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Physiological and pharmacological study of projections from nucleus of the posterior commissure to the… Pettman, Patrick Harold 1970

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A PHYSIOLOGICAL AND PHARMACOLOGICAL STUDY OP PROJECTIONS FROM NUCLEUS OP THE POSTERIOR COMMISSURE TO THE VENTROLATERAL NUCLEUS •IN. THE FELINE THALAMUS by > PATRICK HAROLD PETTMAN B.Se.,i, University of B r i t i s h Columbia,1968 A thesis submitted i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree of MASTER OP SCIENCE i n the department of : v.. PHYSIOLOGY l e accept this thesis as conforming to r . the required standard THE UNIVERSITY OP BRITISH COLUMBIA May,1970 In p resent ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of ~PV\MS\QL_Q€=M The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date Oe -r (p^ [l O ABSTRACT Many neurones in the thalamus, li k e neurones in other parts of the CNS are excited by iontophoretlcally applied acetylcholine (ACh), and i t has been suggested that ACh may be involved in synaptic transmission i n the thalamus. In these experiments, the lontophoretlc technique was employed to investigate the location, and the neurophyslol-g l c a l and pharmacological properites of neurones in the ventrolateral nucleus of the thalamus (VL), which responded to e l e c t r i c a l stimulation of the l p s l l a t e r a l nucleus of the posterior commisure (NPC). Stimulation of the contralateral brachium conjunetivum (BC) was used to confirm the presence of the recording microplpette in VL. Pour drugs were applied in various sequences to most neurones encountered above, in and below VL. BL-homocysteate (DLH) was used for the activation and localisation of quiescent neurones; ACh was applied to test whether the neurones were oholinoceptivej eserine (physostigmine) was used as an anti-cholinesterase; and atropine was used as a muscarinic block-ing agent* Choiinoceptive cells were found above (3*0-6*Oram below the fornix), and In VL (0.0-10.0mm below the fornix), the highest proportion being located ln VL. the majority of cholinoeeptive cells in VL responded to NPC and to BC stimulation, (^holinoceptlve neurones located above VL were not evoked either by fJPC or BC stimulation* while those in the lowest part of VL were evoked by NPC stimulation only. -11 Although atropine "blocked the effect of iontophoretioally applied ACh, It did not affect synaptic responses evoked by stimulation of SJPC and/or B<? fibers. Eserine excited 1 some cells "and'potentiated the actions of ACh. These results indicate that a pathway arising from the UPC projects to the ip s l l a t e r a l VL and that this fiber tract le non-chollnergie. - i i i -TABLE OF CONTENTS INTRODUCTION. . .. * . .... ........ .p. 1 I .Anatomy .............. ................p. 1 a) The anatomy of the Ventrolateral Nucleus (VL) in man.o.c0............................p. 1 b) Extrapyramidal projections to VL in the feline thalamus... p. 1 c) Cerebellar projections to VL in the feline thalamus..*.... p. 4 d) Nucleus of the Posterior Commissure (NPC) projections to VL in the feline thalamus......p. 6 II. Pharmacology* . . . 6 . . . . . . . . . . . * . . . . . . • . .p. 8 a) Acetylcholine....... .p. 8 b) The thalamus.... p.13 III,Microelectrophoresis tIontophoresis) Technique......p.22 IV.Anaesthe tics .p.27 METHODS.....*............*.................... .p.29 I.Surgical preparation of the animal.......... .p.29 II.Iontophoresis technique., ..........*.........p.30 III.Recording technique........... .p.32 IV*His tology. o.« ....p.32 RESULTS. . p . 3 4 I.Effects of ele c t r i c a l stimulation .p»34 II.Effects of chemical s t i m u l a t i o n . . p . 4 2 a) Effects of acetylcholine (Ach) •>.*<• *....p.42 b) Effects of homocysteate (DLH)• p.42 .. -Iv-III .Atropine P-44 IV.Eserine (physostigmine)... P»45 V..Effects of anaesthetic (pento'barD i t b h e ) . . . . . . P* 48 DISCUSSION.. .... ................................P.50 BIBLIOGRAPHY......... '. ill'.-ill ..... i*;.i.ii> ip. 5 5 APPENDIX I*..*.. ................. .P.64 APPENDIX I I . . . . . . . . . . .- . • • • .P»66 APPENDIX III • .p.67 APPENDIX IV.. • • • • .'.".'.V.p.68 APPENDIX V.............. ......... ............ ............ ...P..69 APPENDIX VI . . .V. . . . . . .V.". ... ...... 72 #v*» LIST OP TABLES Table I Excitatory amino acids and related substances p.7 2 Table II Depressant amino acids and related substances p. 7 3 Table III Miscellaneous amines and related compounds p.7^ Table IV Some centrally acting drugs and other substances p. 7 5 Table V Cholinomimetic agents p. 7 6 Table VI Acetylcholine antagonists p.7 7 Table VII Central aotlon of cholinomimetics relative to acetylcholine p. 7 8 LIST OP PLATES Plate 1 Histological verification of the location of an electrode marker in VL p. 69 Plate 2 Histological verification of the location of an electrode marker ln NPC p. 7 0 Plate 3 Histological verification of the location of an electrode marker ln BC - v l i -LIST OF FIGURES Figure I. Dorsolateral section of the thalamus p. 2 Figure 2 Diagram of the major f i b e r connections of the Extrapyramidal, 'Cerebellar and Pyramidal motor systems'. P* 3 Figure 3 I l l u s t r a t i o n of the major afferent pathways to VL p. 5 Figure 4 Schematic diagram of NPC projections to the f e l i n e thalamus. p. 7 Figure 5 Basic p r i n c i p l e of microelectrophoresis. P.23 Figure 6 Tracing of a photograph of a glass f i v e b a r r e l micropipette, with an enlarged view of the t i p . p.24a Figure 7 Plots of the r e l a t i v e s e n s i t i v i t i e s of neur-ones located above, i n and below (Decere-r brate preparation) VL. p.35 Figure 8 Plots of the r e l a t i v e s e n s i t i v i t i e s of neurones located above, i n and below VL (Anaesthetized preparation-pentobarbitone)„ P.36 Figure 9 Table showing t o t a l number of c e l l s tested and the number of c e l l s which could be excited by Ach at various l e v e l s below the fo r n i x . p .37 - v i i i -Pigure 10 Ratemeter recordings of r e l a t i v e s e n s i t i v i t i e s of neurones located above, i n and below VL, P*39 Figure 11 a)Heurone located 9«8mra below the fornix evoked by NPC stimulation (30v, 0.1msec dura-t i o n ) . C e l l no. 6, May 29» 1969. b)Neurone located 9.3mm below the fo r n i x not evoked by BC stimulation (40v, 0.1msec dura-t i o n ) . C e l l no. 6 , May 29» 1969. P.40 Figure 12 Neurone located 9.8mm. below the fornix evoked by NPC stimulation (40v, 0.1msec dura-tion) a f t e r atropine had depressed the neur-onal response to Ach applied iontpphoretlcally*" C e l l no. 6, May 29, 1969. P.41 Figure 13 Two tracea both showing a neurone located 7.1 mm below the forni x evoked by NPC (1st responses 50v 9 0.1 msec duration),delayed 0.5 msec a f t e r BC stimulation (2nd response; 50v, 0.1 msec duration).Cell no. l,June 19,1969? P. 43 Figure 14 C e l l located 8.7mm below the fornix evoked by NPC (40v, 0.1 msec duration) a f t e r atropine had depressed the neuronal response to Ach applied i o n t o p h o r e t i c a l l y . C e l l no. 10, May 16, I969. p.46 Figure 15 C e l l located 9.5 mm below the forni x evoked by BC (40v, 0.1 msec duration) af t e r atropine had depressed the neuronal response to Ach applied i o n t o p h o r e t i c a l l y . C e l l no. 5,May 15*1969 P-47 - l x ^ Figure 16 A neurone located 9.9mm below the fornix f i r i n g in response to physostigmine applied lontophore-t i e a l l y . Cell no. 8, May 16, 1969. p.49a Figure 17 a)Cell located 8.9mm below the fornix evoked by WPG (40v,' 0.1 msec" duration) Cell ho. 17» May 29» 1969. b)Cell located 8,9mm below the fornix evoked by BC (45v, 0.1 msec duration). Cell no. 17, May 29* 1969. p.49 »ix* Figure 16 A neurone located 9.9mm below the fornix f i r i n g i n response to physostigmine applied iontophore* t i c a l l y . C e l l no, 8 , May 16, 1969. p«»^9a Figure 17 a) Cell located 8.4mm below the fornix evoked by NPC (40v, 0.1 msec duration). Cell no, 1 2 , Nov. 2 7 , i960, b) Cell located 8.%im below the fornix evoked by BC (45v* 0 .1 msec duration). Cell no» 1 2 , Nov* 2 7 , I 9 6 8 . — p , 4 9 • \ 1 wish to thank Dr.H.McLennan , Kenneth C. Marshal l and Dr . J .A .Pearson f o r t h e i r t ime, expert guldanoe and construct ive advice on the research and planning of t h i s t h e s i s . To Hr.B.Walker and Mr.K.Henze X owe a debt of g ra t i tude f o r t h e i r exce l lent ass istance i n the f i e l d s of h i s to logy and photography respect ively* 2 would a l so l i k e to express a s p e c i a l thanks to Mrs.Y.Heap and M i s s . E . P e t r a l l f o r t h e i r techn ica l ass istance throughput the year* ANATOMY There i s a reasonable anatomical analogy between the thalamus i n man and cat. The anatomy of the Ventral L a t e r a l Nucleus (VL) and the extrapyramidal input i n man w i l l be discussed, sinoe the d e t a i l s are better understood, then the cereb e l l a r and the Nucleus of the Posterior Commissure(NPC) projections to VL i n the f e l i n e thalamus w i l l be described. a) Anatomy of VL i n man: The VL of the thalamus Is located between the Ventral Anterior Nucleus (VA) and the Ventral Posterior Nucleus (VP) ( f i g . i , p.2). The nucleus i s composed of large c e l l s r o s t r a l l y and medially situated smaller c e l l s . Because VL and VA receive t h e i r main inputs from the centers concerned with motor function* the cerebellum, red nucleus, substantia nigra and globus p a l l i d u s , and through these from the striatum and subthalamic nucleus* and because they project to the motor oortex these n u c l e i are referred to as the 'motor' component of the thalamus. b) Extrapyramidal projections to VL i n man; The striatum (oaudate-putamen complex) projects mainly to both segments of the globus p a l l i d u s and also to the substantia nigra (pars r e t i c u l a r i s ) . Prom the external segment of the pallidum, f i b e r s pass through the capsule to the sub* thalamic nucleus, which sends f i b e r s back to the i n t e r n a l segment ( f i g . 2 , p . 3 ) . This gives r i s e to two well defined f i b e r systems- f a s c i c u l u s l e n t i o u l a r i s , passing medially through the i n t e r n a l * To and f r o m p r e c u n e u s To a n d f r o m t u p . p a r i e t a l l o b u l e I n t e r n a l m e d u l l a r y l a m i n a ( i n t r a - l a m i n a r n u c l e i ) To a n d f r o m p r e f r o n t a l c o r t e x To a n d f r o m i n f . p o r i t t o l l o b u l e a n d a r e a s 18 a n d 19 M e d . g e n i c u l a t e b o d y L a t . g e n i c u l a t e b o d y To l i m b i c c o r t e x M a m i l l o t h a l a m i c t r a c t T o " p r e m o t o r c o r t e x ( a r e a s 6 a n d 8 ) T h a l a m i c f a s c i c u l u s To m o t o r c o r t e x ( a r e a 4 - p r e c e n t r a l g y r u s ) B r a c h i u m c o n j u n c t i v u m D i f f u s e c o r t i c a l p r o j e c t i o n R e t i c u to t h a l a m i c f i b e r s M e d i a l l e m n i s c u s , s p i n o t h a l a m i c t r a c t s R e t i c u l o t h a l a m i c f i b e r s B r a c h i u m o f inf . c o l l i c u l u s a n d l a t e r a l l e m n i s c u s O p t i c t r a c t T o v i s u a l c o r t e x ^ f ( a r e o 17) To a u d i t o r y c o r t e x { a r e a s 41 a n d 4 2 ) T r i g e m i n o t h a l a m i c t r a c t s To s e n s o r y c o r t e x - f a c e a r e a To s e n s o r y c o r t e x - n e c k , t r u n k a n d e x t r e m i t i e s a r e a Figure 1 Dorsolateral section of the thalamus (Strong and Blwyn, 1964) Kir,L RE 9, Diagram of the Major Fiber Connections vf the " Extra-p\inmid/tl," Cerebellar and Pyramidal \1otor Systems. lfbrrxialU'Tn are Q\ follows: C, caudate nucleus; CbCx, cerebellar nntex; CCx, cerebral cortex; D.W dentate nucleus; CP Ext, (,P Jntt the external and internal segments of the globus pallidus; fl,, intra-larmnar nurlet of the thalamus, including the centromedtan nucleus; MRF, midbrain rrtuulur formation; SFF, nucleus oj Foreii field; FX, pontine nuclei; PVT. putamen; RS, red nucleus; SX, sub-stantia nigra; ST, subtliaiamic nucleus; VA, VI., nuclei irntraJis anterior and ventialis lateralis thalami; 04 and 6. motor and premolar cortex. t 2 Diagram of the major f i b e r connections of the Extrapyramidal , •Cerebellar and Pyramidal motor systems' . ( J . B. Carman, 1968) capsule, and the ansa lenticularIs* which loops around the anterior border of the capsule. These two pathways coalesce on the medial aspect of the capsule to form the bulk of Forel's f i e l d H2. Some of the fibers descend to the midbrain reticular formation, but the majority turn dorsally and then la t e r a l l y through Forel»s fi e l d s H and to reach the thalamus, where they send in the anterior half of VL and the caudal part of VA ( f i g . 3 , p, . J T ) . The substantia nigra also projects to the anterior part of V L via f i e l d s H and of Forel. It i s to this same area of the thalamus that the eerebellum and red nucleus project, via B C and (rostral to the red nucleus) the prerubral radiation ( f i g . 3 . P ; .5" ). Not only the extrapyramidal system but also the cerebellar system provide recurrent or 'feedback* pathways from the cortex that pass through the subcortical $® structures to return to the cortex. o) Cerebellar projections to VL in the feline thalamusi The ascending portions of the BC proper arising from the l a t e r a l (dentate) and interpositus nuclei, decussate completely and those fibers proceeding beyond the red nucleus terminate principally in V L . An accessory BC arising In the caudal portion of the f a s t l g i a l nucleus and distributed to the reticular formation i s described as constituting the only uncrossed portion of the BC, these fibers having already decussated within the cerebellum. Cohen, Chambers, and Sprague ( 1 9 5 8 ) and HoCanoe, P h i l l i s and Westerman ( 1 9 6 8 ) also detected an uncrossed ascending limb of the BC and described i t s termination in the Figure 3 Illustration of the major afferent pathways to VL. (J.B.Carman, 1968) midbrain and thalamus, including VL. Voogd (196*0 preferred to d i s t i n g u i s h t h i s t r a c t from the BC proper and described i t as the ascending uncinate t r a c t , with d i s t r i b u t i o n i n p a r t i c u l a r to thalamic n u c l e i . E l e c t r o p h y s i o l o g i c a l studies have, i n general, shown that the SC pathway has a r e l a t i v e l y d i s c r e t e termination i n VL C&ppelberg, 1961s Combs, 1959). Combs, who stimulated the cerebellar surface and tracked the evoked potentials forward to the point where they began to attentuate, found no evidence of an i p s l l a t e r a l p rojection. To date no e l e c t r o -p h y s i o l o g i c a l evidence i n support of i p s l l a t e r a l pathways such as those described by Voogd has been reported. The uncinate t r a c t which Voogd described may a c t u a l l y e x i s t and project i p s i l a t e r a l l y from the cerebellum to VL but i t may a f f e c t such a small portion of c e l l s i n VL that i t s presence would be hard to detect. d) NPC projections to VL in the f e l i n e thalamust Studies involving the le s i o n i n g of NPC have shown degeneration i n various regions of the f e l i n e thalamus (Bowsher, i96?). The f i b e r s project from NPC I p s i l a t e r a l l y to the oentromedlan, paracentralis and eent r o l a t e r a l n u c l e i ; to VL and VPj and to the zona inoerta. Connections to the contra-l a t e r a l nucleus ooour by way of the posterior commissure (fig!**, P£.7*). - 7 Figure 4 Seematle diagram of UPC projections to the feline thalamus. (D. Bowsher, 196?) PHARMACOLOGY a,) Acetylcholines The terms • nicotinic» and 'muscarinic 1 v;ere originally introduced by Dale (1914) to describe the peripheral actions of various oholine derivatives and have since been applied to categorise the receptors withwhich these combine* The receptors have been characterized on the basis of the a c t i v i -ties of substances that either mimic or antagonize the actions of acetylcholine (ACh). Nicotinic characteristics are esiiibit-ed when neurones are excited'"by carbachol and nicotine and. this excitation and that of ACh ban be antagonized by DH-^ -E (dlhydro^-erythroidlne).* Although carbachol excites both nicotinic and muscarinic receptors* i t i s a more potent nico-t i n i c excitant, and for this reason i s used to characterize nicotinic receptors (Table V) .* Muscarinic characteristics Imply that a neurone can be excited by muscarine and a c e t y l ^ -methyl choline and that the excitation i s antagonized by atropine (Table V i ) • Both types of receptors are of course, affected by ACh, Y ' 1 -Benshaw receptors in the spinal cord of the cat have been identified as nicotinic (Curtis & Ryall, 1966a & b)1? The i n i t i a l phase of orthodromic excitation of Benshaw ce l l s by antidromic ventral root stimulation as well as excitation by carbachol i s blocked by iH-^-B but not by atropine (Curtis & Ryall, 19660). Further experiments on Renshaw cells have revealed the presence of two more types of receptors, one of which reacted with acetyl-^-methyl choline and DL-muscarihe, and was spedifically blocked by Intravenous atropine. This musoarinio reoptor was also responsible for the late phase of orthodromic excitation of Renshaw cel l s by antidromic ventral root stimulation (Curtis & Byall, 1966b & c). A third type of receptor was proposed to account for the findings that In the presence of DH-^-E and atropine many cholinomimetics had a depressant action on Renshaw cel l s (Curtis & Ryall, 1966b). The nicotinic action of ACh on Benshaw cel l s has a: r a i i d onset and i s of brief duration after the cessation of ACh application. A proportion of cortical neurones (10$) which are concen-trated In the deeper layers of the cerebral cortex can be excited by ACh and various cholinomimetics (Kmjevl6 & P h l l l i s 1963a1 Krnjevio, 1965* 1966). ACh excites cortical neurones in a characteristic manners the onset of f i r i n g i s slow, and there i s a prolonged after-discharge. ACh inhibition of cortical neurones has also been observed ( P h l l l i s & York, 196?). In general muscarinic compounds such as aoetyl-^-methyl choline, DL-musoarone and DL-muscarine excite cortical neurones while nicotinic agents such as nicotine and butyrlchollne are relatively inactive. A similar pattern i s shown by specific antagonists of the action of AChi antlmusearihio agents l i k e atropine and hyosolne are especially powerful, whereas the ourariform nicotinic antagonists show practically no activity. Most of the ACh In the feline cerebellum i s located in presynaptic nerve terminals i n the molecular and granular layers (Goldberg & MoCaman, 1967). ACh administered topically, intra-a r t e r i a l l y , or iontophoretloally has an excitant action on elements i n the cerebellar cortex. - 1 0 -Purkinje ca l l s situated near the cerebellar surface were generally Insensitive to ACh but the proportion excited c e l l s increased with depth (MoCanoe & P h l l l i s , 1968). Whether this was a result of a direct interaction between ACh and receptors on ParkInJe ce l l s or the result of excitation of c e l l s in the adjacent granular cortical layer (GCL) and a consequent synap-t i c excitation of Purkinje oells by parallel fibers i s not known. In contrast Crawford & Curtis (I966)- found that oells in the cerebellar cortex were generally insensitive to ionto-phoretically applied ACh. An explanation for the different results could be that Crawford and Curtis confined their study to oells situated near the cerebellar surface where HoCance and P h l l l i s fount them to be relatively insensitive to ACh. Some ce l l s i n the granular layer are exolted by lonto-phoretlcally applied ACh (HoCanoe & Phlllis, t^l96^a) • They are also excited by a range of cholinomimetic drugs, including carbaohol, aeetyl-^-methyl choline and nicotine. Unlike Furklnje cells which are often slow to respond, sensitive ceils In the granular layer usually commence to discharge within lOsec of the onset of applications of ACh. Within this layer, the e l e c t r i c a l activity recorded was principally the summed action potentials of groups of c e l l s . Anticholinesterases including 1 eserIne, neostigmine and edrophonium (Tensilon) exhibit powerful excitatory actions. Dihydro-^-erythroidin (DH-^-B) and hexa-methonium are the most active inhibitors of ACh excitation although neither drug abolished spontaneous neuronal activity or evoked cortical potentials (Table VI). Atropine reduces but does not abolish the action of ACh. In contrast to these findings, Crawford, Curtis, Voorhoeve and Wilson (1966) reported that Purklhje oells {?§$)•» but neither granule nor basket oells* were excited by eholinb-mimetios, and the acteyloholine receptors had muscarinic proper!tes. Intravenously administered atropine and DH-^-E did not depress the synaptic excitation Of cerebellar neujpories evoked by impulses in mosey, climbing or pa r a l l e l 'fibers^ How-ever atropine but not DH-^-E depressed the excitation of Purkinje c e l l s by Iontophoretioally applied ACh; These studies indicate that other substances are Involved as synaptic trans-mitters in the cerebellum. ' Mbssy afferent fibers which stain forAcetylcholinesterase (AChE) run in the cerebellar white matter just below the granular layer and extend up Into this layer ( P h l l l l s , I965a,b) AChE has been found to stain more densely in the granular layer of the feline cerebellum than the molecular layer. It has been concluded'"'that there may be two types of granule c e l l Involved; AChE is distributed oh the su^aCe membrane of the more super-f i c i a l cells* whereas It Is present throughout the c e l l body of the deeper c e l l s . Many ce l l s in the deep cerebellar nuclei stain moderately densely for AG'hE. The middle peduncle of a l l mamm-alian species stains for AChE, the density of staining tending to be more Intense in the smaller species. The Inferior and superior peduncles also contained stained fibers, though as a whole they did not stain as densely as the middle peduncle. These studies along with the iontophoretlo studies strongly Indicate that a chollngerlo pathway originating i n the cere-bellum exists. This would suggest that the superior cerebellar pedunclefBC) from the deep cerebellar nuclei to the thalamus -12-(VL) has some cholinergic component. However, other path-ways have yet to be tested, and in view of the recent histo-chemical evidenoe on the distribution of AChE (Shute & Lewis, i 9 & 3 ) . i t i s possible that the cholinergic component of these pathways i s derived from the reticular formation and midline thalamic structures. -*3 b) The Thalamus A large proportion of ventroposterior (VP) neurones of pentobarbitone anaesthetised oats were sensitive to ionto-phoretic&lly applied ACh (Andersen & Curtis I964a,b). The sensitivity of these oells, particularly the thalamocortical relay neurones, to ACh was almost equal to that of lenshaw oells. The excitation of ventrobasal thalamic neurones b y ACh was slightly slower In both onset and stopping than that of Renshaw cells (Table VII), (Andersen & Curtis, 1964a). Carbachol was the most potent cholinomimetic excitant of the ventroposterlor thalamic (VP) neurones, aoetyl-^methyl choline was approximately as potent as ACh, and propionyl-oholine, n~butyrlcholine, nicotine, muscarine and musoarone were weaker (Tabl0 VII), (Andersen & Curtis* 1964b% HoCane©, P h l l l i s & Tebeoia 1968K Anticholinesterases such as physostig-mine, neostigmine and edrophonium enhanced the action of ACh and in addition were excitants. These results Imply that receptors on ventral thalamic neurones are an intermediate type exhibiting both nicotinic and muscarinic characteristics? There are only moderate amounts of ACh* ACJiE, and choline acetyl transferase in the mammalian thalamus, in comparison with the basal ganglia and ventral roots (Burgem & CShlpjaan* 1951* Sebb & Stiver, 19J6) . This suggests that major afferent path-ways to the thalamus are non^ohollnorgic, or that the cholinergic components of these pathways are relatively small,' m the cerebellum* ACh and choline acetyl transferase levels are also low, however AGhE activ i t y is frequently high* though - 1 4 -th ere Is a considerable species v a r i a t i o n (Feldberg & Vogt,1948s Hebb & S i l v e r , 1 9 5 6 ) . Species v a r i a t i o n i n ACh content i s p a r a l l e l e d by the v a r i a t i o n s i n ACh-syntheslzIng a b i l i t y , and i t has been suggested by Hebb, (1961) that as the cerebellum increases i n s i z e and complexity, i t s chol ine content d iminishes, l a r g e l y owing to the a d d i t i o n of pathways which are not c h o l i n -e rg ic connecting the cerebe l la r cortex wi th other areas of the b r a i n . The deep ce rebe l la r n u c l e i possess a higher l e v e l of chol ine a c e t y l t ransferase then the cor tex . The super ior peduncle, which i s considered to be l a r g e l y an e f ferent t r a c t , a l s o has a high l e v e l of a c t i v i t y . This evidence supports the concept of a cho l ine rg ic pathway o r i g i n a t i n g i n some of the neurons i n the deep n u c l e i . ACh s e n s i t i v e re lay neurones could be blocked by DH-$-E i n the absence of a reduotion i n amino a c i d induced f i r i n g (Andersen & C u r t i s , 1964). Iontophoretlo a p p l i c a t i o n of atropine reduced the s e n s i t i v i t y of thalamic neurones to both ACh and DLH or glutamate. Usual ly f i r i n g of neurones Induced by the amino a c i d recovered f a s t e r than that induced by ACh. I t was poss ib le however, to b lock the f i r i n g of these o e l l s by ACh w i t h -out a f f e c t i n g e i t h e r the spontaneous discharge or f i r i n g induced by DLH wi th the a p p l i c a t i o n of benzoqulnonium (mytoIon- neuro-muscular b lock ing agent) , hexamethonium (gangl ionic b locking agent) , d-tubocurarlne (neuromuscular b lock ing agent)* meoamyl-amine (gangl ionic b locking agent) , a t rop ine , hyosolne (scopo l -amine -ant lmuscar ln io drug), and gallamine ( F l a x e d l l -neuro -muscular b locking agent) . However, a p p l i c a t i o n of hexamethonium, d-tuboourar ine, benzoquinonlum and gallamlne was frequently complicated by excitation and. m^eaayiamlae, atropine andxhyoscine sometimes depressed amino acid excitability (-Andersen & durtis #. 1946* -tfoCance,- J t i l l i l s ACh sensitive receptors on thalamic neurones have mixed nicotinic-muscarinic propertiles, as mentioned earlier. Nicotine i s ' v e r y powerful •excltant^of • Eonshaw cells* and muscarine -is comparatively weak (Curtis & Syall, 1966).. In contrast* both nicotine and ©L-ausearine are slightly less potent than ACh as ^©itanis : of tMlamle neurones''' (ISable v ' l D 'V"' ' -n . / : • Intracellular'• roco3*ding from -neurones"In. the ventrobasal- ; complex of the feline thalamus has shown that spindles result from; an interaction between 'phased ©xeli^to.ry\aM;':inh-lblto2^•: inflttenees. This typo of activity has the same basic compo-nents as the evoked rhythmic burst discharges (Andersen & Sears, i9#&i each spindle consisting of groups of spikes or •burets* containing several spikes at a frequency of 100-300/seo and with a group frequency of 5-12/seo. the spindles of a part-icular c e l l tended to recur at intervals of 4-8 see.v , - - 'Neither'• t h @ v spontaneous .•spindle*'' activity of ventre-posterior (V?) neurones, nor the synaptic f i r i n g Induced b^ peripheral cutaneous or cortical stimulation could be biboked in a specif io way by either M-^S or atropine applied ionto-phoretically or systeHilcaily in concentrations adequate to block f i r i n g induced by ACh (Anderson <S? Curtis, 1964a; McGanee, i h i l l l s & ^esterman* 1963); Similar results have been described for the f i r i n g of VL neurones by impulses in SC (Bavis, 19&51 16-McCanbe, P h i l l i s & Westerman, 1968), although Intravenously administered atropine reduced the f o c a l potentials evoked In th i s nucleus by BC stimulation. The e x c i t a t i o n of VL neurones by mesencephalic r e t i c u l a r formation stimulation was reported to be depressed by Iontophoretic a p p l i c a t i o n of atropine & DH«^-E (MoCahce, P h i l l i s & Westerman, 1968). In the LGN ( l a t e r a l geniculate nucleus), ACh s a t i s f i e s many of the c r i t e r i a that have been established f o r the i d e n t i f i c a t i o n of synaptic transmitters In the CNS (McLennan, 1963). I t i s present In moderate amounts In the nucleus as i s the enzyme responsible, f o r i t s synthesis, choline a c e t y l -transferase (Hebb & S i l v e r , 1956), and destruction AChE (Burgen & Chipman, 1951). Histochemical investigations have shown that AChE i s present i n nerve terminals rather than i n the c e l l somata (Shute & Lewis, 1963, 1966). The AChE containing nerve f i b e r s ascend from the b r a i n stem by the dorsal tegmental pathway of Shute & Lewis (1963)* A large number of LGN neurones have been reported to be sens i t i v e to iontophoretically applied ACh ( P h i l l i s , Tebeols & York, 196?bi Curtis & Davis, 1962j Satlnsky ,1967). The exc i t a t i o n of LGN neurones by ACh had a longer latency and a f t e r discharge.than Renshaw c e l l s . Carbachol was the most active of the choline esters, exceeding ACh i n potency. Other choline esters and nicotine were consistently l e s s a c t i v e than ACh. ACh also depressed a small percentage of LGN neurones. Anticholinesterases, eserine, neostigmine and edrophonium potentiated the a c t i o n of ACh, and often themsleves caused e x c i t a t i o n . Atropine and benzoqulnonlum prevented the ACh 17-exeitation of many ce l l s DH-^-E fa i l e d to reduoe the ACh response significantly. The f i r i n g of LGN neurones in re-sponse to optic nerve or visual stimulation was not blocked P h i l l i s , Tebecis & York, 19670). Stimulation of the mes-encephalic reticular formation caused either an enhancement or a reduction in the excitability of ACh-seneltlve neurones In the LGN. This excitation of LGN neurones was suppressed by benzoqulnonlum. One feature common to both the LGN and the ventrobasal thalamus was the sensitivity of neurones to amino aolds (Table II). Thalamic as well as other central neurones Curtis & watkins, 1963; Crawford & Curtis, 1964; Krnjevio, 1965* 1966) were depressed by -amino-n-butyric acid (GABA) and excited by acidic amino aolds such as L-glutamic a d d . Although L-glutamate i s not the most potent excitatory amino acid, i t has a very strong and quickly reversible action and i s found In the brain in large quantity (Waelseh, 1962). GABA is also present In the brain (waelseh, 1962). Excita-tion by acidic amino acids was of considerable assistance in these studies, and iontophoretieally administered DLH was used to test the excitability of thalamic neurones (Table I ) . Thalamic neurones were very sensitive to DLH, N-methyl-D-aspartlo acid, and glutamate. Neurones located deeper than 6.0mm below the fornix in VL were more sensitive to glutamate than DLH and N-methyl-D-aspartle acid (McLennan, Huffman & Marshall, 1968). GABA also blocked the synaptic evoked -18-activity of ventroposterlor (VPL,VPM) thalamic neurones (Andersen & Curtis, 1964b). In some cases, spontaneous, synaptic and chemically evoked f i r i n g were depressed by noradrenaline (NA), adrenaline, and lsoprenallne. NA&adrenallne also had an excitant action in the ventrobasal complex usually being replaced by depression after repeated application (Table i l l ) , Xn contrast dopamine (DA) depressed the excitability of most neurones tested, Irrespective of their location in the thalamus ( P h i l l i s & Tebecis, 196?). A similar depression was also noted i n barbiturate induced anaesthetised cats (Table IV),(Andersen & Curtis, 1964b). Many adrenergic blocking agents had depressant effects similar to that of NA, presumably as a consequenoe of interaction with NA receptors. For example^-adrenergic antagonist aiderlin, ando<-antagonists phentolamlne, dlbenzyl-ine, and chlorpromazine hadpronounced depressant actions on some thalamic neurones. They also excited cells that were excited by catecholamines. In ventroposterlor (VP) neurones of pentobarbitone anaesthetised cats,5-HT (serotonin) depressed the spontaneous spindle activity of neurones and the f i r i n g induced by ACh or glutamate without affecting excitation by cutaneous nerve volleys (Andersen & Curtle, 196 irt>). DA and 4-hydrox>tryptamlne (4-HT) had similar depressant effects. In contrast 5-HT depressed neuronal f i r i n g induced by glutamate ln the more dorsal nuclei, and excited some celis«in VP. The oats were anaesthetised with nitrous oxide, halothane, or methoxyflurane ( P h i l l i s & Tebecis, 196?); -19-MA and DA blocked the synaptic, antidromic and amino acid excitation of many LGN neurones in oats anaesthetised with nitrous oxide and halothane or methoxyflurane ( P h i l l i s , Tebecls & York, 1967c)• In oats anaesthetised with pento-barbitone, DA and NA were comparatively weak depressants of the excitation of LGN neurones by impulses In optic nerve fibers. Depression of f i r i n g by glutamate was not observed (Curtis & Davis, 1962). In oats anaesthetised with pentobarbitone, 5HT and many structurally related indole and lysergic sold derivatives depressed the f i r i n g of LGN neurones by optic nerve impulses without affecting either the sensitivity to glutamate or ACh* or the invasion of the neurones by antidromic impulses (Curtis & Davis, 1962t Curtis,& Davis, 1963). Consequently, the site of action of 5-HT is confined to the excitatory synapse* and there i s either a reduction in the amount of transmitter released or an interference with the post-synaptic action of the transmitter. There are insufficient data available to decide between these p o s s i b i l i t i e s , but a large number of structurally related compounds, including tryptamlne, phenylethylamlne, and lysergic acid derivatives* have been tested on LGN neurones to try and find an excitant, the post-synaptic action of which would be blocked by 5-HT. TO date, any active substance has had the same depressant effect as 5-HT* 2-byomo-lyserglo acid-diethylamlde, methyl-sergide and dibenamine applied iontophoretieally f a i l e d to block the effect of 5-HT. LSD*25, ergometrine and methyl-ergometrine also depressed f i r i n g of LGN neurones* but for -20 longer duration than 5HT which was of short latency and duration. The most potent compounds tested were 4 and 7-hydroxytrypt-amine. In oats anaesthetised with nitrous oxide, halothane, or methoxyflurane, 5-HT blocked synaptic activation of LGW neurones by optic nerve stimulation, depressed the f i r i n g Induced by glutamate or ACh, and occasionally also blocked antidromic invasion ( P h l l l i s , Tebeols &tork, 1967c) These results are in contrast to those reported by Curtis in 1962. A possible explanation for these different results could be that Curtis & Davis anaesthetised their cats with pento-~ barbitone. This anaesthetic has been shown to reduce the sensitvlty of various thalamic neurones to ACh, NA and 5-HT (Andersen & Curtis, 1964as HoCanoe, P h l l l i s & Tebecls, 1968). In contrast to many previous reports, Satlnsky (I967) found both f a c i l i t a t i o n and depression of IXJN neurones for ACh, 5-HT and NA. Principal oells (those that send their axon to the visual cortex) responded with f a c i l i t a t i o n to ACh, f a c i l i t a t i o n to NA, and depression to 5-HT (type 1). Unidentified LGN ce l l s * comprised of prlnoipal c e l l s and short axon cells* showed this f i r s t type of response as well as two ©ther types (type2 $ 3). One of these consisted of v\ depression to ACh and NA and f a c i l i t a t i o n to 5-HT; the other, seen in only one c e l l , consisted of f a c i l i t a t i o n to ACh and 5-HT, with N A untested. Satlnsky suggests that these l a t t e r two types of response may be characteristic of short axon c e l l s . While Curtis and Davis (I96I, 1962 and 1963) reported finding c e l l s that seem to correspond to the principal c e l l s reported here* they f a i l e d to find the other two types (type 2 .4 This may be due to a number of factors. F i r s t , the barbiturate anaesthesia may have selectively depressed type2 and 3 oellsiv Second, Curtis used larger (tip diameter) electrodes (S*lCU*as opposed to 4 - ^ ) , which niay have recorded selectively from larger ceils (type.• •%)• * :\«jBOlu&lng...typ^ . .2',an4 3. Although It la conceivable that these findings are •associated; with theaction of^ $<!*K% dopamine* or-a ^ closely,, : related' substance as <&,&- Inhibitory transmitter in ,th©-1.l$B.1',©;r {.ventrobasal portion, ofth® thalamus,. .it 'is <possible :th|*t'the . efffeOts are due to a non-specific depression of neurone ' eite'i'tfiibility- which • is' unrelated <to- synaptic mechanisms. .." ••22* MICROELECTROPHORESIS (Iontophoresis) The application of ionized substances to a nerve or muscle c e l l passing a current through a micropipette was f i r s t described by Nastuk (1953)• Iontophoresis Is the technique by which pharmacologically active ions are ejected e l e c t r i c a l l y from fine glass mlcroplpettes into the extra-neuronal space (fig.5 . p.23). In this process extra- or Intracellular potentials can be simultaneously recorded from a single c e l l In the immediate v i c i n i t y (approximately 50-10C>^-for extracellular recording) of the electrode t i p . The recording of extracellular potentials from neurones is usually carried out with the NaCl-containlng centre barrel of f i v e , seven, or nine barrel mlcroplpettes. The mlcro-plpettes have an overall t i p diameter of 4-12^-and individual Internal barrel diameters of 0.8 to 2.Q>~(fig.6, p.24a), several other designs in mlcroplpettes have been used, for example a parallel electrode assembly i s one ln which the recording mleroelectrode of 0.5 to 1. O^diameter projects less than 10.0^beyond the multibarrel drug containing mlcro-plpettes. This assembly Is believed to make i t possible to record from smaller oells. Furthermore, with this parallel ,^design, i t may be possible to detect drug-induoed alternations in the f i r i n g of small neurones indirectly by applying drugs iontophoretieally to larger neurones and recording from smaller neurones simultaneously. Either co-axial (concentric) or parallel mlcroplpettes F l G . SI Basic principle of m i c r o e l e c t r o p h o r e s i s + Diffusion Retention Ejection The diffusion of an active cation A + is reduced by a retaining (anionic) current and increased by an ejecting (cationic) current (Nastuk,1953) FlG. €. Tracing of a photograph of a glass five-barrel micropipette, with an enlarged view of the tip (Wastuk, 1953) -24-make extraneuronal drug admin is t rat ion and i n t r a c e l l u l a r recording poss ib le simultaneously. The i n t r a c e l l u l a r e lectrode permits the recording of changes i n r e s t i n g p o t e n t i a l , the measurement of synaptic p o t e n t i a l s , membr-ane e x c i t a b i l i t y and a l te rnat ions of i n t r a c e l l u l a r ion concentrations upon synaptic and drug induced p o t e n t i a l s . The c o - a x i a l electrode cons is ts of a recording micro -electrode contained w i t h i n , and pro ject ing beyond the o r i f i c e of a s ing le drug containing miorppipette . More recent ly recording electrodes have been placed i n the centre of f i v e b a r r e l assemblies, thus permitt ing studies of more than one substance on s ing le neurones (Cur t is & Crawford, 1969). P a r a l l e l electrodes cons is t of s ing le or double b a r r e l recording mlcroelectrodes attached to the e x t e r i o r of s ing le or f i v e - b a r r e l mioropipettes . There are several advantages associated with the use of iontophoresis . Drugs can be app l ied and e x t r a - or I n t r a c e l l u l a r po ten t ia l s can be recorded simultaneously from a s ing le neurone, a por t ion of which i s located w i t h i n the volume of t i ssue a f fec ted by the ejected compound. The exoi tant or depressant act ions of an ejected compound upon a s ing le neurone can be assessed d i r e c t l y from o s c i l l o g r a p h i c or photographic records of a c t i o n p o t e n t i a l s , or Integrated records of mean f i r i n g r a t e . The b ipod -bra in b a r r i e r i s circumvented, and the s i t e s of the drug act ions are r e s t r i c t e d to the immediate v i c i n i t y of the neurone under observat ion. The e lect rophoret ic administ rat ions of exc i tant amino a c i d s , such as DLH or glutamate provides a way of a c t i v a t i n g * s i lent* .25-neurones upon which test excitant and depressants can be compared. Also the effect of drugs and orthodromic or anti-dromic stimulation can be tested simultaneously, giving more Information on where the receptors are located. Iontophoresis also provides a means of marking the approximate place of the neurones under observation by the eleotrophoretlo deposition of HCl (H*). The subsequent tissue destruction where the neurone was looated can be identified after the histological staining processes, (MoCance & P h l l l i s , 1965)o The major disadvantages associated with the lonto-phoretlo administration of drugs fstem from the inab i l i t y to determine drug concentration at nerve c e l l receptors, and the nonuniform distribution of the ejeoted substance within the tissue as a consequence of i t s administration from a *point* source. Although the relationship between the amount of ion ejected from adjacent barrels of a multlbarrel mlcropipette is roughly proportional to the eleotrophoretlo currents (Kmjevlc, Mitchell & Sserb, I963), this relationship varies with d i f f e r -ent substances and different micropipettes. Also, tissue fragments forming •caps' around the open tips of micropipettes may prevent lone from reaching the appropriate receptors on neurones from which records are being obtained (Andersen & Curtis, 1964a). The concentration of the ejeoted ions may actually be higher in the v i c i n i t y of other neurones because of these 'caps 1, and indirectly affect the c e l l from which records are being taken. Also, locally applied drugs may 2 6 -only W a f f o o t i n g the receptors i a tho general vi c i n i t y where recording i s taking place. For example drugs may only be affecting the somal receptors where recording i s taking place, and excluding the dendritic receptors on the same neurone. Under these circumstances when the neurone as an entity is not responding to the applied drug* relatively non-specific actions may be the results ' • • •> Microelectrophoretlo studies can only provide evidence that a substance has a transmitter^!Ike action upon certain neurones, and that compounds acting ln a specific manner near synapses can either antagonize transmitter action or enhance It.: These results must be correlated with the results of Other studies before a transmitter function can be ascribed with some confidence to a given compound*-- 2 7 -ANAESTHETICS In lontophoretlo experiments there i s evidence that the presence of anaesthetics, particularly in concentrations for •surgical* levels of anaesthesia, may contribute to the variab i l i t y of observations made upon single neurones. After systemic administration most of these agents eventually depress the excitability of neurones with reduction in spon-taneous f i r i n g and alternations of f i r i n g patterns i f they can pass through the blood brain barrier. These experiments in VL have yielded similar effects after systemic administration of pentobarbitone and subsequent iontophoretic recordings of single neurones. When pentobarbi-tone was administered intravenously to a decerebrate prepara-tion, the latency and after discharge response of single neurones to ACh increased proportionally while the mean f i r i n g rate decreased proportionally. The mean f i r i n g rate of neurones in response to DLH was also decreased but not as notloeably as the response to ACh. After more than 15mg/Kg of pentobarbitone (more than half the anaesthetic dose), the receptors became insensitive to iontophoretloally applied ACh but s t i l l respond-ed to the amino acid DLH (McLennan, Marshall & Pettman, 1969). In various thalamic nuclei, pentobarbitone reduced the sensitivity of neurones to ACh, EA and 5-HT, with less effect upon the sensitivity of neurones produced by glutamate (Table IV) (Andersen & Curtis, 1964a; McCance P h l l l i s & Tebeeis, 1968) The depressant effects of systemic anaesthetic agents upon the sensitivity of neurones appeared to be minimal in the spinal cord (Curtis, Ryall & Watklns, 1966; Curtis & Ryall,1966). Chloraloee did not affect the sensitivity of Renshaw cells to ACh (Curtis & Ryall, 1 9 6 6 ) . Recent experiments indicate that anaesthetic doses of pentobarbitone have l i t t l e or no effect upon the NA induced depression;©r the WLM induced f i r i n g of spinal interneurones (Curtis and deGroat, 1 9 6 8 ) . Because of the possible complicating effects of anaes-thetics in mlcroeleetrophoretlc experiments, decerebrate or some other unanaesthetised preparations may be more advisable, Previous studies have suggested a possible cholinergic input to v*L. In the present set of experiments, NPC proj-ections to VX were studied and compared to BC Also the neuronal responses recorded from cats anaesthetised with pento-barbitone were compared to those obtained from decerebrate preparations. -29-METHODS a) Surgical Preparation of the animals Adult oats of ei t h e r sex, weighing between 3.0 and 4.0Kg. were used i n these experiments. The procedures of venous cannulation, tracheal Intubation and deeerebration were c a r r i e d out under halothane anaesthesia. In some cats the procedures of venous canhulatlon and tracheal intubation were carr i e d out under anaesthetic by an Intraperitoneal i n j e c t i o n of sodium pentobarbital (30mg/Kg.). In most cases however, the decere-brate preparation was used because the o v e r a l l s e n s i t i v i t y of the c e l l s to ACh appeared to be reduced i n the anaesthetised animals. Respiration was monitored by a transducer (EKEG e l e c t r o n i c s ) , t i e d around the thorax and recorded on a poly-graph. In some experiments heart rate Was monitored by an ECG battery driven transmitter (EKEG e l e c t r o n i c s ) , picked up on an FM receiver (EICO), operating a t 96 megacycles, and displayed on an osc i l l o s c o p e . In most experiments the whole animal was kept a t 37°C * 1°C by an automatically controlled heating pad under i t s abdomen. A f t e r a l i g n i n g the animal i n a stereotaxic frame (Appendix I l i a ) , a deeerebration was performed by sectioning the b r a i n stem along the plane of the bony tentorium (at 60°) beginning just behind the base of the tentorium as described by Bremer i n 1937. Following this procedure, there was a frequent occurence of extensive oedema of the brain, which was sometimes so severe as to require the termination of the experiment. For the deeerebration, a seven electrode bank -30-was used. Each electrode was Insulated with either insul-x or Beldenamel and l e f t uninsulated for 1.0-2.Omm at the ti p . The electrodes were baked In an oven for 1 hour at 70°C or 4 hours at 250°C respectively, depending on which Insulating material was used. The electrodes were mounted 2.0mm apart and held In position by a clamp and epoxy glue. A current of I5.O-25.O mamps for 15 sees was applied between each adjacent pair of electrodes in 7 successive 2.0mm steps from the base of the skull. After the deeerebration, vthecere-bellar cortex was covered with a layer of wax paper to prevent drying and reduce cooling. A craniotomy was then performed, exposing Idle l e f t cerebral cortex between frontal planes A5.0 and A I 5 . 0 , and lateral planes of 0.0-3.0 (Snider & Niemer,l96l). The cortex and subcortical white matter above the fornix was subsequently sucked out. The area l e f t free for the Insertion of the mlcropipette was Irrigated with warm Locke*s solution (37°C). modified by the omission of bicarbonate and reduction in K* concentration (Appendix II). Trephine openings over the right and l e f t hemispheres were made to permit the insertion of concentric bipolar stimulating electrodes, one was directed to the contralateral superior peduncle at co-ordinates P1.0, L2.0, ?9.0s and a second was placed in the ips l l a t e r a l HFC at co-ordinates A6.0, L2.0, V13.0. b) iontophoresis; The multibarrelied pipettes consisted of five hard pyrex glass tubes (outer diameter 6.5mm, bore 4.5mm) fused together. The array of five tubes was heated and drawn out to a fine t ip by a glass electrode puller (Appendix I l l b ) * The shafts of the micropipettes were then broken back to give an overall tip diameter of 4.0-12*0*'. This was done to ensure that the barrels did not have an excessively high e l e c t r i c a l resistance. The micropipettes were f i l l e d by boiling In f11tered-distilled water. After the water had cooled i t was replaced with solutions of 1.0M NaCl, 0.2M DLH (Na-DL-horao-cysteate) ,at pH.8.0 for the activation and localization of quiescent neurones, 0.5M atropine sulfate in Q.9N saline, 1.0M ACh-Br (acetylcholine bromide- slightly acidic pH)»and 0.2M phystigmine sulfate (eserine). The individual tubes were f i l l e d using a syringe and fin© polythene* tube. The micropipettes were then stored for 24-48 hours at 4°C before use, to, allow diffusion of the dissolved substances into the tips. In a l l the micropipettes, the central barrel (which was always wider than the others, with a resistance only about one tenth as high for a given solution) was f i l l e d with a 1.0M NaCl solution. The resistance of each electrode was measured In a 0,9% NaCl solution using a 6.0v battery with a 10m-*- resistance in series with a galvanometer. The measure-ment in nA (nanoamperes:10 amperes) was compared to a previously calculated graphical plot of galvanometer reading against resistance. Drugs were applied as cations with the exception of DLH which was applied as an anion. A retaining potential was applied to a l l drug-containing barrels to prevent the diffusion of actively ionized compounds ( P h l l l i s , Tebecis & York, 1967). The potential was usually adjusted so that the retaining current — 3 2 — was of the order of 6 . 0 - 8 . 0 nA* A larger braking current was associated with a longer latency before the appearance of drug-induced effects, presumably a result of the removal of the drug from the immediate vlolhlty of the electrode tip* It was assumed that the amount of drug released from the electrode tip was proportional to the current applied from the iontophoresis unit (KrnJevieV Mitohell & Szerb, 1963) . c) Recordingt The centre recording electrode was supported by a plastic holding device on a micromanipulator which allowed changes in three dimensions with vertical movements in steps of l . O ^ (Appendix I l i a ) . The neuronal responses were recorded by the centre electrode in V I * , amplified and displayed on an o s c i l l o -scope (Appendix I V ) and were used to trigger a ratemeter and intensity modulator (Appendix la) whose output could be displayed on a paper chart. Spikes; displayed on an o s c i l l o -scope having a slow time base, were photographed. Square wave stimuli of 0.1 msec duration were delivered either to NPC or BC. ' x w d) HIsttology s ~ At the termination of most experiments mioroelectrbde tracks were marked by insertion at the same sites of a steel eleotrode through which a current was passed to deposit iron ( 2 0 M for 20 sec). Similar currents were passed through the stimulating electrodes. The animals were saorifled by an overdose of chloroform or barbiturate injected intravenously into the femoral vein. The thoracic cavity was opened, the -33-descending aorta clamped, the l e f t ventricle was punctured and the ascending aorta was cannulated. The cats were perfused with 500ml of 0.9$ saline then with 500ml of 10i0# formaline containing 5.0g of potassium ferrloyanide; The cats were decapitated and the heads stored in 500ml beakers f i l l e d with 10.0$ formaline solution. After approximately five days the heads were remounted Into the stereotaxic frame and the brains were sectioned Into 3.0mm blocks, each containing a marker (markersa from VL, A1U0* L4;0, Vi2;0j from SPG* AiSiO, L2>0* V13i0j and from BG, PliQ, L2.0, V9.0-ln the atlas of Snider & Nlemer, 1961). The sections were out and then stained with oresyl violet and the markers were subsequently identified by the Prussian blue reaction, (Plates 1, 2, & 3); -34-RE STILTS In these experiments, 128 c e l l s were studied In, above and below VL.The neurones were found eith e r because they were d i s -charging spontaneously or i n response to e l e c t r i c a l stimulation, of BC and/or NPC, or because they were activated by DLH and/ or Ach applied Iontophoretieally. The c e l l s v/ere r e l a t i v e l y consistent i n s e n s i t i v i t y to Ion-tophoretieally applied DLH down to 9.5 mm below the f o r n i x . Their s e n s i t i v i t y to DLH appeared to decrease below this l e v e l ( FlQ.f,, ~:fr 35). In contrast to the r e s u l t s reported by McLennan, Huffman, and Marshall (1968) c e l l s from 3.0 to 6.0 mm below the fornix were found to be sensitive to iontophoretieally applied Ach. However, the s e n s i t i v i t y of c e l l s to Aoh increased with the depth of the micropipette ( f i g . 7, p. 35). and a higher propor-tion of Ach sensitive c e l l s was found from 7.0 to 9«0 mm below the fo r n i x ( f i g . 9, P» 37). The location of c e l l s tested i n these experiments was established by i n s e r t i o n at the same s i t e s of a s t e e l electrode through which a current was passed to de-posit Iron. 1. EFFECTS OF ELECTRICAL STIMULATION: In the present series of experiments, stimulation of BC was used to confirm the position of the electrode In VL. Many exper-iments involving BC and deep cerebellar n u c l e i stimulation have confirmed the termination of the dentatorubrothalamic t r a c t In the f e l i n e thalamus (VL). Two d i s t i n c t p h y s i o l o g i c a l layers of c e l l s could be d i f f e r -entiated on the basis of the response to BC and NPC stimulation. -35-I . 0 -0 . 6 -Decerebrate Preparations • NPC evoked • BC and NPC o not " Ach o Q o o °o • •• 0 . 2 -. 0 -0 . 6 -0 . 2 • o o o o o o o ,o»- r • -D L H o oo o ° o o o o o C q o o • • J © e c e « « > - i 1 1 1 r— 5 7 9 Depth below Fornix (mm) - 1 13 Figure 7 Plots of the r e l a t i v e s e n s i t i v i t i e s of neurones located above, i n and below VL (Decerebrate preparation). 1.0 on the ordinate corresponds to lOnA and 0.0 to llOnA & s t i l l not exci t a b l e . -36-A n a e s t h e t i z e d Preparat ions «> NPC evoked • BC and NPC o not " 1.0 0.6 | 0.2 H c a> 00 CD I .0 > a £ 0.6 H 0.2 Ach o o O DLH f »»»»! o o f — — ,— O O • o ° o o  8 Depth below Fornix (mm.) —i 13 F i g u r e 8 P l o t s o f the r e l a t i v e s e n s i t i v i t i e s o f thalamic neurones i n v e r t i c a l t r a c t s i n c a t s a n a e s t h e t i z e d w i t h Sodium P e n t o b a r b i t a l (Nembutal). A r e l a t i v e s e n s i t i v i t y o f 0 i n d i c a t e s t h a t the neurone was u n a f f e c t e d by the a p p l i c a -t i o n o f DLH or Ach w i t h e j e c t i n g c u r r e n t s up to HOnA, a valu e o f 1.0 th a t the c e l l was e x c i t e d u s i n g a c u r r e n t o f lOnA. The v e r t i c a l i n t e r r u p t e d l i n e i n d i c a t e s the d o r s a l border o f VL i n the f e l i n e thalamus. Anaesthetized Decerebrate Depth below Fornix ( m m ) Number of Cells tested Ach excited Tested Ach excited 3 - 4 3 0 5 2 4 - 5 3 0 5 3 5 - 6 5 0 8 6 6 - 7 4 1 13 9 7 - 8 10 2 7 6 8 - 9 7 4 12 II 9 - 1 0 2 1 18 15 10 - 1 1 1 0 13 5 1 1 - 1 2 1 1 8 4 12 - 13 1 1 2 0 Figure 9 Table showing total number of cells tested and the number of cells which could be excited by Ach at various levels below the fornix. C e l l s situated approximately 6.0 to 9.0 mm below the fornix could usually be evoked by e l e c t r i c a l stimulation of BC and NPC with voltages ranging from 20-60V (figs, 7 & 17, PP. 35&49). In con-trasts c e l l s l y i n g 9»5 to 12.0 mm below the fornix were evoked by NPC stimulation only ( f i g s . 7,8 , & l l s pp. 35,36,&40). This f i n d i n g would indicate that the NPC has a large input to and below VL i n the f e l i n e thalamus. However, because of cerebral edema, It was hard to in t e r p r e t how f a r below the fornix this NPC input a c t u a l l y extended. Neurones i n VL could usually be evoked by both BC (3 msec latency) and NPC (1.2 msec latency) stimulation with 20-60V and a 0,1 msec duration, from 6.0 to 9.5 ram below the fornix ( f i g s . 7&17, PP» 35&49K These neurones always followed r e p e t i t i v e , stimulation at frequencies of 10-20 per second and sometimes the upper l i m i t exceeded 150 per second. These properties of the BC evoked spikes i n VL. neurone's are s i m i l a r to those described by Sakata, Is h i jlma, and Toyoda (1966).In contrast , 9 . 5 to 12 mm below the fornix, c e l l s could no longer be evoked by BC stimula-tion ( f i g s . 7&ll s PP« 35$ 40), and more dorsally situated c e l l s , from 3.0 to 6.0 mm below the fornix, were unaffected by e l e c t r i -c a l stimulation of eithe r BC or NPG ( f i g . 7, p. 35). Concurrent stimulation studies were performed on BC and NPC to investigate a possible occlusion of NPC evoked responses i n VL by BC stimulation. BC stimulation was delayed i n in t e r v a l s from 0.1 to 1.2 msec before NPC stimulation and the responses were recorded. No evidence was found to suggest an occlusion of the NPC response following BC ( f i g . 13, p. 43). * 110 Figure 10 Ratemeter recordings of relative sensitivities of neurones located above in and below VL. Type A neurones; not evoked by NFC or BC. Type B neurones; evoked by both WPG and BC. Type C neurones; evoked by NPC but not by BC. -40-Pigure 11 a) Neurone l o c a t e d 9.8 mm below the f o r n i x evoked , _ j by NPC s t i m u l a t i o n (30v, O.lraseo d u r a t i o n ) Vmnt C e l l no. 6, May 29, 1969. b) Neurone l o c a t e d 9•8mm below the f o r n i x not evoked by BC s t i m u l a t i o n ( 4 0 v , 0.1msec du r a t i o n ) C e l l no. 6, May 29. 1969. -41 I i » Figure 12 Neurone located 9.8mm below the fornix evoked by NPC stimulation (40v, 0.1msec duration), after atropine had depressed the neuronal response to ACh applied lontophoretlcally. Cell no.6, May 29. 1969. H i EFFECTS OF CHEMICAL STIMULATION: a) EFFECTS..'OF ACH: Ach was a p p l i e d i o n t o p h o r e t i e a l l y to t h a l a -mic c e l l s w i t h c u r r e n t s r a n g i n g from 8.0 to 120.0 nA, and neur-ones s e n s i t i v e t o t h i s m a t e r i a l were found throughout the range o f depth, i . e . i n , above, and below VL. The r e l a t i v e s e n s i t i v i t y to the amount o f i o n t o p h o r e t i e a l l y a p p l i e d Ach i n c r e a s e d w i t h depth ( f i g s . 7&10, pp. 3S&39). A r e l a t i v e s e n s i t i v i t y o f 0- i n d i c a t e s t h a t the neurone was u n a f f e c t e d by the a p p l i c a t i o n o f DLH or Ach w i t h e j e c t i n g c u r r e n t s up to 110 nA, a value o f 1.0, t h a t the c e l l was e x c i t e d u s i n g a c u r r e n t o f 10 nA. I t i s r e c o g n i z e d that t h i s measure o f s e n s i t i v i t y Is a f f e c t e d by such p h y s i c a l .factors as the d i s t a n c e o f the e l e c t r o d e t i p from the neurone and the volume i n t o which the Ion i s e j e c t e d . The h i g h e s t p r o p o r t i o n o f c h o l i n o -c e p t i v e c e l l s was encountered between 7.0 and 9.0 mm below the f o r n i x ( f i g , 9* P« 3 7 ) . C e l l s from 3 . 9 to 6.0 ram below the f o r n i x i n the dorsalthalamus were found to be r e l a t i v e l y s e n s i t i v e to a p p l i e d Ach when the decerebrate p r e p a r a t i o n was employed, but n o t In a n a e s t h e t i z e d animals. These l a s t r e s u l t s are i n c o n t r a s t to those r e p o r t e d by McLennan, Huffman and M a r s h a l l (1968), and a l s o d i s a g r e e w i t h the r e s u l t s o f P h i l l i s and Westerman (1966). The l a t t e r authors r e p o r t e d t h a t o n e - f i f t h o f the c e l l s i n the d o r s a l thalamus and o n e - h a l f o f those i n VL were e x c i t e d by i o n -t o p h o r e t i e a l l y a p p l i e d Ach. b) EFFECTS OF DLH: DLH, because of i t s powerful e x c i t a t o r y a c t i o n ( C u r t i s , P h i l l i s & Watkins, I960) was used f o r the a c t i -v a t i o n and l o c a l i z a t i o n of q u i e s c e n t neurones. However i t was mainly used as a t e s t o f n e u r o n a l e x c i t a b i l i t y a f t e r a t r o p i n e had been a d m i n i s t e r e d and Ach no longer had a d e p o l a r i z i n g e f f e c t - 4 3 -\ i Figure 13 Two traces both showing a neurone located 7.1 mm below the fornix evoked b y HFC (1st response; 50v, 0.1 msec duration), delayed 0 .5 msec after 130 stimulation (2nd response; lj0v, 0.1 msec duratIon). C e l l no. 1 , June 19, 1969. - 44 -on the post-synaptic membrane... I f the membrane was s t i l l e x c i t a -ble by DLH a f t e r the a p p l i c a t i o n of atropine, then i t was con-cluded that atropine was s p e c i f i c a l l y blocking the. Ach e f f e c t . Neurones sensitive to DLH were found throughout* above, and below VL i n the f e l i n e thalamus. Maximum s e n s i t i v i t y was found i n c e l l s from 3.0 to 9.5 mm below the f o r n i x . Below the 9.5 mm l e v e l neurones became less sensitive to DLH ( f i g s . 7&10, pp. 35 & 39).DLH was applied iontophoretieally'to thalamic c e l l s with currents ranging from 8 to 80 nA.'.'* III. ATROPINE: . • - V ' Atropine was applied iontophoretieally when cholinoceptive c e l l s which could be evoked by BC and/or NPC stimulation were encountered. When these c e l l s no longer responded to iontophore-t i c a l l y applied Ach, but continued to* respond, to DLH, BC and/or NPC f i b e r s were again stimulated. A l l C e l l s which had previously been evoked by BC and/or NPC stimulation s t i l l responded a f t e r Ach s e n s i t i v i t y had been abolished by atropine ( f i g s . 12, 14 & 15»PP. 41, 46 & 47 ) . This would Indicate that the post-synaptic receptors of the BC and NPC pathways are'non-muscarinic•, and suggest that the pathways are non-cholinergic. Atropine was given intravenously, i n a dose of 1.5 mg/kg, on one occasion without any e f f e c t upon the BC and NPC evoked responses; however in this experiment the c h o l i n o c e p t i v i t y of the neurones was not examined. Atropine was applied with currents ranging from 50 to lOOnA for up to 30 minutes. In most cases a f t e r 10 minutes the cholino-ceptive receptors were no longer sensitive to Ach but continued to respond to DLH, although the l a t t e r response was also s l i g h t l y depressed. This indicated that the cholinergic neuronal receptors were being blocked while other receptors on the c e l l u l a r membrane were s t i l l a c t i v e . However I f atropine was administered with suf-f i c i e n t l y high currents for any considerable lengths of time, the s e n s i t i v i t y of the neuronal receptors to DLH was also eliminated. Atropine had l i t t l e noticeable e f f e c t on the spontaneous back-ground a c t i v i t y usually encountered i n and above VL.Also i n exper-iments where pentobarbitone was used as an anaesthetic, atropine had no noticeable e f f e c t on the spindle a c t i v i t y of thalamic neurones i n VL. These r e s u l t s are i n agreement with McCance, P h l l l i s , and Westerman (1968) who reported that although atropine (100 nA) abolished the excitatory action of Ach on cholinoceptive c e l l s , i t f a i l e d to modify the 'burst 1 response. DH-^-E was not used i n these experiments to exclude the possi-b i l i t y of n i c o t i n i c receptors i n VL. McCance, P h i l l i s and Wester-man (1968) reported that c e r t a i n responses i n VL, evoked by stimu-l a t i o n of BC and the mesencephalic r e t i c u l a r formation were re-duced by iontophoretically applied atropine and DH-£-E, These r e -su l t s with regard to atropine are not i n agreement with those of the present experiments. Atropine had no noticeable e f f e c t on either BC or NPC evoked neurones except when applied i n excess-i v e l y high concentrations for more than 30 minutes. I V . ESERINE (physostigmlne)s Eserine was tested on a l l c e l l s when encountered whether or not they were cholinooeptive. The usual response of thi s a n t i -cholinesterase vras to increase neuronal s e n s i t i v i t y to ionto-ph o r e t i c a l l y applied Ach. However i t also had an excitatory action on several thalamic neurones when i t was applied alone ( f i g . 16, p» 49a). These r e s u l t s are In agreement with those of McCance, P h l l l i s , Tebecife and Westerman (1968) who observed that eserine 1 1 3 m s i t — \o\o4tr Vr^ce. C e l l located 8.7mm below the f o r n i x evoked by NPC (40v, O.lmaeo duration) a f t e r atropine had depressed the neuronal response to ACh applied iontophoretieally. C e l l no.10, May 16, I969. - 4 ? Figure 15 C e l l located 9.5mm below the forn i x evoked by BC (40v, 0 .1msec. duration) a f t e r atropine had depressed the neuronal response to ACh applied lontophoretloally. C e l l no.5* May 15* 19&9. frequently induced f i r i n g in the absence of an application of Ach. The neuronal response to iontophoretieally applied eserine had a longer latency of onset and offset when compared with Ach. i V. EFFECTS OF ANAESTHETIC (pentobarbitone): As was mentioned in the Introduction, different anaesthetics have been reported to influence the results of experiments signi-ficantly. Neurones in the ventrobasal complex of cats anaesthe-tized with pentobarbitone were frequently characterized by rhyth-mic spontaneous spindle discharges. In the f i r s t set of experiments performed on cats anaesthe-tized with pentobarbitone, thalamic neurones were much less sen-sitive to Ach than In the second set of experiments where the deeerebration technique had been employed (figs. 7 , 8 &9»PP» 3 5 , 3 6 & 3 7 ) * In the former set, pentobarbitone depressed the neur-onal responses to Ach mainly on the neurones which could be evoked by both NPC and BC ( f i g . 8, p. 3 6 ) . In the latter set, the neur-onal response to iontophoretieally applied Ach disappeared after half an anaesthetic dose of pentobarbitone had been injected in travenously (15J mg/kg). These neurophysiological and pharmacological results would therefore confirm the existence of the pathway arising in NPC and projecting to the i p s i l a t e r a l VL (Bowsher, 1 9 6 8 ) , and fur-ther show that this fiber tract Is 'non-muscarinic*. Figure 16 A neurone located 9.9mm below the fornix firing in response to physostigmine applied iontophoretieally. Cell no.8, May 16* 1969. -49 • l ' lms«c — \ o w t c Vv*.ce Figure 17 a) Cell located 8.4mm below the fornix evoked by NPC (40v, 0.1 msec duration). Cell no.12, Nov. 27. 1968. b) Cell located 8.4mm below the fornix evoked by BC (45v, 0.1 msec duration). Cell no.12, Nov. 27 . 1968. - £ 0 -DISC USSION The Ach receptors on VL neurones have been reported not to be Identical either with those at nicotinic synapses on Renshaw cells or with the muscarinic receptors on deep pyramidal cells i n the neocortex, but to occupy a more intermediate pharmacological position, as do those of the L G N (McCahce, P h i l l i s & Westerman, 1966). The response toiontophoretieally applied Ach on VL neurones is intermediate to the rapid onset and short duration of the excitation produced at Renshaw cell s , and the slowly developing, prolonged action of Ach on deep pyramidal c e l l s . It Is hard to ascertain, however, whether the delayed response of VL neurones to Ach i s a typical feature of thalamic neurones or merely a reflection of a low density of Ach receptors on the c e l l surface. Thus the action of Ach on neurones lying i n and above VL tends to be slowly developing and the prolonged excitation after application has ceased could also be the result of a diffusional Impediment and to the f a i l i n g of local AchE to remove the relatively high local concentrations of the substance which occur during iontophoretic application. An Interesting feature of these experiments was the finding that most of the neurones i n VL were sensitive to Ach and DLH, and that the response to Ach was blocked by iontophoretieally applied atropine. This would Indicate that Ach Is not the exitatory synaptic transmitter released at B C and N P C terminals in VL by orthodromic volleys. In contrast Prlgyesi and Purpura (1966) reported that intra carotid administration of atropine,(up to 1.0 rag/kg) had a marked effect on the magnitude of B C evoked Fields i n the thalamus, depressing the post-synaptic components of the VL responses evoked by B C stimulations The fact that iontophoretieally applied atropine f a i l e d to block the excitatory effect of B C stimulation, while Intracarotid adminis-51-tratloa of atropine depressed the BC evoked potential, would seem to indicate either that atropine i s not reaching the neurone l n sufficient concentration when applied iontophoretieally or that some other system i s affected by the intraearotid injection which in turn Influences the neurone under observation. It i s to be noted however that' the Ach receptors upon the neurones were blocked by the iontophoretic application of atropine. Andersen and Curtis (1961*) reported that DH-JJ-E depressed Ach excitation of a l l the thalamic neurones on which i t was tested. DH*j8-E was not used in these experiments to exclude the possibility of nicotinic receptors in VL and therefore the results of the experiments indicates only that the receptors are'non-rauscarinlc«. A further study on the effects of DH-jQrE on VL neurones which are evoked by HPC would be necessary to confirm or exclude the possibility of nicotinic receptors i n VL. There i s biochemical and histological evidence for the presence Of Ach, Ach E and choline acetyltransferase In the feline thalamus (Burgen and Chipmah, 1951 # 19525 Hebb and Silver, 1956I Gereutzoff, 1959). Taken with the evidence that Ach excited a high percentage of neurones above and In VL, this would suggest that one or more afferent thalamic pathways do have cholinergic components. As stated above although Ach excited a high percentage of BC and HPC evoked thaiamie neurones, atropine was Ineffective i n blocking BC and HPC evoked synaptic f i r i n g . However, i t was completely success-f u l i n abolishing the neuronal response to Ach applied Iontophoretie-a l l y . The presence of a limited proportion of nicotinic cholinergic fibers i n the BC and HPC projecting to most of th© BC and HPC evoked neurones In VL, but generally non-cholinergic BC and NPC fibers would explain these results• An alternative explanation i s that cholinergic fibers- from other areas of the brain maintain VL neurones In a state of sub-threshold depolarization, which f a c i l i t a t e s the response to BC or NPC stimulation. The results with mesencephalic reticular form-ation stimulation support the idea that cholinergic fibers project either to BC and IPC pathways or directly to the thalamus (Shute & Lewis, 19631 P h l l l i s , 1967). In addition to NPC fibers terminating i n VL, NPC projections appeared terminate below VL. Although the base of VL is 10.0 mm below the fornix, c e l l s sensitive to Ach and evoked by NPC were also found between 12.0 to 13.0mm. These cells would be located i n the l a t e r a l hypothalamus according to the atlas of Snider and Nieraer (1961). Another interesting observation In these experiments was a change i n average sensitivity of neurones to Ach and DLH as the micropipette dlscended through the thalamus. Cells in the dorsal thalamus 3.0mm below the fornix were relatively sensitive to applied Ach. These results, which d i f f e r from those of McLennan, Huffman and Marshall (1968), and to a lesser degree from those of McCance, P h l l l i s and Westerman (1966), were obtained using the decerebrate preparation, which could explain these differences. Ceils sensitive to Ach were also found throughout and below VL with the relative sensitivity to Ach increasing with depth. This increase i n sensitivity could Indicate that more cholinoceptive receptors are present on the neurones lying at deeper levels i n VL. In these experiments the majority of c e l l bodies were encountered i n the lower three quarters of VL - especially those neurones which could be evoked by NPC. The relative sensitivity to DLH was very consistent as far as the ventral border of VL. Below this level sensitivity to DLH - 5 3 -appeared to decrease. These r e s u l t s are i n agreement with those of McLennan, Huffman and Marshall (1968), although the r e s u l t s of these experiments Indicate a more marked change In r e l a t i v e s e n s i t i v i t y . * As mentioned i n the r e s u l t s , eserine was tested on a l l c e l l s encountered whether or not they were cholinoceptive.A potentia-tion of Ach e x c i t a t i o n by this cholinesterase Inhibitor was ob-served i n VL. This also occurs In the LGN ( P h i l l i s , Tebecis & York, 1967),. at the n i c o t i n i c synapse on the Renshaw c e l l s (Cur* t l s , P h i l l i s and Watkins, 1961a) and the granule layer of c e l l s of the cerebellar cortex (McCance & P h i l l i s , 1964). I t was not however observed on muscarinic synapses of cholinoceptive c e l l s i n the cerebral cortex (Krnjevic & P h i l l i s , 1963b). I t i s there-fore conceivable that the potentiation of Ach action by c h o l i n -esterase i n h i b i t o r s i s c h a r a c t e r i s t i c of n i c o t i n i c synapses although this point remains to be established. Eserine also had an excitatory action on several thalamic neurones when i t Was applied alone,presumably by Inactivating AchE in the synaptic c l e f t , , leaving Ach i n the c l e f t to activate the post-synaptic receptor. In a number of d i f f e r e n t papers,. Ach has been reported to exert an i n h i b i t o r y action on some neurones i n the thalamus (McCanoe, P h i l l i s & Westerman, 1968; Andersen & C u r t i s , 1964b). In these experiments no neurones were encountered which were Inhibited by Ach. There has been much controversy as to the v a l i d i t y of the r e s u l t s obtained from experiments involving the use of b a r b i -turate a anaesthetics.In this series of experiments, s e n s i t i v i t y of the receptors to Ach, and to a lesser degree to DLH, was depressed by intravenously injected pentobarbitone. This Is - 5 4 -partly in agreement with Crawford and Curtis (1966) who claimed that barbiturates depressed the response of cerebrocortlcal neurones to excitant amino acids and Ach to an equal extent, but not with the experiments reported by Krnjevic and P h i l l i s (1963 a,b) in the cortex and by McCance, P h i l l i s and Tebecis (unpublished observations) in the thalamus which indicated that barbiturates selectively depressed neuronal responses to Ach, In the present experiments when'the decerebrate preparations were employed instead of anaesthetized ones, the proportion of thalamic neurones which responded to Ach in VL Increased. In summary, there are only moderate amounts of Ach, AchE and cholineacetyltransferase in the mammalian thalamus in comparison to the basal ganglia and spinal ventral roots; how-ever, some neurones in VL are extremely sensitive to Ionto-phoretieally applied Ach. Histological investigations have shown that AchE-eontaihing nerve fibers project from mesencephalic reticular formation to the thalamus (Shute & Lewis, 1963*1967)• As yet however, no pathway excluding the reticular formation networks,has been found to converge upon the thalamus which could definitely be considered at least in part cholinergic* - 5 5 -BIBLIOGRAPHY Andersen, P., C. JtcC. Brooks, and J, C. Eoeles, 1964. Ele c t r i c a l responses of the ventrobasal nucleus of the thalamus. Progress In Brain Research, 5:100-113. Andersen, P., and D. H. Curtis, 1964a. The excitation of thalamic neurones by ACh. Acta Physiol. Soand. 61:35-99. Andersen, P.. and D. R. Curtis, 1964b. The pharmacology of the synaptic and ACh-Induced excitation of ventrobasal thalamic neurones. Aota. Physiol. Soand. 61:100-120. Andersen, P., and T. A. Sears, 1964. The role of inhibition In the phasing of spontaneous thalamo-oortloal discharge. J . Physiol. i?3»459-480. 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W, P h l l l i s , i960. The a c t i o n of procaine and atropine on s p i n a l neurones. J . P h y s i o l . 153*17*34. -58* C u r t i s , D. R, , J . W. P h l l l i s , and J . C. Watkins, 1959* The Depression of S p i n a l Neurones by -amino n -butyr io ao ld and 6 - a l a n i n e . J . P h y s i o l . 146$185-203. C u r t i s , D. R., J . W. P h l l l i s , and J . C. Watkina, i960. The chemical e x c i t a t i o n of s p i n a l neurones by c e r t a i n a c i d i c amino a c i d s . J . P h y s i o l . 150(656-682. C u r t i s , D. R., and R. W. R y a l l , 1966a. The e x c i t a t i o n of Renshaw o e l l s by chol inomimetics. S x p t l . B r a i n Res. 2:49. C u r t i s , D. R. , and R. W. R y a l l , 1966b. ACh receptors of Renshaw c e l l s . E x p t l . B r a i n Res. 2)66 -80. C u r t i s , D. R. , and R. W. R y a l l , 19660. Synaptic e x c i t a t i o n of Renshaw c e l l s . E x p t l . B r a i n Res. 2*31-96. Dale , H. H . , 1914. The a c t i o n of c e r t a i n esters and ethers of chol ine and t h e i r reac t ion to muscarine. J . Pharmacol. 6:147-190. Davis , R. , 1966. ACh-sens l t i ve neurones i n VL nucleus. Ins The Thalamus, Ed. by D. p. Purpura and H. D. Yahr. Columbia Un ivers i t y Press , New York. p.193. Eooles, jr. C , 1966. Propert ies and func t iona l organizat ion of o e l l s In the ventrobasal complex of the thalamus. Ins The Thalamus. Ed. by D.P. Purpura and M. D. Yahr. Columbia Un ivers i t y Press , New York. p.129. 59-F r l g y e a i , T. L. and D.P.Purpura, 1966, Acetylcholine s e n s i t i v i t y of thalamic synaptic organizations activated by brachium conjunctlvum Stimulation. Archs. i n t . Pharmaoodyn. Ther. 163»110-132. Gallndo, A., K. Krnjevlc and S. Schwartz, 1967. Micro* iontophoretic studies en neurones i n the cuneate nucleus, j . Physiol* 192s359*377. Gerebtzoff, M. A,, 1959* Cholinesterases, Pergamon Press, Oxford. Goldberg, A. M. and R. E. MeCaman, 196?. A quantitative microchemlcal study of choline a c e t y l transferase and acetylcholinesterase i n the cerebellum of several species. L i f e S e l . 6$1493*1500. Goodman, L. S. and A. Oilman, 1965. The Pharmacological Basis of Therapeutics. Third E d i t i o n , Maomillan Go., New York. Hebb, C. 0 . , and A. S i l v e r , 1956. Cholineaoetylase i n the central nervous system of man and some other mammals. J . P h y s i o l . 134*718*728. Krnjevio, K., 1965. Actions of drugs on single neurones In the cerebral cortex. B r i t i s h Med, B u l l . 21*10-14. •60-Krnjevio, K. and J. P. Mitchell and J. C. Szerb, 1963. Determination of lontophoretlo release of acetylcholine from micropipettes. J. Physiol. 165*421-436. Krnjevio, K. and J. W. P h l l l i s , 1963a. lontophoretlo studies : of neurones in the mammalian cerebral cortex. . . J . Physiol. 165«274-304. Krnjeyic, K. and J. M. P h i l l l s * 1963b. Acetylcholine sensitive c e l l s In the cerebral cortex. ^. Physiol. 1669296-327. Lewis, P. R., and C. C. D. Shute, 1967. The ascending cholinergic reticular system* Neooortloal, ©Ifaotory, and subcortical projections* Brain 90*497-520. Lewis, P. R., and C. C. D. Shute, 1967a, The cholinergic limbic systami Projections to Hlppocampal Formation, medial cortex* nuclei of the ascending cholinergic reticular system, and the subfornical organ and supra-optlo crest. Brain 9 0 * 5 2 1 - 5 3 9 . HoCanoe, X. and J . W. P h l l l i s , 1964. Discharge patterns of elements In cat cerebellar cortex and their responses to lontophoretlcally applied drugs. Nature 204(844-846. McCance, J• and J. W. P h l l l i s , 1968. Cholinergic mechanisms In the cerebellar cortex. Int. J. Neuropharmaool. 7*447. McCance, I., J. W. P h l l l i s , A. K. Tebecls and R. A. Westerman, 1963. The pharmacology of ACh-exoltatlon of thalamic neurones. B r i t . J. Pharmacol. 3 2 : 6 5 2 - 6 6 2 . -61* McCanoe, J . , J . W. P h l l l i s and R. A. westerman, 1966. Responses of thalamic neurones to lontophoretlcally applied drugs. Nature 209:715-716. McCance, J., J . W. P h l l l i s and R. A. Westerman, 1968. Acetylcholine s e n s i t i v i t y of thalamic neurones: i t s r e l a t i o n s h i p to synaptic transmission. B r i t . J . Pharmacol. 32»635-651• McCance, J . , J . W. P h l l l i s and R. A. Westerman, 1968a. P h y s i o l o g i c a l and Pharmacological studies on the Cerebellar Projections to the F e l i n e Thalamus. Experimental Neurology 21:257-269. McLennan, H., R. D. Huffman, and K. C, Marshall, 1968. Patterns of E x c i t a t i o n of thalamic neurones by amino acids and by acetylcholine. Nature 219:387-388. McLennan, H., K. C. Marshall, and P. H. Pettman, I969. Unpublished observations. M i t c h e l l , J . F., 1963* The spontaneous and evoked release of acetylcholine from the cerebral cortex. J . Physiol. 165:98-116. Nastuk, W. L., 1953* Membrane p o t e n t i a l changes a t a single end plate produoed by t r a n s i t o r y a p p l i c a t i o n of a c e t y l -choline with an e l e c t r i c a l l y controlled mlorojet. Fed. Proc. 12:102. P h l l l i s , J . W., 1965a. Chollnesterase In the oat cerebellar, cortex, deep n u c l e i , and peduncles. Experienta 21:266-268. *62-P h l l l i s , J. W., 1965. Cholinergic meohanisms in the cerebellum. British Medical Bulletin 21:26-29. P h i l l i s , J . W., and A. K. Tebecis, 1967, The responses of thalamic neurones to iontophoretieally applied mono-amines. J . Physiol. 192i715-745. P h i l l i s , J . W., A. K, Tebecis and D. H. York, 196?. The inhibitory action of mono amines on lateral geniculate neurones. J, Physiol. 1901563-581. P h i l l i s , J. W., A. K. Tebecis and D. H. York, 1967a. A study Of oholinooeptlve c e l l s in the late r a l geniculate nucleus. J . Physlol 192:695*714. P h i l l i s , J. W.. and D. H. York, 1967. Cholinergic inhibition in the cerebral cortex. Brain Res. 5*517-520. Pokrovsky, M. V,* I960. Apparell a transistors destine a rendre l a luminance du trace sur l'eoran d*un o s c i l l o -scope independent de l a Vitesse de deplaoement ve r t i c a l . Ed. C. N. Smyth. Medical Electronics, p.85. Sakata, H., T. ishljlma and Y. Toyoda, 1966. Single unit studies of ventrolateral nucleus of the thalamus in the cats i t s relation to the cerebellum, motor cortex, and basal ganglia. Jap. J . Physlol. 1.4*42-60• Satlnsky, D., 1967. Pharmacological responsiveness of Lateral Geniculate Nucleus neurones. Int. J . Neuropharraacol. 6t387-397. -6> Steiner, P. A.*., 1968. Influence of Mioroelectrophoretieally Applied Acetylcholine on the Responsiveness of Hippooampal and Lateral Geniculate Neurones. Pflugers Arch. 303:173. Snider, H. S., and Nleraer, W. T., 1961. A Stereotaxic atlas of the cat brain. University of Chicago Press, Chicago. Strong, 0. a., and A. E. Elwyn, 1964. Human Neuroanatomy. Third Edition. Williams and Wllklns Co. p.408, Voogd, J., I964. The Cerebellum of the Cat. Van Gorcum and Co. Assen, Holland. Waelsch, H., 1962. Neuroohemistry. In: Neuroohemlstry. Ed. by"K. A. <]f. E l l i o t , 1. H, Pa^e and J. H. Quastel. Thomas, Springfield, p.288. •64~ APPENDIX I M \ A Y I ^ V r- ^ A r / > H ' ~ ^ [ / 1 T%£I | [ ^ ™ ^ \*<*mx | I A Control © 3 CgthoO. HU<ck digram ol r; + BV MPF03 »" ZN3702 2N3702 2N3702 2 N37D2 MPF103 MPFI03 Input*—| Mot*: R«aietanc«t Intwm.ty in ModuWran fo arid GapocitiM tn>uF Intensity Modulation to Cathode Drive to Drive to PoteMiomctnc Low-imp»oonca Recorder Recorder in , . 2. C'iRiiii diagram nf fi J a) Circuit diagram of rate meter and Intensity modulator. 65-APPEMDIX I b) Reoordlng apparatus ln the shielded room. -66-APPEND IX I I Locke's Solution (-^modified) IgCl .858 mraoles/llter 2 KC1 4.208 mmoles/llter CaCl 2.302 ramoles/liter 2 NaGl 1.462 ramoles/liter Glucose .013 mmoles/liter # modified by the omission of NaHCO and a reduction 3 i n the concentration of KC1 (to equal that In the cerebrospinal f l u i d ) . Stereotaxic apparatus and electrode holder. Glass electrode puller "•68"" APPENDIX IV DC Iontophoresis: Supply Unit =1 Schematic diagram of Iontophoresis c i r c u i t . 69 of an electrode marker In VL. - 7 0 Plat* 2 Histological verification of the location of an electrode marker ln NPC. •71--Plate 3 H i s t o l o g i c a l v e r i f i c a t i o n of the l o c a t i o n of an electrode marker In BC. -72-APPENDIX VI (fables) I TABLE I. Excitatory amino acids and related substances (In each group, substances are In approximate descending order of potency.) Compound Action DL-Homocysteic N-Methyl-DL-aspartic L-Glutamic L-Cysieic DL-Cysteic L-Aspartic D-Aspartic N-Methyl-L-aspartic Strong p-Methyl-DL-aspartic D-Glutamic DL-a-Aminoadipic DL-a-Aminopimelic Weak L-Asparagine D-Asparagine L-Glutamlne D-Glutamlne Variable o-Oxogluuric Succinic Fumaric L-Malic D-Malic liethionic None - .- J (K. Krajevlc, 1965) -73 T A B L E II. Depressant amino acids and related substances Compound Action • p - A m i n o n - b u t y r i c S t r o n g , w i t h TAmi n o - ^ - . - h y d r o x y b u t y r i c c o m p a r a b l e o ' - A m i n o-n - v a l e r i c p o t e n c y v - A m i n o c r o t o n i c 3 - A m i n o p r o p a n e s u l p h o n i c 3 - G u a n i d i n o p r o p i o m c G u a n i d i n o a c e t i c fl-A l a n i n e D L - ' i - A m i n o i s o b u t y r i c W e a k N - M e t h y l - y - a m i n o b u t y r l c N N - D i m e t h y l - v - a m i n o b u t y r i c M - M e t h y l - ? a l a n i n e N N - D i m e t h y l - f J - a l a n i n e D l - i - A l a n i n e T a u r i n e G l y c i n e Y - B u t y r o b e t a i n e f > - P r o p i o b e t a i n e C i t r u l l i n e C r e a t i n i n e L - H i s t i d l n e DL - P h e n y l a l a n i n e t - A m i n o c a p r o i c M i x e d c w - A m i n o c a p r y l i c v - G u a n i d i n o b u t y r i c DL - T r y p t o p h a n T y r o s i n e L - S e r i n e M o s t l y n o n e L - L e u c i n e r - A m i n o b u t y r y l c h o l i n e t - C a p r o l a c t a m (K. K rn jev lb . 19$5) «»y 4"*" T A B L E III. Miscellaneous amines and related compounds C o m p o u n d D e p r e s s a n t i c t i o n I m i d a z o l y l a c e t i c ac id Q u i c k ( in a p p r o x i m a t e T r y p t a m i n e d e s c e n d i n g o r d e r of 5 - H y d r o x y t r y p t a m i n e p o t e n c y ) D o p a m i n e A d r e n a l i n e N o r a d r e n a l i n e P r o c a i n e A t r o p i n e H y o s c i n e N u p e r c a i n e C e t y l t r i m e t h y l a m m o n i u m M e c a m y l a m i n e E p h e d r i n e D - L y s e r g i c a c i d d i e t h y l a m i d e E r g o m e t r i n e S h e l l - f i s h t o x i n R e l a t i v e l y I ' O W and p r o l o n g e d ( m o s t l y c f c o m p a r a b l e p o t e n c y ) (K. Krnjevlc, 1965) - 7 5 T A B L E IV. Some centrally acting drugs and other substances ( T h e s i g n i f i c a n c e o f t h e s y m b o l s i s a s f o l l o w s : + : e x c i t a n t ; — : d e - -p r e s s a n t ; H — : m i x e d e f f e c t s ; ( ) : t r a c e s o f a c t i v i t y ; a n d 0 : n o a c t i v i t y . ) Compound A c t i o n G e n e r a l a n a e s t h e t i c s B a r b i t u r i c a c i d 0 B a r b i t o n e s o d i u m 0 P e n t o b a r b i t o n e s o d i u m D i a l l y l b a r b i t u r i c a c i d — T h i o p e n t a l s o d i u m -C h l o r a l o s e — C o n v u l s a n t s a n d c e n t r a l s t i m u l a n t s S t r y c h n i n e ( + ) B r u c i n e 0 P e n t y l e n e t e t r a z o l ( M e t r a z o l ) -4 f P i c r o t o x i n ( > ' ) T e t a n u s t o x i n — M c N e i l 4 8 1 * T h i o s e m i c a r b a z i d e ( * - - ! > L o b e l i n e — r C a f f e i n e ( + ) I m i p r a m i n e — A n t i c o n v u l s a n t s a n d c e n t r a l d e p r e s s a n t s D i p h e n y l h y d a n t o i n ( + ) B u l b o c a p n i n e ( t ) T e t a n u s a n t i t o x i n — C h l o r p r o m a z i n e 0 R e s e r p i n e 0 T h a l i d o m i d e — G l u t e t h i m i d e - + P y r i d o x l n e ( + ) M o r p h i n e ( - ) P r o s t a g l a n d i n E | 0 (K. Krnjevlo. 19$5) TABLE V. Cholinomimetic agents Agent ACn-lika ni'on A c e t y l c h o l i n e r x - M u j c a r o n e D L - M u s c a r i n e A m b t n o n i u m A c e t y l - 3 - m e t h y l c h o l i n e S t r o n g ( i n a p p r o x i m a t e o r d e r o f p o t e n c y ) P r o p i c n y l c h o l i n e O r b a m i n y l c h o l i n e S u c c i n y l c h o l i n e C h o l i n e W e a k A c r y l y l c h o l i n e N i c o t i n y l c h o h n e U r o c a n y l c h o l i n e B e n i o y l c h o l i n e D e c a m e t h o n i u m A r e c o l i n e P i l o c a r p i n e O x o t r e m o r i p . e D i m e t h y l p h e n y l p i p e r a 2 i n i u m 2 - C a r b a m e t h o x y e t h y l t r i m e t h y l a m m o n i u m B u t y r y l c h o l i n e M a i n l y n o n e C r o t o r y l c h o l i n e 3 - J i - D i m e t h y l a c r y l y l c h o l i n e I s o v a l e r y l c h o l i n e N i c o t i n e P e n t e n y l c h o l i n e T e t r a m e t h y l a m m o n i u m E s e r i n e P o t e n t i a t e d a n d P r o s t i g m i n e p r o l o n g e d E d r o p h o n i u m ( T e n s i l o n ) L (K. Krnjevie, 19^5) -77-TABLE VI. ACh antagonists A n u j o n i i c A c t i o n H y o s c i n e ; A t r o p i n e G a l l a m i n e B e n a c t y r i n e P r o c y c l i d i n e ( K e m a d r i n ) S t r o n g ( i n a p p r o x i m a t e o r d e r o f p o t e n c y ) T r i m e t h a p h a n ( A r f o n a d ) B e n z h e x o l ( A r t a n e ) C a r a m i p h e n C y c r i m i n e ( P a g i t a n e ) A d i p h e n i n e ( T r a s e n t i n ) T e t r a e t h y l a m m o n i u m H e x a m e t h o n i u m W e a k o r v a r i a b l e D i h y d r o - 2 - e r y t h r o i d i n e T o x i f e r > ,r n r ' ^ o c u ; a r i n e i . m e t h y l - D - t u b o c u r a r i n e C u r a r e e x t r a c t N e g l i g i b l e (K. Krnjevlc, 1965) •78* Central action of cholinomimetics relative to acetylcholine Substance Renshaw cell Thalamic neuron© Acetylcholine + J- + + + + Propionylcholine + + + n-Butyrylcholine Acetyl-p-methylcholine + + + + + Carbamylcholine + + + + + -f + + Tetramethylammonium + •!- + + + Nicotine + + + + DL-Muscarine -f- + + + signs show the degree of excitation compared with that rf ~"Zf^vlc^o1'ne ~h ~t~ "fr-Table VII Central a c t i o n of cholinomimetics r e l a t i v e to acetylcholine* (D. H. Curtis* 1 9 6 5 ) . 

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