations within the muscle as a whole. The present study, however, employed two monopolar needles with one acting as the t r i g g e r i n g electrode. The paired needles were used to probe for synchronous MU a c t i v i t y i n the masseter muscle cross-section, and also along additional axes because of the p o s s i b i l i t y of underestimating MU t e r r i t o r y by cross-sectional probing alone due to the multipinnate nature of the muscle. In addition, because the needle recording t i p s could be located s t e r e o t a c t i c a l l y i n three dimensions of space, the location of these s i t e s could be displayed within a three-dimensional reconstruction of the masseter muscle unlike previous studies of masseter MU t e r r i t o r y (Stalberg and Eriksson, 1987). Discrete SMU CAPs were re a d i l y recorded by the t r i g g e r i n g electrode and MUs maintained regular f i r i n g at very low force l e v e l s . The SMU CAPs were consistent i n amplitude and shape which f a c i l i t a t e d continuous t r i g g e r i n g of an oscilloscope sweep. The recording of synchronous SMU a c t i v i t y by the second electrode proved to be r e l a t i v e l y simple i n most instances by systematic probing of the muscle cross-section i n d i f f e r e n t parts of the muscle. However there was a potential systematic error inherent in the technique which occurred, i n part, because of the need to l i m i t the number of needle penetrations at any one experiment as a means of maintaining patient compliance. At each recording session the second electrode was i n i t i a l l y located approximately 20mm from the t r i g g e r i n g s i t e (as measured on the skin surface). However, i f no synchronous EMG a c t i v i t y was recorded i n the muscle 187 cross-section at that p a r t i c u l a r s i t e , the needle was moved progressively closer to the t r i g g e r i n g s i t e . I f no a c t i v i t y was recorded a f t e r 2-3 probes the needle was then moved to a po s i t i o n 5-7mm from the t r i g g e r i n g electrode where a synchronous CAP could usually be recorded. This may lead i n some instances to an underestimation of the l i m i t s of synchronous MU a c t i v i t y along the axis defined by the l i n e j o i n i n g the two recording t i p s . The distances between paired recording s i t e s i n the masseter muscle were markedly greater than the mean SMU diameter (3.7±0.6mm) reported i n the i n f e r i o r part of the muscle by Stalberg and Eriksson (1987) , and i n fact may be even larger since the radius of pick-up of the needle electrode was estimated to be 2mm. The fo c a l MU t e r r i t o r i e s i n the pig masseter muscle described by Herring et a l (1989) were also s i g n i f i c a n t l y smaller. Distances between paired recording s i t e s i n the medio-lateral d i r e c t i o n were however comparable with the t e r r i t o r i e s defined by Stalberg and Eriksson (1987) who used transverse cross-sectional scanning. The t e r r i t o r i e s recorded by the present technique suggest that masseter unit t e r r i t o r i e s cannot be measured accurately, and may be consistently underestimated, by un i a x i a l cross-sectional scanning alone. Although most SMU diameters described by Stalberg and Eriksson (1987) were less than 5mm, there were however f i v e out of 32 recordings i n which the diameter ranged from 5.0-12.5mm, and three of these appeared to traverse a large part of the muscle cross-section. Likewise i n the present study a small number of paired recordings (six out of 32) measured i n the medio-lateral 188 d i r e c t i o n ranged from 5-10mm. This observation i s at variance with the s i t u a t i o n i n the human limb and most animal muscles where MU f i b r e s are d i s t r i b u t e d over only part of the muscle cross-section (e.g. Buchthal & Schmalbruch, 1980; Stalberg & Antoni, 1980; Stalberg et a l , 1976), and concurs with Stalberg and Eriksson's (1987) finding that a small number of masseter units may have quite large t e r r i t o r i e s . Motor unit t e r r i t o r i e s i n the masseter muscle, although larger than previously reported, do not i n most instances appear to extend widely throughout the muscle, and may i n fact be r e s t r i c t e d to s p e c i f i c regions. The r e s u l t s also suggest that there i s a preferred axis of orientation i n the antero-posterior d i r e c t i o n between pairs of recording s i t e s . This i s consistent with the notion of "cigar-shaped" MU t e r r i t o r i e s arranged i n layers throughout the muscle, rather than the circular-shaped t e r r i t o r i e s proposed by Stalberg and Eriksson (1987) which were based on masseter muscle cross-sectional scans. The diagrams of the masseter muscle i n Figure 4 4 are redrawn from Schumacher (1961). They show the separation of successive layers of muscle f i b r e s by connective t i s s u e septa from the s u p e r f i c i a l to the deep part of the muscle. The width of the f i b r e aggregations i n the antero-posterior d i r e c t i o n i s greater than along other axes. The preferred orientation of the masseter SMU t e r r i t o r i e s appears consistent with the anatomical compartments described by Schumacher (1982, 1961). 189 F i g u r e 44. I n t e r n a l a n a t o m y o f t h e h u m a n m a s s e t e r m u s c l e . The separation of successive layers of muscle f i b r e s (shaded) by connective t i s s u e septa are depicted from the s u p e r f i c i a l to the deep part of the muscle, (from above down). These diagrams are redrawn from Schumacher (1961). 190 The locations of paired recording s i t e s and the distance between them do not themselves indicate the whole t e r r i t o r y of a MU. The locations from which recordings are made do however reveal the minimum extent of unit t e r r i t o r y along the axis of orientation defined by the l i n e j o i n i n g the two recording t i p s provided t h e i r radius of pickup i s known (in t h i s instance 2mm). By placing more than two electrodes intramuscularly, i t would be possible to probe for additional f i b r e s i n multiple axes of o r i e n t a t i o n within the muscle, and to r e l a t e the positions of the recording t i p s r e l a t i v e to one another within the reconstructed muscle. Thus a more comprehensive picture of the l i m i t s of the MU t e r r i t o r y could be determined within the whole muscle volume. The present method of estimating masseter muscle MU t e r r i t o r y r e l i e s on the absence of MU synchronization within the muscle. In normal muscles MUs are presumed to r e c r u i t asynchronously (Loeb et a l , 1987), and during the ensuing voluntary contraction units f i r e independently of one another (Milner-Brown et a l , 1975). In human muscles, synchronization has been described i n subjects with neuromuscular disorders (Milner-Brown et a l , 1974). Synchronization has also been observed within the muscles of normal subjects who regularly exert large, b r i e f forces (e.g. w e i g h t l i f t e r s and manual labourers), and i s generally observed to increase with the duration of muscle contraction and p a r t i c u l a r l y with muscle fatigue (Milner-Brown et a l , 1975; Person & Kudina, 1968) . 191 In the human masseter muscle, Yemm (1977) was unable to detect any tendency towards synchronization of MUs using the r e c t i f i e d EMG technique described by Milner-Brown et a l (1973). Likewise Goldberg and Derfler (1977) , using c r o s s - c o r r e l a t i o n histograms, reported no tendency towards masseter MU synchrony. More recently Miles and Nordstrom (1988) reported only a very weak tendency towards synchronization of masseter MUs which f i r e d consistently for periods of 15 minutes, using cro s s - c o r r e l a t i o n i n t e r v a l histograms on masseter MU spike t r a i n s . In the present study masseter MUs f i r e d for only short periods of time and at low force thresholds; therefore, any tendency towards synchronous MU f i r i n g within the muscle, although possible, was un l i k e l y . The r e s t r i c t i o n of MUs to certa i n regions of the masseter muscle provides a possible substrate for d i f f e r e n t i a l motor control of discrete regions of the muscle as has been proposed previously for the human masseter muscle (Stalberg & Eriksson, 1987) and reported i n the pig and rabbit masseter muscles (Herring et a l , 1989; 1979; Weijs & Dantuma, 1981). Thus d i f f e r e n t i a l contraction of units i n separate parts of the muscle would then permit m u l t i - d i r e c t i o n a l contraction of in t e r n a l connective tissue septa, and therefore provide a functional basis for the heterogenous behaviour of the human masseter muscle suggested by Belser and Hannam (1986) and Eriksson et a l (1984). The wide d i s t r i b u t i o n of a small number of MU t e r r i t o r i e s in the muscle cross-section, however, suggests that muscle 192 compartments may not be based s o l e l y on the i n t e r n a l anatomical features of the muscle, and that the compartments may not i n fact have s t r i c t l y defined boundaries, or some unit t e r r i t o r i e s may traverse more than one compartment as proposed by Stalberg and Eriksson (1987). Previous studies by Herring et a l (1989) i n pig masseter muscle suggest that there may not be clear-cut anatomical compartments but rather, less d i s t i n c t functional subdivisions. The extent to which t h i s p u t a t ive " f u n c t i o n a l compartmentalization" applies to the human masseter i s presently uncertain. Certainly the concept of "regional s p e c i a l i z a t i o n " has been proposed for the human masseter muscle by Stalberg and Eriksson (1987), but i t i s equivocal without further i n s i g h t into the s p e c i f i c behaviour patterns of SMUs i n d i f f e r e n t known regions of the muscle. B. MOTOR UNIT TASK PROFILES IN DIFFERENT REGIONS OF THE MASSETER MUSCLE The s t e r e o t a c t i c method of locating needle electrode recording s i t e s employed i n t h i s study permitted MU behaviour to be studied systematically i n known regions of the masseter muscle, in cont r a d i s t i n c t i o n to previous studies i n which the precise recording location within the muscle was unknown (e.g. Belser and Hannam, 1986; Eriksson et a l , 1984; Goldberg and Derfler, 1977; Yemm, 1977). The unique a b i l i t y of human subjects to perform highly s k i l l e d voluntary motor tasks made the present study of masseter MU 193 behaviour f e a s i b l e . The subjects who took part i n t h i s study were adept at f i r i n g masseter MUs slowly and consistently using auditory and v i s u a l feedback. Each subject was dentally-trained and therefore highly cognisant of the d i f f e r e n t i n t r a o r a l strategies used to activate i n d i v i d u a l masseter MUs, and p a r t i c u l a r l y the subtle tasks involving tooth clenching on v e r t i c a l and i n c l i n e d planes. The l e v e l of s k i l l of the subjects was such that unit a c t i v a t i o n strategies could be changed i n a matter of seconds with unit f i r i n g usually maintained by jaw muscle coactivation during the b r i e f t r a n s i t i o n between tasks. The SMU EMG recording technique yielded SMU compound action potentials (CAPs) of consistent shape during the performance of multiple i n t r a o r a l tasks, and only occasional minor repositioning of the needle t i p was required i n order to maintain the EMG signal amplitude. Despite minor a l t e r a t i o n s i n the jaw position predicated by the d i f f e r e n t unit a c t i v a t i o n strategies, there was no s i g n i f i c a n t discomfort during or a f t e r the recording session. Continuous f i r i n g of a unit and simultaneous v i s u a l i z a t i o n of the SMU CAP on an oscilloscope screen was considered mandatory during the performance of d i f f e r e n t tasks i n order to di s p e l any uncertainty with regard to consistent recording from the same MU, which was a p o s s i b i l i t y i f there were s i g n i f i c a n t pauses i n unit f i r i n g i n combination with minor needle electrode movement. As an additional safeguard, plots of averaged CAPs for each unit task were compared v i s u a l l y for consistency of waveform features which e f f e c t i v e l y eliminated the chance of recording from another unit. 194 The 13 i n t r a o r a l tasks used to e l i c i t MU a c t i v a t i o n i n t h i s study do not represent the t o t a l number of i n t r a o r a l tasks that may be performed by any one subject. They were however selected as a representative sample of jaw muscle coactivation strategies and a d i s t i n c t range of contact positions along the tooth row which may be performed e a s i l y by dentally-aware subjects. Tooth contact positions were selected on both sides of the dental arch, i n part, to explore possible differences i n EMG a c t i v i t y with changes i n cross-arch contacts, because d i f f e r e n t i a l EMG a c t i v i t y has been observed previously i n d i f f e r e n t parts of the human masseter muscle when i p s i l a t e r a l versus c o n t r a l a t e r a l tooth clenching was performed (Belser and Hannam, 1986; Greenfield and Wyke, 1956). The present stereotactic technique of locating recording s i t e s presented a unique opportunity to probe for unit a c t i v i t y i n d i f f e r e n t known parts of the muscle during clenching in b i l a t e r a l l y symmetrical tooth-clenching positions, and thus c l a r i f y the proposed functional d i f f e r e n t i a t i o n of the human masseter muscle into s u p e r f i c i a l and deep parts (Belser and Hannam 1986). From c l a s s i c a l anatomical studies i t has been assumed that the s u p e r f i c i a l f i b r e s of the masseter muscle are active during a n t e r i o r l y directed jaw closure whereas the deep part of the muscle i s involved i n jaw elevation with r e t r u s i o n (Brand and Isselhard, 1986; Du Brul, 1980). In the present study, however, the 13 disparate i n t r a o r a l tasks used to t e s t f o r MU a c t i v a t i o n could be performed by at least one MU i n both s u p e r f i c i a l and deep 195 parts of the muscle, a l b e i t with d i f f e r e n t prevalences. This suggests that MUs i n both parts of the muscle may contribute to a c e r t a i n task but that the r e l a t i v e contribution i n terms of t o t a l MU a c t i v i t y may be d i f f e r e n t . In support of t h i s contention, Belser and Hannam (1986) have described quantitative changes in EMG a c t i v i t y i n s u p e r f i c i a l and posterior deep parts of the masseter muscle depending on the po s i t i o n of the b i t e point along the tooth row and the d i r e c t i o n of e f f o r t at that point. This notwithstanding, SMUs which could be activated by such tasks as jaw retrusion with and without tooth contact appeared to be p r e f e r e n t i a l l y located i n the deep part of the muscle where the major orientation of the muscle f i b r e s was most favourable for those tasks. Likewise more units could be activated by a n t e r i o r l y directed jaw c l o s i n g tasks i n the s u p e r f i c i a l part of the muscle. In the s u p e r f i c i a l part of the muscle, units could be activated by i p s i l a t e r a l molar clenching only i n the superior part of the muscle which i s i n agreement with Greenfield and Wyke's (1956) findings using a surface EMG technique. Units activated by co n t r a l a t e r a l molar clenching, however, were located i n the anterior superior part of the muscle i n both s u p e r f i c i a l and deep parts. A l l 50 masseter MUs studied were capable of performing more than one task and i n some instances up to f i v e or s i x tasks. This finding compares favourably with the multi-task units described previously i n the human masseter muscle (Eriksson et a l , 1984), the adductor p o l l i c i s brevis muscle (Thomas et a l , 1987; 1986), 196 the f i r s t dorsal interosseous muscle (Desmedt, 1983; Thomas et a l , 1986), and the biceps b r a c h i i muscle (ter Haar Romeny et a l , 1982). However, Eriksson et a l (1984) and t e r Haar Romeny et a l (1982) also described t a s k - s p e c i f i c units within the human masseter and temporalis, and biceps muscles respectively, whereas in the present study a l l masseter units were capable of performing multiple tasks. The predominance of "dental" (tooth contact only) units i n the posterior s u p e r f i c i a l part of the muscle suggests that t h i s region may be involved mainly i n the generation of b i t e f o r c e , with l i t t l e or no r o l e i n jaw posture. The histochemical composition of t h i s region supports t h i s contention because there are reportedly more type 11 f i b r e s i n t h i s region than any other part of the muscle (Eriksson and Thornell, 1983). Each masseter unit could perform at l e a s t two of the 13 "test" tasks but the p a r t i c u l a r tasks performed varied from unit to unit. This observation concurs with Eriksson et a l ' s (1984) findings in masseter and temporalis muscles where not a l l MUs could perform two designated tasks, v i z . intercuspal clenching and jaw retrusion, and i s i n agreement with the observation by English (1985) that many MUs are not necessarily active during contraction of the parent muscle. The d i v e r s i t y of tasks performed by d i f f e r e n t masseter MUs i s at variance however with studies i n the adductor p o l l i c i s brevis and f i r s t dorsal interosseous muscles where there was no evidence for the s e l e c t i v e a c t i v a t i o n of units for d i f f e r e n t tasks (Thomas et a l , 1987; 1986). 197 Masseter MUs i n the present study were activated s e l e c t i v e l y to perform c e r t a i n i n t r a - o r a l tasks. This suggests that d i f f e r e n t populations of motoneurons were activated within the trigeminal motoneuron pool, possibly as a consequence of the d i s t r i b u t i o n of input a c t i v i t y to the pool varying according to the task at hand (ter Haar Romeny et a l , 1982). This proposal of MU subpopulations i s apparently at variance with the c l a s s i c a l " s i z e p r i n c i p l e " of MU recruitment whereby inputs to the motoneuron pool are di s t r i b u t e d homogenously throughout the pool and thus an i n j e c t i o n of current to the pool excites most or a l l motoneurons which in turn permit motoneuron recruitment i n a predictable order from small to large somata on the basis of each c e l l ' s input impedance (Henneman et a l , 1965a; Henneman et a l , 1974) . However modification of t h i s s t r i c t hierarchy by muscle s t r e t c h reflexes, suprasegmental commands which designate the use of multifunctional muscles i n d i f f e r e n t ways, uneven d i s t r i b u t i o n of sensory inputs to the motoneuron pool, and changes i n synaptic e f f i c i e n c y appear to permit a ce r t a i n amount of f l e x i b i l i t y i n motoneuron patterning which may r e s u l t i n segments of the motoneuron pool being used for p a r t i c u l a r functional requirements (Freund, 1983; Kanda et a l , 1977; Luescher et a l , 1979). This p o t e n t i a l for limited modification of muscle MU combination appears to be r e s t r i c t e d to composite, multifunctional muscles (Freund, 1983) . Such variations in MU combination may be observed only i n humans when the MU population output i s studied over a spectrum of goal-directed movements (Freund, 1983; Stephens et a l , 1978), and not 198 d i s c e r n i b l e i n the experiments of Henneman et a l (1965a,b) which were ca r r i e d out on decerebrate or p a r t i a l l y de-efferented cats. Motor unit f i r i n g frequency was r e a d i l y c o n t r o l l e d by means of auditory and v i s u a l feedback during the performance of a p a r t i c u l a r task. Each subject routinely practiced f i r i n g the MU under study at the lowest sustainable f i r i n g frequency (LSFF) for each task p r i o r to the recording of an array of ISIs. The LSFF or f i r i n g rate below which prolonged interruption i n f i r i n g occurred was analogous to that defined by Petajan (1981) , and to the " l i m i t frequency" described by Eriksson and Stalberg (1984) . The method of slowly increasing then decreasing the u n i t f i r i n g rate a number of times p r i o r to recording a spike t r a i n appeared to give the subject an inherent " f e e l " for the LSFF of the MU for a p a r t i c u l a r task, and permitted the operator (ASM) to record an ISI array in a standardized manner between subjects. The c a l c u l a t i o n of the mean inter-spike i n t e r v a l (ISI) and standard deviation (SD) for any SMU spike t r a i n may be subject to random fluctuations depending on the number of ISIs used for the c a l c u l a t i o n , and to bias i f , for instance, i n d i v i d u a l spikes are omitted during the EMG recording of the spike t r a i n . Andreasen (1980) has previously shown that the error rate for the c a l c u l a t i o n of the mean i s usually below 1% f o r a series of 200 ISIs recorded consecutively; therefore, a comparable array of ISIs were recorded i n the present study. The mean inter-spike inte r v a l s (ISIs) recorded at the lowest sustainable f i r i n g frequency (LSFF) for the s e r i e s of masseter 199 Table 111. Human muscle motor unit mean inter-sp i k e i n t e r v a l s recorded at the lowest sustainable f i r i n g frequency: Muscle ISItlSD (ms) Fro n t a l i s 102129 Orb i c u l a r i s o r i s 70119 Deltoid 116123 Biceps 124121 Triceps 132136 Brachiora d i a l i s 116122 Pronator teres 132138 F i r s t dorsal interosseous 142139 Multifidus 152133 Vastus l a t e r a l i s 126130 Gluteus maximus 128130 T i b i a l i s anterior 124126 Biceps femoris 132129 Medial gastrocnemius 156129 Extensor digitorum brevis 138129 data abstracted from Petajan (1981) 200 units concur with the findings of Freund (1983), Petajan (1981) and Clamann (1970) that MUs i n general cannot be driven consistently below 6Hz, even by h i g h l y - s k i l l e d normal subjects using audiovisual feedback. In the present ser i e s of masseter MUs, the LSFF for many units f e l l within the range 5-8Hz described by Eriksson et a l (1984) although some units could not be driven consistently below 8-10Hz as reported previously by Nordstrom et a l (1989), and some units f i r e d consistently above 10Hz. Thus the observation by Petajan (1981) that the LSFF f o r f a c i a l muscle MUs was greater than that for limb muscles was not consistently borne out i n the present series of masseter units, at l e a s t for cer t a i n tasks. In fact, many masseter MUs had s i m i l a r LSFFs to limb muscles. Mean ISIs recorded at the LSFF for a s e l e c t i o n of human limb muscle MUs derived from Petajan (1981) are presented i n Table 111 for the purpose of comparison with masseter muscle means ISIs in Table 11. There was considerable v a r i a t i o n i n the mean ISIs recorded at the LSFF for d i f f e r e n t tasks i n single masseter units which i s in agreement with previous work i n t h i s muscle (Eriksson et a l , 1984), but i n con t r a d i s t i n c t i o n to the findings i n adductor p o l l i c i s brevis muscle units where mean ISIs recorded for d i f f e r e n t tasks were si m i l a r (Thomas et a l , 1987). The f i r i n g frequency of SMUs usually increases monotonically as the force produced by the muscle as a whole increases (De Luca, 1985; Miles, 1987). Motor unit f i r i n g patterns i n general are determined by the i n t r i n s i c properties of the parent motoneurons 201 and by d i s t r i b u t i o n of central and peripheral inputs to these motoneurons (Henneman and Mandell, 1981). The f i r i n g pattern during voluntary contraction varied between tasks f o r a l l single masseter units studied, which indicates changes i n excitatory drive and hence va r i a t i o n s i n afferent or descending input to the trigeminal motoneuron pool according to the task performed even although subjects consistently attempted to maintain unit f i r i n g at the LSFF. Peripheral sensory receptors i n the o r o - f a c i a l region include muscle spindles, cutaneous, j o i n t and periodontal mechanoreceptors, and free nerve endings (Dubner et a l , 1978). The evidence i s not compelling for the presence of Golgi tendon organs i n human jaw muscles (Sessle, 1981). Periodontal mechanoreceptors have been shown to be d i r e c t i o n a l l y s e n s i t i v e (Hannam, 1982), therefore i t i s l i k e l y that t h e i r output i s modulated depending on the r e l a t i v e angulation of opposing tooth contacts as well as t h e i r p o s i t i o n along the tooth row. Periodontal mechanoreceptors appear to have a role i n i n h i b i t o r y feedback mechanisms to jaw elevator muscles (Kidokoro et a l , 1968; Van Steenberghe and Devies, 1978), and a greater density of these receptors has been located i n the anterior segment of the dental arch (Hannam, 1982). Muscle spindles are common in the human masseter muscle (Dmytruk, 1974; Smith and Macarian, 1967), and t h e i r projections to trigeminal motoneurons although reportedly weaker than for limb muscles, appear to be graded i n a s i m i l a r manner with stronger inputs to functional type S, than type FR or FF motoneurons 202 (Taylor, 1981). Masseter muscle spindle afferent f i b r e s are also reported to reach the cerebral cortex v i a the i p s i l a t e r a l trigeminal mesencephalic nucleus (Dubner et a l , 1978; Hoffman and Luschei, 1980). Type l a afferents project to motoneurons of the parent muscle and also to the motoneurons of functional synergists (Burke, 1981). In the o r o - f a c i a l region there i s considerable interplay between s y n e r g i s t i c muscles during jaw movements and isometric tooth contact, for example the paired masseter, medial pterygoid, and temporalis muscles during jaw elevation (Dubner et a l , 1978; MacDonald and Hannam, 1984). I t i s conceivable that during the performance of d i f f e r e n t tasks by a single masseter MU that there i s d i f f e r e n t i a l l a input from sy n e r g i s t i c muscles on to masseter motoneurons depending on the contribution of these synergists to the p a r t i c u l a r task, which may lead i n turn to v a r i a t i o n s in peripheral excitatory drive to the motoneuron pool. Binder et a l (1976) have demonstrated that a SMU twitch produces a l t e r a t i o n s i n the discharge patterns of muscle spindle afferents i n limb muscles, although spindle receptors do not necessarily respond to the twitches of a l l SMU twitches. In the cat posterior t i b i a l i s muscle, Binder and Stuart (1980) demonstrated that muscle spindle discharge was highly v a r i a b l e i n response to contraction of SMUs within the muscle. The response of a p a r t i c u l a r spindle to a SMU twitch appeared to be cl o s e l y related to the anatomical arrangement of the spindle with respect to the SMU t e r r i t o r y . Based on these findings, sensory 203 p a r t i t i o n i n g of the MU population within a muscle has been proposed, with muscle spindles servicing groups of MUs within the muscle (Binder and Stuart, 1980). Although s p a t i a l d i spositions of muscle spindles within the human masseter muscle are uncertain, "sensory p a r t i t i o n i n g " presents an a t t r a c t i v e model with which to explain, at l e a s t i n part, the differences i n excitatory drive exhibited by the same MU for d i f f e r e n t tasks. Given the r e l a t i v e l y l imited masseter MU t e r r i t o r i e s and t h e i r association with putative MU compartments (McMillan and Hannam, 1989), i t i s possible that the d i f f e r e n t muscle l i n e s of action produced according to the task performed may d i f f e r e n t i a l l y activate the muscle spindles present i n that p a r t i c u l a r muscle region causing variations i n l a inputs to higher centres of motor contr o l . The central neural connections of o r o - f a c i a l afferent inputs are not so well established as those for spi n a l afferents (Dubner et a l , 1978; Hoffman and Luschei, 1980). O r o - f a c i a l muscle spindle afferent stimulation appears to evoke c o r t i c a l and extrapyramidal a c t i v i t y as part of a jaw muscle regulatory mechanism (Dubner et a l , 1978; H e l l s i n g , 1987; Hoffman and Luschei, 1980). Periodontal mechanoreceptors also seem to modulate c o r t i c a l c e l l discharge during c e r t a i n jaw movements (Hoffman and Luschei, 198 0) . However, muscle spindle deafferentation does not appear to s i g n i f i c a n t l y a f f e c t isometric b i t i n g force i n monkeys (Goodwin et a l , 1978) which suggests the greater importance of central than peripheral mechanisms to jaw motor control. 204 During the performance of the d i f f e r e n t tonic voluntary i n t r a o r a l tasks i n the present study, subjects used auditory and v i s u a l feedback as a means of c o n t r o l l i n g unit f i r i n g frequency. There was therefore also constant central neural processing of v i s u a l and v e s t i b u l a r feedback i n addition to the processing of peripheral afferents. The central control of jaw m o t i l i t y i s les s well documented than for limb movements. Cortico-bulbar pathways have been traced which extend from both motor and sensory cortex to the trigeminal main sensory nucleus, supra- and inte r - t r i g e m i n a l areas, the spinal trigeminal nucleus, and the adjacent r e t i c u l a r formation (reviewed by Lund and Enomoto, 1988). Hoffman and Luschei (1980) have observed the responses of c e l l s i n the motor cortex i n non-human primates during voluntary b i t i n g , and concluded that the motor cortex i s important in the regulation of jaw closing movements. Of p a r t i c u l a r note were the v a r i a t i o n s i n f i r i n g rate of c o r t i c a l c e l l s during controlled jaw b i t i n g i n which some c e l l s increased t h e i r f i r i n g rates, whereas others decreased f i r i n g but could increase f i r i n g during jaw opening, while the f i r i n g rate of some c e l l s did not appear to be modulated during voluntary b i t i n g . This p o t e n t i a l for variations i n output of some c o r t i c a l c e l l s according to task has also been observed i n limb muscles. Directionally-tuned corticomotoneuronal c e l l s serving reaching have been i d e n t i f i e d i n non-human primate motor cortex and somatosensory association cortex (Georgopoulos, 1987; Kalaska et a l , 1983). The frequency of impulses from these c e l l s to the 205 motoneuron pool i s highest i n the neuron's preferred d i r e c t i o n . The p o s s i b i l i t y that corticomotoneuron output may be modified by a p a r t i c u l a r muscle function i s i n t r i g u i n g because i t may begin to explain the differences i n LSFF observed i n masseter SMUs, for example intercuspal clenching versus jaw muscle coactivation, with intercuspal clenching y i e l d i n g a higher LSFF because corticomotoneurons projecting to masseter motoneurons may f i r e faster than for jaw muscle coactivation, and cause increased excitatory drive i n c e r t a i n motoneurons which i n turn may cause increased SMU f i r i n g for that p a r t i c u l a r task. In addition to c o r t i c a l control of jaw movement the cerebellum and basal ganglia appear to orchestrate the sequencing of muscle a c t i v i t y and s e l e c t i o n of p a r t i c u l a r muscles to be used for any movement (Hellsing, 1983). The ISI v a r i a b i l i t y recorded i n the present s e r i e s of masseter units was i n general greater than that recorded i n limb muscles (Petajan, 1981, and Table 111) and concurs with previous studies of masseter MU ISI v a r i a b i l i t y (Derfler and Goldberg, 1978). In addition, histogram plots of ISIs for each of the 50 masseter units were p o s i t i v e l y skewed with a right-handed t a i l which i s consonant with the previous findings of D e r f l e r and Goldberg (1978) i n the masseter muscle. However the population of masseter SMUs studied i n t h i s experiment were a l l low threshold units recruited at l e s s than approximately 10% maximum voluntary contraction, and Clamann (197 0) and Freund et a l (197 5) have shown that low-threshold units tend to display the l a r g e s t range of 206 steady f i r i n g frequencies. In any spike t r a i n the ISI v a r i a b i l i t y may be due to changes in synaptic output and/or fluctuations i n the mechanism by which synaptic current i s converted into spike t r a i n s (Calvin and Stevens, 1968). Chance fluctuations i n the mechanics of spike-generation such as f i r i n g l e v e l fluctuations have been implicated in ISI v a r i a b i l i t y . However, "synaptic noise" or random fluctuations i n membrane potentials of motoneurons apparently due to impulse a c t i v i t y a r r i v i n g in the presynaptic terminals appears to be the most s i g n i f i c a n t source of ISI v a r i a b i l i t y (Calvin and Stevens, 1968) . The increased ISI v a r i a b i l i t y of many of the masseter units compared with limb muscles may be due to the absence of Renshaw i n h i b i t i o n i n trigeminal motoneurons (Derfler and Goldberg, 1978) as recurrent c o l l a t e r a l s are implicated in the maintenance of consistent f i r i n g patterns of s p i n a l motoneurons (Freund, 1983). A number of the lowest f i r i n g units i n the present series showed p a r t i c u l a r l y large ISI v a r i a b i l i t i e s . These may have been due to s i g n i f i c a n t pauses occurring between spikes as the l e v e l of subject e f f o r t f e l l below a c r i t i c a l point r e s u l t i n g in interruptions of MU f i r i n g . Freund (1983) has observed that MU f i r i n g v a r i a b i l i t y increases exponentially when the f i r i n g rate f a l l s below a c r i t i c a l value. Likewise a small number of units exhibited an apparent LSFF of greater than 15Hz, and s i g n i f i c a n t reductions i n ISI v a r i a b i l i t y for c e r t a i n tasks. In these instances the units may i n fact have been f i r i n g consistently 207 above the LSFF, as ISI v a r i a b i l i t y i s reported to diminish when units f i r e consistently above t h e i r tonic a c t i v a t i o n threshold (Freund, 1983). In general there was considerable functional heterogeneity within the masseter muscle, with differences i n regional a c t i v a t i o n depending on the jaw position, the b i t e point along the tooth row, and the d i r e c t i o n of e f f o r t . This f i n d i n g i s consonant with previous studies i n the human masseter (Belser and Hannam, 1986; Eriksson et a l , 1984; Greenfield and Wyke, 1956), the pig masseter (Herring et a l , 1989; 1979), and the rabbit masseter muscle (Weijs and Dantuma, 1981). C. MOTOR UNIT INHIBITORY REFLEX BEHAVIOUR IN THE MASSETER AND LATERAL PTERYGOID MUSCLES The recording technique used i n t h i s experiment was both sensi t i v e and f o c a l , but nonetheless represented a random probe. Masseter SMU recordings were obtained from the middle t h i r d of the muscle i n both s u p e r f i c i a l and deep parts. In the case of the l a t e r a l pterygoid muscle, the needles most l i k e l y penetrated the lower half of the i n f e r i o r head of the l a t e r a l pterygoid muscle (Wood et a l , 1986). Insertion of a precurved needle into the i n f e r i o r head of the l a t e r a l pterygoid muscle required a fastidious technique analoqous to a l o c a l anaesthetic posterior superior alveolar nerve block. Because of i t s f l e x i b i l i t y , the needle did not penetrate the muscle on a small number of occasions and was required to be reinserted. In 208 retrospect, a r i g i d needle would have permitted more predictable insertions into the muscle, although probably with more discomfort because of the increased diameter and bevel of the needle. A possible complication of the needle i n s e r t i o n technique in the l a t e r a l pterygoid muscle i s a bleeding episode i n the region of the pterygoid venous plexus. On one occasion a blood vessel in t h i s region was pierced during needle i n s e r t i o n which caused a l a t e r a l f a c i a l swelling and subsequent haematoma formation. The r a p i d i t y of the swelling suggested that the bleeding was most l i k e l y a r t e r i o l a r i n nature, most probably from a branch of the posterior superior alveolar artery. The a p p l i c a t i o n of an ice-pack to the area controlled the swelling, and prophylactic oral a n t i b i o t i c s minimized further sequelae. Masseter units were activated mainly by tooth contact tasks, for example intercuspal and i n c i s a l clenching, and also by coactivation of jaw opening muscles. Like the masseter SMUs, the l a t e r a l pterygoid units recorded were capable of a c t i v a t i o n by more than one strategy, i n many instances f i r i n g with jaw opening, but on occasion acting s y n e r g i s t i c a l l y with jaw c l o s i n g muscles. When i t occurs, such jaw muscle coactivation, representing a common drive between SMUs of agonistic and antagonistic muscles, i s probably s i m i l a r to that demonstrated i n human extensor and flexor p o l l i c i s longus when voluntary coactivation i s used to s t i f f e n the interphalangeal j o i n t (De Luca and Mambrito, 1 9 8 7 ) . The minimum controlled f i r i n g frequency of i n d i v i d u a l masseter and l a t e r a l pterygoid SMUs changed fo r d i f f e r e n t tasks. 209 Differences i n threshold f i r i n g frequency f o r varyinq types of a c t i v a t i o n have been reported previously for the human masseter and temporalis muscles (Eriksson et a l , 1984). However, in these muscles the lowest rate of SMU f i r i n q which could be kept constant was 6-8Hz i n the case of masseter MUs, whereas i n the l a t e r a l pterygoid muscle the lowest continuously maintainable f i r i n g rate was 8-10Hz. In a l l subjects SMU f i r i n g frequency i n the masseter muscle could be controlled at 10 or 15Hz although some units could be f i r e d more e a s i l y at 15Hz, whereas i n the l a t e r a l pterygoid muscle units were more e a s i l y controlled at the faster (15Hz) frequency. These features indicate subtle task-dependent behavioural changes within the masseter and l a t e r a l pterygoid motoneuron pools. By constraining the f i r i n g frequency of the masseter and l a t e r a l pterygoid SMUs during the prestimulus period i t was assumed that the respective masseter and l a t e r a l pterygoid motoneuron pools were subject to a source of r e l a t i v e l y constant e x c i t a t i o n based on the asynchronous a r r i v a l of multiple excitatory post-synaptic potentials (Granit et a l , 1963). Under these conditions, single-pulse i n t r a o r a l e l e c t r i c a l stimulation apparently produces only an i n h i b i t o r y e f f e c t on both the masseter and l a t e r a l pterygoid motoneuron pools and delays the f i r i n g of motor units i n both muscles. The increased duration of the peristimulus ISI i n these units may be explained i n part by the model of masseter motoneuron i n h i b i t i o n proposed by Miles et a l (1987). During normal constrained f i r i n g of a motoneuron, the 210 afterhyperpolarization (AHP) phase of each action p o t e n t i a l i s of a r e l a t i v e l y constant form and duration. However, i f an exteroceptive stimulus i s delivered during t h i s recovery phase, an i n h i b i t o r y p o t e n t i a l i s produced which e f f e c t i v e l y drives the AHP towards a more hyperpolarized state. In the case of both masseter and l a t e r a l pterygoid muscles, such a r e s u l t i n g prolonged AHP could induce the motoneurons, and consequently the motor units, to f i r e l a t e r than expected and thus account for the observed increase i n the peristimulus ISI. Although the degree of i n h i b i t i o n evoked by the exteroceptive stimulus was q u a l i t a t i v e l y s i m i l a r i n a l l masseter MUs f i r i n g at 10Hz, there were s i g n i f i c a n t differences i n r e f l e x responses (as judged by the duration of peristimulus intervals) when d i f f e r e n t tasks were performed by the same unit, even although the pre-stimulus f i r i n g frequency and the stimulus i n t e n s i t y were matched. Turker et a l (1989) have described s i m i l a r quantitative changes in masseter SMU i n h i b i t o r y r e f l e x responses at d i f f e r e n t degrees of jaw opening. Likewise t h e i r unit r e f l e x responses were reproducible when repeated at the same jaw opening which concurs with the present findings i n which a s i m i l a r i n h i b i t o r y response was obtained when the r e f l e x paradigm was repeated for the same task by the unit under t e s t . The reasons for the apparent modulation of masseter i n h i b i t o r y r e f l e x behaviour according to task i s not e n t i r e l y c l e a r . The rigorous nature of the experimental paradigm permits the l e v e l of pre-stimulus e x c i t a t i o n of a masseter motoneuron to be kept 211 constant over numerous t r i a l s by constraining the pre-stimulus MU f i r i n g frequency (Turker and Miles, 1989) . During the present study a small series of control t r i a l s were performed i n which no s t i m u l i were delivered. As can be seen i n a t y p i c a l example i n Figure 45, the "peri-stimulus" i n t e r v a l s are very s i m i l a r to the two pre-stimulus control ISIs, i n d i c a t i n g a consistent net l e v e l of e x c i t a t i o n of the MU during the peri-stimulus period, i n the absence of an exteroceptive stimulus. Because the e x c i t a b i l i t y of the masseter MU was controlled p r i o r to d e l i v e r y of the exteroceptive stimulus, the observed differences i n r e f l e x i n h i b i t i o n according to task suggests that the i n h i b i t o r y r e f l e x has been modulated by d i f f e r e n t i a l afferent input occurring at some location between the peripheral o r o - f a c i a l region and the trigeminal motoneuron pool i n the brainstem. The supratrigeminal nucleus, the trigeminal s p i n a l nucleus, and adjacent r e t i c u l a r formation have been implicated as s i t e s of interneurons involved i n i n h i b i t i o n of jaw c l o s i n g muscle reflexes (Sessle, 1981), and both pre- and post-synaptic events have been c i t e d as part of the mechanism of i n h i b i t i o n (Dubner et a l , 1978) . Lik e l y sources of peripheral sensory input are muscle spindles, j o i n t a r t i c u l a r receptors, periodontal mechanoreceptors, and free nerve endings i n the oral mucosa. There are differences i n jaw v e r t i c a l dimension and thus muscle length during MU f i r i n g by such tasks as intercuspal clenching versus jaw muscle coactivation which may cause changes in the a c t i v i t y of peripheral o r o - f a c i a l receptors, which i n turn 212 F i g u r e 45. R a s t e r d i s p l a y o f s p i k e s and i n t e r - s p i k e i n t e r v a l s f o r a s i n g l e m a s s e t e r m o t o r u n i t , f i r i n g a t a c o n t r o l l e d p r e s t i m u l u s f i r i n g f r e q u e n c y o f 1012Hz, w i t h no e l e c t r i c a l s t i m u l i d e l i v e r e d t o t h e g i n g i v a l t i s s u e s . Spikes are aligned as i f a stimulus had been given. Each spike immediately preceding the "sham" stimulus i s aligned at time zero. Spikes occurring during a period of 500ms before and a f t e r these spikes are shown. Seventy eight increasingly delayed "sham" sti m u l i are depicted by a diagonal l i n e , and arranged sequentially from above down, (see tex t ) . 213 may modulate masseter i n h i b i t o r y interneurons, as has also been postulated by Turker et a l (1989) . Sensory afferents from periodontal mechanoreceptors which relay i n the trigeminal sensory nucleus have also been implicated i n r e f l e x i n h i b i t i o n of jaw closing muscle a c t i v i t y (Hannam, 1982). I t i s conceivable that the sensory afferent input from these receptors may vary depending on the number of tooth contacts and t h e i r l o c a t i o n along the tooth row, both of which vary according to task, and thus contribute to the observed differences i n unit i n h i b i t o r y r e f l e x behaviour according to task. In the present study, the nature of the l a t e r a l pterygoid i n h i b i t o r y p o t e n t i a l i s uncertain. A diphasic stimulus-induced i n h i b i t o r y p o t e n t i a l has been demonstrated i n masseteric motoneurons of the cat (Kidokoro et a l , 1968), and a s i m i l a r diphasic i n h i b i t i o n has been postulated i n the human masseter muscle, based on evidence of re f l e x i n h i b i t i o n i n motor unit data expressed as peristimulus histograms (Miles et a l , 1987). I f the l a t e r a l pterygoid was affected by a conventional i n h i b i t o r y post-synaptic p o t e n t i a l (IPSP) with a 15-20ms duration the delay in the appearance of the post-stimulus spike would be expected to be of s i m i l a r magnitude, i . e . a prestimulus ISI of 100ms would be associated with a 120ms peristimulus ISI. However, the increase in the peristimulus i n t e r v a l was s i g n i f i c a n t l y longer; i n some instances the i n t e r v a l was 180ms i n duration. The duration of the presently hypothesized i n h i b i t o r y p o t e n t i a l would seem to be of the order of 8 0-100ms which suggests that i t was not a simple 214 IPSP, but consisted of a more complex hyperpolarizing response akin to that associated with the i n h i b i t i o n of masseteric motoneurons (Miles et a l , 1987). The l a t t e r i s assumed to be an IPSP, followed by a b r i e f reversal i n the depolarizing d i r e c t i o n , followed by a second long hyperpolarization of approximately 100ms duration. Lateral pterygoid motoneurons ( f i r i n g at 10±2Hz) were r e l a t i v e l y unaffected by a stimulus injected l a t e r than 3 5ms af t e r SMU f i r i n g . Due to motor-nerve conduction and neuromuscular delay there would have been a delay of at least 3ms between motoneuron f i r i n g and the occurrence of a prestimulus SMU spike. The e a r l i e s t stimulus was then delivered 1ms af t e r the SMU spike. The afferent conduction time for t h i s mildly noxious stimulus (involving AS fibres) would have been approximately 6ms, followed by a probable intraneuronal transmission time of 2ms. Any i n h i b i t o r y p o t e n t i a l therefore would have arrived at the motoneuron pool approximately 12ms a f t e r motoneuron f i r i n g , during the early-mid phase of the AHP, only then t r i g g e r i n g a hyperpolarizing p o t e n t i a l change, and s h i f t i n g the motoneuron membrane potential away from the threshold for i n i t i a t i n g a propagated p o t e n t i a l . A stimulus delivered 35ms aft e r the prestimulus spike would not have affected the motoneuron pool u n t i l 47ms a f t e r f i r i n g , i n the mid-late phase of the AHP, and any stimulus delivered l a t e r than 3 5ms probably would have reached the motoneuron pool i n the la t e AHP-early r e p o l a r i z a t i o n phase when the motoneuron membrane was much les s susceptible to hyperpolarizing changes. When the constrained f i r i n g frequency of 215 the l a t e r a l pterygoid motoneuron pool was 15Hz, there was no obvious i n h i b i t o r y e f f e c t except when the stimulus was delivered less than 5ms a f t e r the prestimulus spike. Due to the shorter ISIs (54-67ms) at t h i s frequency, s t i m u l i occurring at 5ms or l a t e r would have affected only the very l a t e stages of the AHP and presumably would have had a minimal e f f e c t on the l a t e r phase of the AHP. When the present data from masseter and l a t e r a l pterygoid units were reconfigured to peristimulus histograms, two periods of apparent i n h i b i t i o n separated i n a number of instances by apparent e x c i t a t i o n were observed. Inhibitory periods were of longer duration i n masseter MUs, with the two periods merging in some instances (see Figure 27), as described previously by Miles et a l (1987) . Periods of apparent e x c i t a t i o n have also been reported i n previous studies of the human l a t e r a l pterygoid muscle (Widmer, 1987) c a r r i e d out with interference electromyography and in masseter SMUs (Miles et a l , 1987). The apparent e x c i t a t i o n seen in binned histograms i s most l i k e l y due to resumption of motor unit a c t i v i t y which occurs when motor units recommence f i r i n g at t h e i r prestimulus frequency a f t e r a period of i n h i b i t i o n . Any tendency of units to resume f i r i n g near the same time i s further accentuated by the nature of the binning process. Thus the apparent e x c i t a t i o n observed may be considered more a product of experimental method rather than a manifestation of e x c i t a t i o n . Our experimental findings i n both the masseter and l a t e r a l pterygoid muscles concur with the 'frequency p r i n c i p l e ' of motor 216 unit i n h i b i t i o n described by Miles and Turker (1986) whereby for a constant stimulus strength, SMUs are r e f l e x l y i n h i b i t e d to an extent that i s determined by t h e i r prestimulus f i r i n g frequency. Units f i r i n g at a lower frequency are i n h i b i t e d more r e a d i l y . The i n t e n s i t y of the exteroceptive stimulus required to e l i c i t i n h i b i t i o n i n masseter MUs was consistently non-noxious and less than that required to i n h i b i t l a t e r a l pterygoid MUs. This suggests that components of the masseter i n h i b i t o r y r e f l e x may be involved in i n t r a o r a l feedback mechanisms during normal o r a l functions such as suckling and mastication, as has been proposed by Bratzlavsky (1972) , Yu et a l (1973) , and Cadden and Newton (1989) . The higher, near-noxious threshold for l a t e r a l pterygoid MU i n h i b i t i o n suggests a possible role for t h i s muscle i n nociception, possibly to minimize o r a l trauma during jaw clos i n g . The extent to which events i n t h i s experiment were influenced by stimulus frequency i s unknown. Given the rigorous experimental paradigm and the d i f f i c u l t y of f i r i n g SMUs slowly for prolonged periods of time i n human subjects, i t was impractical to stimulate at frequencies low enough for phenomena such as habituation to be addressed. I t remains possible, though u n l i k e l y , that the frequency of stimulation suppressed an excitatory response i n the l a t e r a l pterygoid motoneuron pool. I t i s noteworthy that under the pr e v a i l i n g experimental conditions, e x c i t a t i o n was never observed i n l a t e r a l pterygoid units following the f i r s t stimulus i n a series . It i s reasonable to expect that the l a t e r a l pterygoid muscle 217 should not behave as a t y p i c a l jaw depressor, since i t s p r i n c i p a l function i n man i s to e f f e c t mandibular t r a n s l a t i o n . The muscle also frequently coactivates with the masseter muscle for some oral functions, for example i n c i s a l tooth clenching. Thus a si m i l a r r e f l e x organization to masseter muscle, at le a s t f or some functional conditions, might be anticipated. D. MOTOR UNIT TWITCH TENSIONS IN THE MASSETER MUSCLE The extent to which jaw biomechanical linkage and muscle contraction strategies bias spike-triggered averaged (STA) measurements i n single masseter muscle MUs has not been addressed previously. The spe c i a l configuration of the midline i n c i s a l point force transducer employed i n t h i s study, with i t s capacity for controlled a l t e r a t i o n of tooth lever arms without a f f e c t i n g jaw position, f a c i l i t a t e d the manipulation of jaw biomechanics and muscle contraction as a means of studying t h e i r e f f e c t on masseter muscle STA twitch p r o f i l e s . The d i s t r i b u t i o n of spike-triggered measured tension (STMT) contraction times i n t h i s study was comparable to those reported by Yemm (1977) and Goldberg and Derfler (1977) . Our range of peak STMTs were also s i m i l a r to the putative twitch tensions (PTTs) reported by Yemm (1977) for low threshold units, but smaller than those reported by Goldberg and Derfler (1977) who measured t h e i r PTTs i n a predominantly high threshold population. The f i r i n g frequency of masseter muscle SMUs (<10Hz) during the STA procedure used i n the present study was lower than the mean f i r i n g 218 frequencies reported by Yemm (1977) and Goldberg and Derfler (1977) , and may have contributed to our smaller peak STMTs due to the known e f f e c t of decreased unit f i r i n g frequency on the amplitude of masseter PTTs (Nordstrom et a l , 1989). When the orientation of the b i t e point was altered, there were differences i n unit peak STMTs, but not the systematic increases or decreases that would have been expected from the corresponding decreases or increases i n the tooth moment arms. Taken with the d i s p a r i t i e s between STTORs measured under the same conditions, the resul t s suggest that e f f e c t i v e SMU moment arms may have varied with task. A multipinnate muscle l i k e the masseter has the potential for multiple l i n e s of action, p a r t i c u l a r l y i f SMU f i r i n g i s not uniform throughout the muscle. This i s apparent from our observation that units could be activated by voluntary strategies other than clenching on a midline b i t e point. In such instances of non-uniform SMU behaviour, the l i n e of action of an active part of the muscle may be quite d i f f e r e n t from that of i t s remaining parts (Weijs, 1980). In addition, a l l SMUs within a muscle are not necessarily active during a p a r t i c u l a r muscle function (English, 1985). Four masseter muscle SMUs activated i n the present study could not be driven by isometric clenching on a b i t e point, and Eriksson et a l (1984) have noted s i m i l a r differences i n SMU behaviour. This suggests that the basic assumptions of constant single working l i n e s of action and uniform contraction throughout the masseter muscle, universally common i n previous STA studies, may have been too s i m p l i s t i c (Goldberg and Derfle r , 1977; Miles 219 et a l , 1987; Nordstrom et a l , 1989; Yemm, 1977). Thomas et a l (1987, 1986) have shown that there i s a l i n e a r re l a t i o n s h i p between twitch tensions measured i n f i r s t dorsal interosseous and adductor p o l l i c i s brevis SMUs activated by d i f f e r e n t contraction strategies. At f i r s t glance, the present data appear to d i f f e r from t h e i r s i n that there were no s i g n i f i c a n t associations between STMTs measured for the two tasks performed. I t should be noted however that the present data were plotted on a l i n e a r scale while t h e i r s were plo t t e d on logarithmic scales. Inspection of the plotted data of Thomas et a l (1987, 1986) reveals that considerable differences i n estimated twitch tensions can occur for d i f f e r e n t tasks; i n many cases the same unit's tensions can vary f i v e - f o l d or more according to the d i r e c t i o n of e f f o r t . The apparent scatter i n the present data would l i k e l y be considerably less i f the data were plotted on logarithmic scales, and i n fact few i f any of our units demonstrated f i v e - f o l d differences i n absolute estimated tension. Whatever r e l a t i o n s h i p exists between averaged forces recorded d i s t a n t l y from the unit i n question when a muscle i s activated by two d i f f e r e n t contraction strategies, i t w i l l r e f l e c t the lever biomechanics peculiar to that subject and the i n t e r n a l structure of the p a r t i c u l a r muscle. Such a r e l a t i o n s h i p , i f indeed one exists which can be described s t a t i s t i c a l l y by a curve, i s also presumably dependent upon the s i t e s of the units sampled. I f the units making up the sample are located i n muscle regions which d i f f e r f u n c t i o n a l l y with respect to a given task, the scatter of 220 data evident i n plots such as that i n Figure 41 i s l i k e l y to increase. Factors such as these indicate the care which must be exercised when comparing ostensibly s i m i l a r data from d i f f e r e n t muscles, and reinforce the need to standardize both the task and the recording s i t e when attempting to ca l c u l a t e twitch tension measurements with other variables such as recruitment thresholds and rank order. The complex anatomical arrangement of the connective tissue within the masseter muscle provides an e l a s t i c , s t r e s s - t o l e r a n t system. Even within a p a r a l l e l - f i b r e d muscle, the length-tension curves obtained from isol a t e d muscle f i b r e s d i f f e r from those recorded i n whole muscle preparations due to the e l a s t i c i t y of the connective t i s s u e i n whole muscle (Borg and C a u l f i e l d , 1980). The mechanical compliance of tissues interposed between a SMU and the force transducer may be modified by changing st r a t e g i e s within a muscle (Burke, 1981). The events occurring within the masseter muscle during the contraction of other jaw muscles, and t h e i r e f f e c t s are uncertain. In t h i s study, although the jaw was i n the same, fixed p o s i t i o n at both orientations of the b i t e point, i t i s conceivable that d i f f e r e n t i n t e r n a l muscle contraction strategies were involved, p a r t i c u l a r l y when coactivation occurred. Any changes i n the ar c h i t e c t u r a l arrangement of the muscle would have considerable e f f e c t on the length tension rela t i o n s h i p , shortening v e l o c i t i e s and force generation of the muscle (Loeb et a l , 1987). The apparent differences i n STMTs we recorded for ind i v i d u a l units may therefore be at t r i b u t a b l e , at l e a s t i n part, 221 to i n t e r n a l changes i n tissu e compliance. Muscle coactivation, although d i f f i c u l t to assess, appears to have affected the peak STMTs measured during the short duration of our STA paradigm. SMUs were reported as f i r i n g slowly and consistently for periods of f i v e minutes during STA recordings by Yemm (1977) and for 15 minutes by Nordstrom et a l (1989) . In these studies i t i s probable that at least some muscle coactivation occurs during the prolonged recording sessions. It i s also possible that some of the variance we observed i n indi v i d u a l STMTs was due to synchronization of SMU discharge. However Nordstrom et a l (1989) and Yemm (1977) f a i l e d to show synchronization of SMU discharge i n human masseter muscle SMUs, and i n the present study, SMUs were only permitted to f i r e slowly for short periods of time, and a l l had low force thresholds. Since, i n addition, there were no gross aberrations i n the peak STMTs measured for the present series of low threshold units, i t i s probable that the occurrence of SMU synchronization, though possible, was un l i k e l y . The findings suggest that any given STMT measurement of a masseter muscle SMU may represent a range of STMTs which depend, inte r a l i a , on the inter n a l muscle set and degree of muscle co-act i v a t i o n p r e v a i l i n g at that p a r t i c u l a r recording session. At best, the STMTs recorded i n t h i s study simply represent the current tension contributions of masseter muscle SMUs to i n c i s a l b i t e force, and even these can be modified by subtle changes in the conditions of the experiment. The use of STA as a method for 222 determining r e l i a b l e unit tensions i n a muscle such as the human masseter, however, would seem inappropriate, given i t s complex inter n a l muscle architecture and the e s s e n t i a l l y uncontrollable e f f e c t s of coactivation by other jaw muscles. 223 111. General Conclusions The present studies of voluntary and r e f l e x SMU behaviour i n the human masseter muscle aimed to expand the l i m i t e d knowledge of the functional organization of t h i s complex muscle, as a basis for c l a r i f y i n g the putative relationship between the masseter muscle's peripheral functional and anatomical organization. The ubiquitous use of h i g h l y - s k i l l e d subjects who were capable of a c t i v a t i n g and dr i v i n g masseter SMUs by performing a var i e t y of postural and tooth contact tasks provided a unique experimental s i t u a t i o n i n which subtle, v o l i t i o n a l changes i n SMU behaviour could be studied i n depth. Such conditions do not e x i s t i n highly stereotyped animal studies, except perhaps to a very limited extent i n behavioural studies involving non-human primates. The development of a stereotactic l o c a t i n g technique permitted the c o r r e l a t i o n of physiological SMU EMG recordings and t h e i r anatomical locations i n three dimensions of space, in con t r a d i s t i n c t i o n to previous studies in the human masseter muscle (e.g. Eriksson et a l , 1984; Stalberg and Eriksson, 1987). Subsequent s t e r e o t a c t i c masseter MU mapping revealed evidence for MU t e r r i t o r i e s which were more extensive than previously described in the human masseter (Stalberg and Eriksson, 1987) or the pig masseter (Herring et a l , 1989; 1979), although i t i s l i k e l y the experimental method employed by Stalberg and Eriksson (1987) in the human masseter consistently underestimated the l i m i t s of MU t e r r i t o r y due to the multipinnate nature of the muscle. The 224 t e r r i t o r i e s had a preferred axis of orient a t i o n within the muscle, and appeared for the most part to be r e s t r i c t e d to muscle subvolumes which resembled the anatomical compartments described by Schumacher (1982, 1961). The r e s t r i c t i o n of MU t e r r i t o r i e s to certain parts of the muscle indicates a p o t e n t i a l f o r se l e c t i v e , regional motor control of the muscle. The systematic probing of voluntary SMU behaviour patterns in known locations i n the masseter muscle discl o s e d regional differences i n SMU behavioural p r o f i l e s . Although units were ostensibly multifunctional, they had c e r t a i n task s p e c i f i c i t i e s which varied i n some instances according to the p a r t i c u l a r muscle region, which suggests a certa i n degree of regional s p e c i a l i z a t i o n . There was however no d e f i n i t i v e evidence for the location of s p e c i f i c SMU task groups e x c l u s i v e l y i n discrete regions of the muscle. Given the s e l e c t i v e a c t i v a t i o n of d i f f e r e n t masseter MUs according to task, i t may be i n f e r r e d that there i s sel e c t i v e a c t i v a t i o n of subpopulations of motoneurons within the masseter motoneuron pool depending on the p r e v a i l i n g muscle behavioural set, as has been proposed previously for other multifunctional human muscles (Freund, 1983; Kanda et a l , 1977; Luescher et a l , 1979) . Such presumptive f l e x i b i l i t y of MU combination i s impossible to discern i n animal models, because i t requires that MUs be driven v o l u n t a r i l y and t h e i r mode of ac t i v a t i o n altered behaviourally. When the s p a t i a l dispositions of masseter MU t e r r i t o r i e s are considered i n combination with the p l a s t i c i t y of MU behaviour, i t 225 i s l i k e l y that the complex task-dependent contractions of various parts of the muscle involve m u l t i - d i r e c t i o n a l contraction of intern a l connective tiss u e septa, which i n turn lead to s i g n i f i c a n t v a r i a t i o n s i n the working l i n e of action of the muscle according to task. Even i n an experimental s i t u a t i o n where the jaw posi t i o n was apparently fixed, such as during the present measurements of twitch tensions, d i f f e r e n t i n t e r n a l muscle contraction strategies were possible, and muscle l i n e s of action appeared to vary, e s p e c i a l l y when antagonistic jaw muscle coactivation was involved. This being so, the c l a s s i c a l concept of a single l i n e of action for the human masseter becomes untenable. This finding i s s a l i e n t when the mechanical properties of muscle such as MU twitch tensions are measured i n whole muscles. I t also has important implications for jaw muscle biomechanical analyses, p a r t i c u l a r l y for both s t a t i c and dynamic modelling of the jaws. The control of voluntary f i r i n g of masseter MUs at t h e i r lowest sustainable f i r i n g frequency was highly task-dependent, despite auditory and v i s u a l means of constraining u n i t f i r i n g , in contrast to limb muscle MUs (Thomas et a l , 1987; 1986). Likewise r e f l e x i n h i b i t i o n of masseter units was highly task-dependent, even when unit pre-stimulus f i r i n g frequency and exteroceptive stimulus magnitude were c a r e f u l l y matched. This indicates s i g n i f i c a n t changes i n descending drive to the masseter motoneuron pool most probably due to variations i n the magnitude of central and peripheral afferent inputs to the motoneuron pool according 226 to task. Descending inputs from the motor and somatosensory cortex to the masseter motoneuron pool appear l i k e l y to a f f e c t the f i r i n g pattern of voluntary driven units depending on the task at hand. Previous work i n non-human primates (Hoffman and Luschei, 1980) has revealed modulation of c o r t i c a l drive depending on the p a r t i c u l a r jaw movement pattern. I f the corticomotoneuronal c e l l s are indeed directionally-tuned, the frequency of impulses of these c e l l s are l i k e l y to be gated depending on the movement strategy. In contrast, the c o r t i c a l drive to the masseter motoneuron pool was rendered e s s e n t i a l l y constant i n the pre-stimulus period during the r e f l e x experiments due to voluntary clamping of the unit f i r i n g frequency. P a r t i c u l a r peripheral receptors implicated i n the modulation of task-dependent voluntary and r e f l e x behaviour patterns are muscle spindles, and a r t i c u l a r and periodontal mechanoreceptors. The p o s s i b i l i t y that muscle spindles i n the masseter may respond p r e f e r e n t i a l l y to the a c t i v i t y of MUs i n close proximity to the receptors i s an in t r i g u i n g , although as yet untested scheme with which to explain differences i n excitatory drive of MUs according to task. The use of spike-triggered averaging to educe twitch tensions in the human masseter muscle appears to be inappropriate given the modulating e f f e c t s of heterogeneous voluntary SMU behaviour, tiss u e compliance, muscle s t i f f n e s s , and jaw muscle coactivation, none of which are re a d i l y controllable. Spike-triggered averaging 227 may be apposite as a technique for quantifying the mechanical contribution of SMUs to a p a r t i c u l a r act, provided antagonistic jaw muscle coactivation can be controlled. 228 IV. Future Directions The burden of evidence i s i n favour of some form of compartmentalization within the human masseter muscle based on the present knowledge of functional and anatomical organization of the muscle. Although during the present experiments the s p a t i a l relationships of SMU t e r r i t o r i e s were disclosed s t e r e o t a c t i c a l l y i n three dimensions of space, there was no s p e c i f i c knowledge of t h e i r r e l a t i o n s h i p to internal anatomical features of the muscle under study because the resolution of the Magnetic Resonance (MR) technique precluded accurate i d e n t i f i c a t i o n of i n t e r n a l muscle features. High resolution MR imaging of the masseter muscle, (for example with 1.5 Tesla imagers), and subsequent three-dimensional reconstruction of inter n a l architecture including connective tissue septa, i n addition to st e r e o t a c t i c l o c a t i o n of EMG recording s i t e s , would permit the question of anatomical compartmentalization to be addressed more rigorously. Functional data on human masseter SMU t e r r i t o r y and heterogeneous SMU behaviour i n combination with d e t a i l s of intern a l anatomy i n the same muscle would also c o l l e c t i v e l y provide a data base for the development of a biomechanical model of the in t e r n a l mechanics of the muscle. This would permit current hypotheses of muscle pinnation and d i f f e r e n t i a l motor control of the complex masseter muscle to be tested and t h e i r implications extrapolated to other complex jaw muscles. Ultimately such data could be used to address functional and anatomical interactions 229 i n dysfunctional masseter muscles, i n p a r t i c u l a r SMU EMG studies of muscle t r i g g e r points i n combination with t h e i r s p e c i f i c three-dimensional anatomical locations. The e f f e c t of i n t r a o r a l e l e c t r i c a l stimulation on SMU a c t i v i t y of the i n f e r i o r head of the l a t e r a l pterygoid muscle appears q u a l i t a t i v e l y to be si m i l a r to that of the masseter muscle. Previous multi-unit r e f l e x studies i n the d i g a s t r i c , jaw-opening muscle using exteroceptive stimuli have revealed equivocal excitatory responses. The present rigorous SMU experimental paradigm, i n which both unit f i r i n g frequency and stimulus p o s i t i o n are controlled, would be an appropriate technique with which to address putative excitatory r e f l e x a c t i v i t y i n the human d i g a s t r i c muscle. 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