FIRING PROPERTIES AND Na -DEPENDENT PLATEAU POTENTIALS OF NEURONS IN NUCLEUS PRINCIPALIS TRIGEMINI OF THE GERBIL + VLADISLAV MICHAEL SANDLER A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Neuroscience Program) We accept this thesis as conforming (\o n the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1996 © Vladislav Michael Sandler, 1996 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 written permission. Department of Q^txplM^-^t ^t^u^LLt^s. The University of British Columbia Vancouver, Canada Date DE-6 (2/88) lS^Q£ fif^t, J33£ 11 ABSTRACT We in investigated the e l e c t r o p h y s i o l o g i c a l properties the nucleus principalis trigemini (PrV), using whole-cell recordings in in vitro slice preparations of brainstem. three groups mainly by their spontaneously doublets active or bursts; firing and able properties: to discharge We i d e n t i f i e d type 1 neurons w e r e action type 2 neurons, d e p o l a r i z e d fired action potentials in a nonadapting (tonic) of n e u r o n s potentials by current pulses, and the less c o m m o n l y encountered type 3 neurons also fired in s u c h patterns but with and biphasic reconstruction between types dendritic trees pattern; in afterhyperpolarizations. Neurobiotin did not reveal s i g n i f i c a n t morphological 1 and 2 neurons distributed mainly which were staining differences multipolar, along one axis. with Type 3 n e u r o n s had more e x p a n s i v e a n d circular dendritic arborizations. Hyperpolarization potential due to beyond -75 current pulse mV or down to the injection, resulted K in + reversal an inward rectification which w a s e x p r e s s e d a s a s a g in the voltage r e s p o n s e s of types 1 and type 2 neurons. A rebound depolarization or spike burst w a s evident on t e r m i n a t i o n In type 1 neurons, the application of C s + subthreshold of a p u l s e . (2 mM), a blocker of a Ill hyperpolarization-activated voltage cation current (l ), H eliminated the sag and the dependence of the rebound spike-latency on membrane voltage, but did not alter the main features of the rebound response. We attribute the inward rectification an l -like current. H Depolarization by current pulse injection hyperpolarized "plateau with potentials". DC to prevent This feature, neurons, consisted of an initial that decreased in amplitude, firing, 2 + oscillatory and then free media, with burst of 3 or 4 s p i k e s plateaued (TEA) 2+ for a variable We always observed pulse injection or without antagonists, C o o r C d , and during external 2 + evoked not observed in types 2 or 3 these voltage shapes on depolarizing current Ca of into type 1 neurons, occasionally duration, followed by an abrupt repolarization. perfusion with to the activation during the C a - c h a n n e l 2+ tetraethylammonium application. An analysis of the depolarizing voltage responses evoked by current pulses in type 1 neurons during blockade of persistent transient and N a conductances with TTX (600 nM) and K conductances + + with T E A (10 mM) and 4-aminopyridine presence of inward rectification. (4-AP; 0.5 mM), revealed the This had a peak activation near iv the plateau itself and was completely blocked by N i T h e s e o b s e r v a t i o n s are consistent with the activation Ca -conductance. Hence, we 2+ conductance mechanism propose controls the that a 2 + (600 uM). of a t r a n s i e n t Ca -dependent 2+ generation of the K + plateau potential. T h e application of T T X , as low a s 0.6 nM, i n c r e a s e d the to onset a n d d e c r e a s e d the duration of the plateau potential, greatly affecting manner, action potentials. T T X enhanced the descended towards concentrations potential an negative abrupt terminal [Na ]-perfusion, plateau potentials and fast of the plateau, + plateau genesis. Low reduced the a m p l i t u d e s Evidently, N a conductance can produce Higher a b o l i s h e d the blocking action potential spikes. as i t repolarization. 6 min) however, s i m u l t a n e o u s l y + without concentration-dependent slope of T T X (e.g., 60 nM for before c o m p l e t e l y persistent In a latency small marked of c h a n g e s in a c h a n g e s in firing current pulses behavior of type 1 neurons. A producing long-lasting the suprathreshold hyperpolarization plateau potential. depolarization also evoked a h y p e r p o l a r i z a t i o n in C a followed Indeed, 2 + free subthreshold A C S F with at the offset Co 2 + of the current (1 or mM) pulse. This hyperpolarization varied with was blocked by TTX (5 nM and 300 nM) and changes in the duration semiquantitative analysis hyperpolarization depended on the conclude, therefore, that revealed Na + entry of the plateau that the potential. magnitude of A the neuronal depolarization. We during a depolarization can increase a K conductance in type 1 neurons. + From represent our the studies, we conclude contributions conductances, high threshold of + persistent plateau and potentials transient Ca -dependent rectification, 2+ as C a - and Na -dependent K 2 + that + conductances. The ability neurons of primary sensory nuclei. In nucleus principalis burst responses to mechanical stimuli neurons that messenger likely regulation. are subject + as w e l l to bursts as part of Na -dependent plateaus is an unusual property + Na fire in trigemini, represent a normal output of to intra- and extracellular - vi TABLE OF CONTENTS ABSTRACT i i TABLE O F CONTENTS v i LIST O F F I G U R E S ix ACKNOWLEDGEMENTS x i 1. INTRODUCTION 1 2. M E T H O D S ....4 3. R E S U L T S 7 3.1 7 IDENTIFICATION O F N U C L E U S PRINCIPALIS TRIGEMINI ( P R V ) N E U R O N S 3 . 2 PHYSIOLOGICAL PROPERTIES OF P R V NEURONS 3.2.1 10 Inward rectification in type 1 neurons 12 3.2.2 Contribution of inward rectification to spike generation in type 1 neurons 12 3.2.3 Firing patterns of type 2 neurons 14 3.2.4 Firing patterns of type 3 neurons 16 3 . 3 P L A T E A U POTENTIALS IN T Y P E 1 N E U R O N S 18 3 . 4 IONIC MECHANISM O F PLATEAU POTENTIAL GENERATION IN T Y P E 1 N E U R O N S 20 Vll 3.4.1 Relationship of plateau potential firing to extracellular Ca 20 3.4.2 Involvement of a persistent Na 3.4.3 Sensitivity of plateau potentials to TTX. 25 3.4.4 Extracellular replacement of Na 27 3.4.5 Involvement of Ca -dependent rectification 3.4.6 Involvement of Ca -activated K 2+ conductance + + with choline 29 2+ 2+ + conductance 3 . 5 T H E P O S T - P U L S E HYPERPOLARIZATION ( P P H ) 4. 23 DISCUSSION 4.1 DEPOLARIZING R E S P O N S E S IN T Y P E S 2 AND 3 NEURONS 31 3 3 3 7 37 4 . 2 HYPERPOLARIZING C U R R E N T PULSE INJECTIONS INTO P R V NEURONS 3 8 4 . 3 CLASSIFICATION O F T Y P E S 1 A N D 2 N E U R O N S 3 9 4 . 4 B U R S T FIRING IN T Y P E 1 NEURONS 3 9 4 . 5 IS T H E BURST FIRING PATTERN O F T Y P E 1 NEURONS REPRESENTATIVE O F P R V NEURONS IN V I V O ? 40 4 . 6 T H E PLATEAU POTENTIALS O F T Y P E 1 NEURONS 41 4.6.1 Influence of cation currents 4.6.2 Repolarization of plateau potential 4 . 7 P O S T - P U L S E HYPERPOLARIZATION ( P P H ) 4 . 8 FUNCTIONAL CONSIDERATIONS IN T Y P E 1 NEURONS 42 43 44 4 6 Vlll 5. R E F E R E N C E S 5 0 6. 5 8 APPENDIX 1 6.1 D A T A ACQUISITION A N D P R O C E S S I N G P R O G R A M V M S 5 8 ix LIST OF FIGURES FIGURE 1 C A M E R A LUCIDA DRAWINGS OF NEUROBIOTIN-STAINED NEURONS 8 FIGURE 2 CHARACTERISTIC FIRING BEHAVIOR OF T Y P E 1 NEURON 11 FIGURE 3 INWARD RECTIFICATION, EVOKED BY HYPERPOLARIZATION, A N D REBOUND FIRING IN T Y P E 1 NEURON 1 3 FIGURE 4 CHARACTERISTIC TONIC FIRING BEHAVIOR O F T Y P E 2 NEURON 15 FIGURE 5 CHARACTERISTIC TONIC FIRING BEHAVIOR O F T Y P E 3 NEURON 17 FIGURE 6 T Y P E 1 N E U R O N S GENERATE PLATEAU POTENTIALS DURING C A - F R E E PERFUSION 1 9 2 + FIGURE 7 T I M E COURSE FOR DEVELOPMENT OF PLATEAU POTENTIAL UNDER CA -FREE 2 + CONDITIONS WIRH 1 M M C O I N T H E A C S F 22 2 + FIGURE 8 B L O C K A D E OF PERSISTENT N A + CONDUCTANCE IN A T Y P E 1 NEURON ELIMINATES PLATEAU POTENTIAL A N D P O S T - P U L S E HYPERPOLARIZATION ( P P H ) FIGURE 9 REDUCTION IN SLOPE OF PLATEAU DUE TO T T X 2 4 BLOCKADE OF PERSISTENT N A C O N D U C T A N C E IN A T Y P E 1 NEURON UNDER C A - F R E E CONDITIONS WITH 1 M M 2 + FIGURE 1 0 EFFECTS OF REDUCED EXTRACELLULAR [ N A ] + DURING PERFUSION WITHOUT [ C A ] AND WITH C O 2 + CO 2 + + 26 ON PLATEAU POTENTIALS EVOKED 2 8 2 + F I G U R E 1 1 HIGH-THRESHOLD C A - A C T I V A T E D K C O N D U C T A N C E IN A T Y P E 1 NEURON.. 3 0 2+ FIGURE 1 2 B L O C K A D E OF K + + CONDUCTANCE BY T E A APPLICATION (1 M M ) TRANSFORMS SPIKE BURST FIRING OF A T Y P E 1 NEURON INTO PLATEAU POTENTIAL GENERATION 3 2 FIGURE 1 3 NA -ACTIVATED K + + CONDUCTANCE IN A T Y P E 1 WITH C A - F R E E A C S F A N D C O 2 + 2 + (1 M M ) FIGURE A 1 MAIN PANEL NEURON DURING PERFUSION 3 6 5 9 FIGURE A 2 SUBPANEL "CONTINUOUS D A Q " 60 FIGURE A 3 "BUILD P R O T O C O L " VI 61 FIGURE A 4 "BUILD PROTOCOL" VI GENERATION FIGURE A 5 IN THE MODE OF THE C H I R P - T Y P E STIMULUS 62 "BUILD PROTOCOL" VI GENERATION IN THE MODES OF THE S I N E W A V E - T Y P E STIMULUS 63 FIGURE A 6 " C H A R T R E C O R D E R " VI 6 4 FIGURE A 7 " P R O T O C O L D A Q " VI 6 5 FIGURE A 8 " P L A Y BACK" VI FIGURE A 9 " D S P " VI 67 "FIT" VI 6 9 FIGURE A 10 FIGURE A 11 " D X / D T " VI 6 6 70 xi ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Dietrich W. F. Schwarz without whose support and encouragement this work would not have been possible. I would like to thank Dr. Ernest Puil for his invaluable advice and his endless time on direction preparation of this thesis. during process of experiments and I 1. INTRODUCTION The nucleus principalis station trigemini (PrV) is the primary in the lemniscal pathway mediating somatosensory s i g n a l s from facial regions to the cortex. In mammals, the spinal trigeminal nucleus, consisting of subnuclei oralis, constitutes cortex central interpolaris, and c a u d a l i s , a second neuron system in the sensory pathway to the (Olszewski 1950). defined and distinct In the gerbil, the PrV is large, from the spinal nucleus, situated the brainstem and just rostrally to the bifurcation well- laterally in of the t r i g e m i n a l root (Ramon y Cajal 1910). The PrV ascending information receives fibers of mostly ventroposteromedial the by the primary fifth way nerve of the nucleus of the Torvik 1957; Williams et al. 1994). sensory input from short and, as an output, sends medial thalamus lemniscus to the (VPM; Jones 1 9 8 5 ; The PrV also receives afferent fibers from sensorimotor regions of the cerebral cortex (cat: Brodal et al. 1956), red nucleus (cat: Edwards 1972), periaqueductal and dorsal raphe nuclei (rat: Li et al. 1993). gray Hence, neuronal 2 operation and sensory transduction in the PrV are likely subject to conveys a various modes of control. The peripheral input, predominance of tactile although multimodal, sensory afferents the dorsal column nuclei. to the PrV, analogous to In the PrV of rodents which have w e l l - developed whiskers, barrelets occupy a large portion of the neuropil, representing patterns afferents convey from individual information vibrissae. about topographically in a point-to-point V P M , en route to the cortical vibrissal projection, barrel Their field discharge deflection, to the barreloids (Belford in and K i l l a k e y 1979a; Ma and Woolsey 1984; Van der Loos 1976; Woolsey 1970). Developmentally, such maps are present first in PrV at birth, and later in V P M and cortex (Belford and Killakey 1979b; Erzurumlu and Killakey 1983; Killakey and Belford 1979). Peripheral stimulation neurons that electrical involve stimulation produces single unit responses in P r V a variety of the phasic and tonic Smith 1960). properties. of the skin, the patterns rapidly or slowly adapting afferent for firing Following of impulses from fibers, however, cannot account response modes of PrV neurons (Darian- The input-output transformations, therefore, infer the 3 e x i s t e n c e of different cell c l a s s e s . nonmonotonic s t i m u l u s - r e s p o n s e certain neurons, showing beyond the strength the spike rate. that Darian-Smith an i n c r e a s e an inhibition ('surround' in receptive projection neurons and interneurons hand, repertoire of stimulus fields). membrane that reduction and s p a c e a p r e s e n c e of (Mountcastle patterns properties, of types the of above, the neurons. morphology, connectivity, PrV, is there a On t h e reflect according to various a P r V should contain neuron's knowledge development and spike firing properties absence of its of functionally information neurons. are not stations. conductance For e x a m p l e , an interaction with Ca 2 + and K + of conductances in the work, we primary properties sensory a persistent is the about In this known to occur in neurons of other of patterns report on three distinct cell types with certain m e m b r a n e relay distinct 1984). can Despite a detailed surprising electrophysiological that reduced this adaptation) This implies response probability, is (phasic intensity of voltage- a n d ligand-gated c o n d u c t a n c e s . In view different over time different contributions in interpretation represents other described curves for the p h a s i c r e s p o n s e s o f needed for maximum firing A classical (1960) Na + particularly interesting because it endows the capability limited by a long-lasting PrV neuron with a bursting inhibition. 2. M E T H O D S Using isoflurane, (Meriones unguiculatus) we deeply anesthetized Mongolian gerbils aged between 11 and 17 days (P11 to P17). After decapitation, the brain was removed and immersed in i c e - c o l d artificial cerebrospinal fluid (ACSF) for 1-2 min. into two with a quasi-sagittal The brain was cut (i.e., a 20 to 30° horizontal deviation from the ideal sagittal plane) incision and further trimmed to form a block containing brainstem and caudal cerebellum. For making slices (with a Vibratome), oriented rostro-laterally to ventro-caudally, we used the the tissue block brainstem. The cut thickness. Slices, trigeminal nucleus temperature that contained slices were containing (PrV), the between a visually were submerged larger portion 300 and 350 identifiable into u,m in principal A C S F at (23 °C) and allowed to recover for at least A C S F contained of room 1 h. The (in mM): NaCI, 125; KCI, 2.5; CaCI , 2; MgCI , 1; 2 2 glucose, 25; NaHC0 , 25; N a H P 0 , 1.25 and was saturated with 95% 3 0 2 4 and 5% C 0 which maintained the pH near 7.4. Low [Na ] s o l u t i o n s + 2 2 were made by partially substituting 'Na-deficient' NaCI with choline chloride. perfusion, the [NaCI] in the 1/4, 1/8, For and 1/16 NaCI solutions were, respectively, 57.5, 42.1, and 34.1 mM. For C a f r e e 2 + A C S F , 2 mM CaCI was omitted or substituted 2 with 1 mM CoCI , or 1 2 mM CdCI . 2 For whole-cell recording (Blanton et al. 1989; Strohmann et a l . 1994), we used patch electrodes, borosilicate glass with filament pulled from (WP Instruments) thin-walled and filled with a solution containing (in mM): K-gluconate, 115; ethylene glycol-bis(paminoethylether) A/,A/,A/',A/'-tetraacetic acid hydroxyethylpiperazine-A/'-2-ethanesulfonic (EGTA), acid 10; A/-2- (HEPES), 10; MgATP, 4; NaGTP, 0.3; and KCI, 20. The pH was adjusted to 7.25 w i t h KOH. The signals, recorded in the current-clamp 2B, Axon Instruments), kHz with Instruments were filtered a data acquisition custom-made data acquisition developed with Labview Instruments) (Appendix 1). at 3 kHz and sampled at board (16 Corp.) in a Macintosh mode ( A x o c l a m p - bit resolution, Quadra 950 computer National running and processing programs which instrumentation software 5-10 were (National The electrode resistances ranged from 7 6 to 9 MQ and access resistances were <100 MQ. All experiments were conducted at room temperature (22-24 °C). In eleven experiments, pipette solution (5 mg/rhl). was withdrawn from the neurobiotin was added to the At the end of the recording, the pipette neuron and the slice was immediately, with 4% paraformaldehyde in phosphate buffer (pH = 7.2) containing 20% sucrose. The fixed neurobiotin were visualized using an avidin 1988). cells under observations microscope using a camera lucida attachment (Zeiss). kit, tetrachloride (Sigma) as a chromogen (Horikawa and Armstrong from were biotinylated A B C Elite Vector Laboratories) and 0.05% 3,3'-diaminobenzidine reconstructed solution Neurons f i l l e d horseradish peroxidase (HRP) complex (Vectastatine were fixed, brain slices frozen and resectioned at a thickness of 75-90 urn. with patch Stained a light 7 3. Results 3.1 Identification of nucleus principalis In quasi-sagittal principalis light as trigemini an dorsoventral cerebellar was rostral spinal opaque, pale direction. Its the brainstem, ovoid location Anatomical structure, to the that and ventral to the PrV, as well nucleus which caudally the PrV (Figure 1, A-C). the inferior its nerve w h i c h as the cigar shaped, descended in the these brain (n = 6 ) , to confirm identity of the PrV and surrounding structures. of recorded neurons provided confirmation in helped in We also studied frozen sections, cut from slices and Nissl-stained with cresyl violet nucleus or r e f l e c t e d elongated was ventral landmarks neurons the were the sensory root of the trigeminal trigeminal brainstem. of (PrV) appeared under translucent peduncule. identification sections trigemini (PrV) the Neurobiotin s t a i n i n g of their location within Figure 1. Camera lucida drawings of neurobiotin-stained neurons in thenucleus principalis trigemini. A: type 1 neuron. B: type 2 neuron. C: type 3 neuron. Scale, 100 u.. 9 We recorded from 62 neurons in the three types on the basis of neurobiotin PrV and distinguished staining and firing modes. The initial membrane potentials were similar, falling range (-52 of the neurons w e r e to -58 mV). More than one half spontaneously active, frequently (type 1; Figures 1A, 2; n = 33). firing action into a narrow potentials in bursts Neurons of another group fired repetitive action potentials only in response to depolarizing pulses (type 2; Figures 1B, 4A; n = 23). types 1 and 2 neurons afterhyperpolarizations (AHPs). large, long-lasting morphological features. Both somata and dendritic trees extending mainly along one axis (cf. Figure 1A,B). fired of It was not possible to d i s t i n g u i s h types 1 and 2 neurons had multipolar "tonically" current The action potentials included type 1 from type 2 neurons by their neurons single action potentials Another group of with fast AHPs in response to depolarizing current pulses (type 3; Figures 1C, 5A; n = 6). Type arborizations 3 neurons had more expansive, than types 1 or 2 neurons. dendritic In each type, the soma usually was elongated, sometimes triangular about 10-15 (im (long axis). radial in shape, measuring 10 3.2 Physiological properties Firing patterns of PrV neurons of type 1 neurons. In response to depolarizing current injection type 1 neurons started firing with a burst consisting of doublets, i.e., a superposition of two action on a slower hump (n = 33). Because spontaneously active (Figure 2B), we injected hold cells at potential (V ) of -60 investigation injections. Figure 2 shows that depolarization pulses elicited responses to to mV for current a pulse to threshold with one or two bursts of action potentials. increase in the depolarizing current initial burst, were a constant current systematic current their potentials neurons mV, -65 mV, or -70 h of these pattern, An decreased the latency to the as well as the duration of the hump and the amplitude of the AHP. The primary spike burst remained steadfast secondary doublets eventually disappeared and the firing while the of s i n g l e action potentials increased (Figure 2A), with an increase in current pulse magnitude. Eleven type 1 neurons fired a burst of three s p i k e s ("triplet") at the onset of a depolarizing current pulse, followed a doublet and/or single spike firing. by Only one neuron fired a burst of four action potentials. For all type 1 neurons, the burst duration w a s <21 ms and the intraburst frequency was between 100 and 250 Hz. Figure 2. Characteristic firing behavior of type 1 neuron(holding potential, V = -60 mV). h A: voltage responses evoked by 500 ms current pulses of increasing amplitude (right). B: spontaneous activity of a type 1 neuron at its "resting" membrane potential ( V = 0). Note that the neuron fired mostly doublet and triplet action potentials h (not shown) and that variations in amplitude reflect low sampling rate (500 Hz). 12 3.2.1 Inward rectification in type 1 neurons The injection duration) into exhibited a large depolarizing of type 1 hyperpolarizing neurons depolarizing current such a s l H evoked (Figure eliminated the voltage current-voltage 3C), c o m p l e t e l y b l o c k e d the pronounced r e c t i f i c a t i o n -70 m V , a n d developed fully mV. K rectifier + 1994) s e e m e d unlikely in our e x p e r i m e n t s reversal potential w a s -98 mV. C s - b l o c k a d e , w e c o n c l u d e d that + produced the inward H rectification and - 1 0 0 m V in type 1 neurons. H this of C s (2 mM) + A s evident Cs in t h e application + that a c t i v a t e d IR near ( V ) of - 1 0 0 m ( l ; Travagli and G i l l i s b e c a u s e the c a l c u l a t e d K From the a c t i v a t i o n an l a (McCormick and by a membrane potential A contribution of an inward of We investigated 1 neuron. (Figure that an a c t i v a t i o n the a p p l i c a t i o n s a g in a type relationships 3A). (500 m s responses blocker of l + Figure 3 B s h o w s that pulses voltage s a g , implying possibility by applying C s , a s e l e c t i v e P a p e 1990). current w a s the major in a voltage + voltage a n d current that range between - 7 0 Figure 3. Inward rectification, evoked by hyperpolarization, and rebound firing in type 1 neuron (V = -60 mV). A,B: voltage responses evoked by hyperpolarizing h current pulses of increasing amplitude under control conditions (A) and during C s application (B) show blockade of inward rectification and increased latency to rebound firing. C: current-voltage relationships for early and late responses (measured as indicated in A,B) to current pulse injections show blockade of inward rectification by + Cs . + D: dependence of latency to first action potential of rebound on amplitude of current pulse amplitude under control conditions and during C s application. + Os abolished the voltage-dependence of the latency to rebound firing. + Note that 14 3.2.2 Contribution of inward rectification to spike generation in type 1 neurons A brief, hyperpolarizng depolarizing current hump pulses. remained at This "rebound the offset of response" led to a burst of action potentials and often a second burst or a single a c t i o n potential (Figure 3). The latency to the rebound response became progressively shorter with current pulse injections of increasing amplitude (Figure 3D). Extracellular C s application (2 mM) did not + significantly rebound change the amplitude (Figure 3B). However, or the number of spikes in the Cs + eliminated the voltage- dependence of the latency along with the sag (Figure 3C). Evidently, an l -like tail current did not greatly contribute to the amplitude of H the depolarizing rebound response but was an important contributor to the latency of firing emerging from hyperpolarization. 3.2.3 Firing patterns of type 2 neurons Figure 4 shows the characteristic depolarizing potentials current pulses, generating with slow AHPs. firing of a type 2 neuron t o a tonic pattern of a c t i o n As in the case of the bursting type 1 neuron, an increase in the current pulse injected into a type 2 neuron 15 A B 20 0 - 0 > If > E -20 E > E -40 -60 20- Amm > ^ 55 pA -20-40-60-80-100- 18 8 i—H 0.0 0.2 1 r 0.4 0.6 T T r 0.8 1.0 0.0 0.2 t (s) T T T T 0.4 0.6 0.8 1.0 t (s) Figure 4. Characteristic tonic firing behavior of type 2 neuron (V = -58 mV) h evoked by current pulses of increasing amplitude (right). A: depolarizing voltage responses. B: hyperpolarizing voltage responses show evidence of inward rectification. 16 produced a decrease in the latency to the first action potential, interspike interval and amplitude of the A H P (Figure 4A). Similarly, hyperpolarizing depolarizing membrane current voltage pulse displacement sag, resulting potential hyperpolarizing 4B). We did not produced a - 1 5 mV activated a In all type produced larger consisting of a single action potential hump. of that in a new steady-state (Figure pulses injections investigate level 2 neurons, rebound of the larger responses, on top of a slow depolarizing the ionic mechanism of the depolarizing sag or rebound responses in type 2 neurons. 3.2.4 Firing patterns of type 3 neurons Figure 5 shows the characteristic neuron depolarized distinguishing by current of fast firing injection feature of the action potential was a biphasic A H P , consisting Figure 5C). pulse tonic of a type 3 (n = 6). A in this type of neuron and slow components (cf. Type 3 neurons exhibited little, if any, adaptation during the tonic firing to depolarizing current pulses of 0.5 s duration and no voltage sag in the hyperpolarized range (e.g., to V = -100 mV). m We did not observe rebound responses at the offset of current in type 3 neurons (Figure 5B). pulses t (s) 0.0 C 0.2 0.4 0.6 t (s)" 0.8 1.0 -35-, 0.28 0.32 . 0.36 t (s) 0:40 Figure 5. Characteristic tonic firing behavior, biphasic afterhyperpolarization, and hyperpolarizing voltage responses of type 3 neuron (V = -58 mV) evoked h by current pulses of increasing amplitude (right). A: depolarizing responses. B: hyperpolarizing responses. C: faster time scale shows afterhyperpolarization with fast and slow components, from an action potential marked by the asterisk in A. 18 3.3 Plateau potentials in type 1 neurons During the course of these (n=3) found that a depolarizing of three or four action until the towards potential potentials plateaued with for r e - a p p e a r e d , abruptly 6B). W e also we occasionally pulse produced an o s c i l l a t o r y observed burst d e c r e a s i n g peak a m p l i t u d e s a variable the end of the plateau, a s i m i l a r amplitude (Figure investigations, period (-100 ms); oscillation of i n c r e a s i n g ending in c o m p l e t e repolarization a replacement of initial burst r e s p o n s e s to injected p u l s e s by s u c h "plateau potentials" a n d then, a resumption of the regular type 1 firing behavior. In contrast, the plateau potentials were not apparent in the types 2 and 3 n e u r o n s . W e a s s u m e d that the ability of type plateau 1 neurons, with potentials may relate to bursting a s p e c i a l role in P r V function, p r o c e e d e d to investigate their ionic m e c h a n i s m . and 1 20 - Control 0 > E -20- E > -40-60- \ A —AA w I F J I 0.08 i i 0.12 0.16 1 t B 1 V i 1 0.20 i 0.24 (S) 200 - > -20- £ > -40-60- 9 i 0.0 1 1 1 1 0.2 0.4 0.6 0.8 pA r~ 1.0 (s) t 120 CO l 0.0 1 1 0.2 0.4 1——I 0.6 t (s) 0.8 r~ 1.0 E 100H o c CD H—* co 80H 60' 800 400 t (s) 2+ Figure 6. Type 1 neurons generate plateau potentials during C a -free perfusion. A: time course for plateau potential development (V = -63 mV). The records to h the same current pulse (27 pA) were obtained at the indicated times, before and 2+ after starting C a -free perfusion. B: current pulse-evoked plateau potential response in a different neuron under normal ionic conditions (inset is on faster time scale). C a reduction in latency to the first spike in the burst accompanies 2+ the development of the plateau potential during time of C a -free perfusion (as in A). The plot shows the latency as a function of time after initiation of perfusion without C a . 9 20 3.4 Ionic mechanism of plateau potential generation in type 1 neurons 3.4.1 Relationship of plateau potential firing to extracellular C a On perfusion of Ca -free A C S F to reduce C a 2+ 2 + 2+ influx (n = 4), w e observed a dramatic change in the firing pattern of the type 1 neuron evoked by depolarizing current pulses. [Ca ], the neuron fired 2+ potential (Figure a doublet 6A, Control). During perfusion with 2 mM followed During by a single the C a - f r e e 2+ action perfusion, however, we observed a progressive reduction in the latency of the initial action potential, potential (Figure 6A,C). enhanced bursting, After and onset of a plateau ~2 min. of perfusion with the C a 2 + free A C S F , the neuron fired triplets followed by a doublet 6A, 130 s). By 200 s, the neuron fired an initial burst comprised of four spikes and AHPs of decreasing amplitude followed burst and an action potential neuron transformed (Figure 6A). The initial into a plateau at V (Figure 6A, 370 s), although m (Figure by another burst of the = ~-20 mV for - 1 0 0 ms the plateau duration varied greatly between experiments. We performed similar experiments using blockade of conductance by perfusion of Ca -free ACSF containing C o 2+ 2+ Ca 2 + (1 mM; n 21 = 16). Figure 7 shows the effects changes in firing of this blockade which produced behavior of a type 1 neuron, similar to those of C a - f r e e perfusion but with a much more rapid transition from 2+ normal firing min., mode to the plateau potential. we observed a well-developed - 2 0 0 ms at V m the Thus, in less than 3 plateau potential, lasting for = - - 2 0 mV (Figure 7, 160 s), and in less than 4 min., its duration increased to nearly 500 ms (Figure 7, 200 s). neurons, application of Co 2 + (1 mM) in C a - f r e e A C S F for 2+ minutes resulted in plateau potentials duration of the depolarizing current conducted a set of experiments [Ca ] (0 mM) in the A C S F with C d 2+ (n = 6; not shown). In s e v e r a l that were longer than pulse (not using partial 2 + shown). substitution (1 mM) with very similar 6-8 the We a l s o of the results 2+ Figure 7. Time course for development of plateau potential under Ca -free and 1 mM C o conditions (V = -60). Note prominent oscillations on plateau during perfusion for 200 s. 2+ h 23 3.4.2 Involvement of a persistent N a + conductance Based on the data obtained during C a - f r e e perfusion without 2+ and with Co persistent or C d , we hypothesized that 2 + 2 + Na motoneurons + conductance, (Llinas such and Sugimori as Purkinje 1980a,b; 1980), produced the plateau potentials. potentials in of a cells Schwindt and and C r i 11 As a test, we evoked plateau by applying C o i n C a - f r e e A C S F and then, tetrodotoxin 2 + 2+ (TTX) to block the persistent N a conductance. After + duration, plateau potential in <1 min. evoking a l o n g - (Figure 8A, Control), the application TTX (600 nM) annihilated the plateau potential potentials the activation (not shown). and the fast We also of action applied lower concentrations of TTX, bearing in mind that lower concentrations the hydrophilic toxin would take longer to saturate the tissue. of The application of 60 nM TTX caused, after 40 s, a division of the plateau potential into two shorter depolarizations (Figure 8A). subsequent 3 min., there was a progressive reduction and duration, and after 6-7 min., an elimination in amplitude of the potential, leaving behind a burst of two action potentials reduced amplitude (Figure 8A). Over the plateau of s l i g h t l y With this concentration, then, it w a s possible to annihilate plateau potentials without markedly affecting 24 ->—r 300 19 pA 0.0 0.2 0.4 0.6 0.8 1.0 t (s) Figure 8. Blockade of persistent Na+ conductance in a type 1 neuron eliminates plateau potential and post-pulse hyperpolarization (PPH). A: time course of T T X application 2+ 2+ (60 nM) under C a -free and 1 mM Co conditions shows splitting of plateau potential into two (40 s), a shortening of its duration (160-240 s) and complete blockade (340 s). After 420 s, TTX application blocked the burst of 3 action potentials (incompletely resolved) present at 340 s. B: changes in temporal sum of P P H ( W ^ , see Eq. 1 and text) measured in same neuron as a function of time of T T X application. 25 the neuron's ability to fire action potentials. slope of the rising phase of the fast action potential after a total blockade of the plateau potential potentials disappeared 5-6 A reduction min. after was apparent with TTX. The action TTX application. findings show that small changes in the persistent due to TTX application in the can produce dramatic These N a conductance + changes in the f i r i n g behavior of the neuron. 3.4.3 Sensitivity of plateau potentials to T T X . We investigated TTX blockade the sensitivity by performing of the plateau potentials concentration-response (Figure 9). First, we changed the firing perfusion of Ca -free solutions with C o 2+ potentials then with depolarizing current applied TTX at different experiments mode of type 1 neurons by 2 + (1 mM) and evoked plateau pulses (500 ms, duration). concentrations for long-lasting effects. criterion For quantification behavior, we used a ratio TTX and control conditions. We 6-10 min. Application of TTX for 10 min. allowed observations of full which we considered an important to recovery for assessment of i t s of effects on f i r i n g of slopes of the plateau potential under We obtained the slope from a linear f i t of the voltage points between the local minimum after the second 26 t (s) Figure 9. Reduction in slope of plateau due to T T X blockade of persistent N a 2+ + 2+ c o n d u c t a n c e in a type 1 neuron under C a -free and 1 m M C o conditions. A : superposition of plateau potentials s h o w s the more rapid descent of the plateau, o b s e r v e d at - 1 0 min of T T X application in 0.6, 1.2, and 1.8 n M concentrations. Solid lines are a linear fit to voltage response between the local minimum after the s e c o n d spike at the start of the plateau and the local minimum before the plateau terminated in a rapid repolarization. B: concentration-response relationship for T T X effects on the s l o p e of the plateau (as in A). E a c h point (± S E ) represents 8 to 11 m e a s u r e m e n t s taken from e a c h neuron (n = 5) and solid line represents a Langmuir m o d e l fit. 27 action potential in the initial oscillation, and the local minimum just before termination of the plateau potential. Figure 9A shows that TTX application increased the latency to the first spike and decreased spike magnitude and duration dependent manner. amplitude of the plateau itself, as well However, the slope of the plateau decay provided of TTX action. shows that the concentration-response relationship a Langmuir the in a c o n c e n t r a t i o n - a more sensitive, reproducible indicator with as model, representing the binding Figure 9B is of consistent TTX to N a + channels. 3.4.4 E x t r a c e l l u l a r replacement of N a As additional confirmation N a conductance, we investigated + replacement with choline + with choline for an involvement the effects during blockade of C a mM C o . 2+ 2 + In contrast the plateau (Figure 10). 2+ to TTX, the + As pulses to induce the plateaus effects of extracellular 1 Na + Thus, a sequential reduction of the N a + caused corresponding reductions in the amplitude potentials Na currents with C a - f r e e A C S F containing deficiency were unspecific. concentration persistent of extracellular on plateau potentials before, we used depolarizing current of as well as action potentials. of These 28 200 - > Control -20- E > -40-60- [Ca ] = 0 mM, 2+ [Co ] = 1 mM 2+ Choline CI Replacement 1/4 [NaCI] 24 pA l 0.0 1 1 0.2 0.4 1 1 T 0.6 0.8 1.0 - t (s) Figure 10. Effects of reduced extracellular [Na ] on plateau potentials e v o k e d 2+ 2+ during perfusion without [ C a ] and with 1 m M C o . Choline CI w a s used to replace NaCI in the A C S F . All measurements were m a d e at - 1 0 min after exchanging the perfusion. Note the systematic reduction in plateau (and spike) amplitude, in contrast to the reduction in duration o b s e r v e d under T T X (cf. Figure 8). + 29 observations are in good agreement with the expectations based on the calculated change in the N a reversal potential due to the change + in extracellular [Na ]. + At low N a concentrations, the duration of the + plateau was determined by the injected current pulse (cf. Figure 1 0 , 1/8 and 1/16 [NaCI]). Therefore, the terminal plateau may require a critical extracellular repolarization [Na ]. + In summary, the marked reductions in the amplitude of plateau potentials deficiency in the extracellular TTX provide strong [Na ] and their + evidence for a Na due to a complete blockade by involvement, + of the likely a persistent N a conductance. + 3.4.5 Involvement of Ca -dependent rectification 2+ From our results it seemed likely that activation of Ca 2 + currents in type 1 neurons did not cause and, indeed, prevented the formation of plateau potentials. why a blockade of C a 2 + holding the neuron at V m influx We investigated supported their possible reasons generation. While near -60 mV, we applied TTX (600 nM), 4 - aminopyridine (4-AP; 0.5 mM) and tetraethylammonium (TEA; 10 mM) in order to unmask this possible C a in Figure 11 A, depolarizing 2 + current conductance (n = 5). As shown pulses evoked voltage during the initial 200-300 ms of the responses. humps The current-voltage 2+ ' + Figure 11. High-threshold C a -activated K c o n d u c t a n c e in t y p e l neuron. A : voltage r e s p o n s e s evoked by 500 ms depolarizing current pulses of increasing amplitude (36, 4 1 , 54, 5 8 , 6 3 , 68 and 72 pA) during N a and K c o n d u c t a n c e b l o c k a d e by application of T T X (0.6 m M ) , T E A (10 mM) a n d 4 - A P (0.5 m M ) . B: current-voltage relationships for peak and steady voltage r e s p o n s e s (as in A ) . 2+ C : a s in A , but during application of Ni (0.6 mM) which completely blocked inward rectification. + + 31 relationships for the peak and steady voltage responses (Figure 11B) show that this application rectification of N i 2 + activated at - 4 0 mV. E x t r a c e l l u l a r (600 mM) completely blocked the r e c t i f i c a t i o n (Figure 11C). These data are consistent transient, high-threshold Ca contribute to the initiation 2 + with conductance. the activation of a This rectification may of the plateau potential and have some bearing on its limitation. 3.4.6 Involvement of Ca -activated K 2+ + conductance We considered the possibility that a C a the influx necessary for activation 'controlling conductance provided 2 + of a K conductance and hence, a + A H P ' . For. example, a plausible plateau potential is that the blockade of C a activation of a K conductance that normally + explanation 2 + for the entry prevented the repolarized the neuron. As a check, we applied TEA (1 mM) to see if a K -channel blocker + also produced a propensity caused by a blockade of C a course of effects mM). The first for plateau generation, similar 2 + influx. Figure 12 illustrates produced by extracellular application sign of TEA action on the firing neuron depolarized with a current to t h a t the t i m e of T E A (1 behavior of the pulse was a conversion of the initial burst of two spikes into a broader hump crowned by fast 32 20H 38 pA 0.0 0.2 0.4 0.6 0.8 1.0 t (s) Figure 12. B l o c k a d e of K conductance by T E A application (1 mM) transforms s p i k e burst firing of type 1 neuron into plateau potential generation ( V = -57 m V ) . T h e s a m e pulse amplitude w a s used for the recordings before and after the indicated times after initiation of T E A - a p p l i c a t i o n . + h 33 spikes (Figure repolarization, potentials. the 120 s). of 12, repolarization, spikes resulted from a blockade 160 s). By as reflected transformed action this exaggerating time, the initial TEA application burst delayed in a 20-30% increase in the duration of the burst (not shown). into plateau potentials the bursts (Figure 12, 260 s). of Such bursts whereas doublets later, bursts and plateaus, replaced the single action potentials followed of depolarizing hump became broader and increased, the second action potential slowly This also evident from the reduced AHPs of single Later, the initial number (Figure 12, These effects and that of TEA are comparable to the changes in the firing pattern of the same types of neurons deprived of external C a the concept that a C a potential 3.5 activated (cf. Figures 6 and 12) and support K conductance regulates + plateau expression. The post-pulse An 2 + 2 + abrupt hyperpolarization repolarization (PPH) terminated the plateau potentials in type 1 neurons (Figures 7, 8). for the sudden repolarization remains unclear. initial resting potential Na -dependent, + The exact reason The undershoot of the resulted in a long-lasting hyperpolarization 34 after termination the of the stimulus PPH occurred blockade of activation K during blockade conductances + pulse (PPH; Figure 8A). of Ca influx 2 + by TEA, we but Because not hypothesized during that the of a Na -dependent K conductance (cf. Bader et al. 1 9 8 5 ; + + Schwindt et al. 1989) produced the PPH. In order to estimate the effect of the N a conductance blockade on the P P H size, we measured + the temporal sum of the P P H amplitude ( W PPH ) which is (1) where V is an initial value and V (t) is membrane potential h given time. The integral was taken from the point where repolarized to V (t = 0), to the end of the recording («>). h reduction of Na + influx due to TTX blockade corresponding reduction in the P P H (n = 4). course of the reduction roughly to the at a m in the V m Indeed, a nM) caused a Figure 8B shows the time of the normalized W decrease (60 the plateau PPH which corresponded potential during TTX application. A long-lasting hyperpolarization dependent manner, following absence of a plateau hyperpolarization a depolarizing potential. at the offset also occurred, in a v o l t a g e current We used Eq. 1 to of depolarizing pulse in quantify pulses that the the evoked 35 sub- and suprathreshold responses under conditions of blockade of Ca 2 + influx, before and after TTX application (n = 4). For this s e r i e s of experiments, we only partially blocked N a conductances with l o w + concentrations (e.g., 5 nM) of TTX. At 15-20 min. of the TTX application, the neuron did not generate a plateau potential but s t i l l was able to discharge one or more action potentials. Figure 13 shows the results of such an experiment in a type 1 neuron where V = ~-70 mV. In this neuron, a substantial h of the PPH was apparent during TTX application pulse amplitude exceeded 25 pA (Figure 13A.B). TTX had little or no effect subthreshold voltage effects on the of TTX were pronounced with when the mV (Figure larger current evoked plateaus, bursts, and single action potentials. also observed these effects current The application hyperpolarization responses below -60 reduction of following 13C). The pulses t h a t Note that when the voltage responses were we still under the threshold for a plateau potential (—48 mV) in this neuron. These observations provide strong support for a N a activated conductance mechanism in the P P H . + K + 36 0.7 0.8 1.0 0.9 1.1 1.2 t (s) -70 -65 V m -60 (mV) Figure13. N a - a c t i v a t e d K conductance in a type 1 neuron during perfusion 2+ 2+ with C a -free A C S F and C o (1 m M ) . A : temporal s u m of post-pulse hyperpolarization (Wp^, s e e Eq.1 and text) a s a function of current pulse amplitude during control conditions and T T X application (5 nM). B: e x a m p l e s of hyperpolarizations s h o w n at high gain in Control (1 in A) and T T X (2 in A) conditions. C : d e p e n d e n c e of Wp-,. on the level of subthreshold depolarization. + + 37 4. DISCUSSION In this principalis first study trigemini of the electrophysiology neurons, we distinguished neurons (types 1, 2, and 3), according to their current-pulse repetitive injections. (tonic) classes firing in response to depolarizing strength. of This currents, in receptive fields (cf. of at stimulus-response of which, classically, is the basis for quantifying neurons of responses to DC and may represent a simple rate code for the intensity sensory stimuli responses three nucleus In all neurons, we observed a pattern rates that depended on current relationship of the thalamo-cortical levels, Mountcastle 1984). 4.1 Depolarizing responses in types 2 and 3 neurons The simplest transformation count / intensity depolarization pattern. of an input current into a s p i k e - code was evident with a ramplike in type 3 neurons. slope preceded Because this slope increased with a regular current orderly inverse relationship resulted between stimulus latency. A slow firing magnitude, an strength and The responses of type 2 neurons to depolarizing currents 38 were similar, except that they exhibited slow AHPs and, therefore, somewhat lower firing rates. In type 3 neurons, the biphasic AHPs led to complex depolarizing slopes that preceded repetitive firing of single rate action potentials and seemed to control the and regularity of firing (e.g., Figure 5). 4.2 Hyperpolarizing current pulse injections into PrV neurons Types 1 and 2 neurons exhibited a rectifying, in their type responses to hyperpolarizing current depolarizing sag pulses. 3 neurons responded to such pulse injections changes that appeared passive, possibly influenced dendritic neurons tree. After a hyperpolarizing responded with potentials a rebound occurred at shorter In c o n t r a s t , with by a radiating pulse, both type depolarizing voltage hump. 1 and 2 Action latencies on top of the hump a f t e r larger hyperpolarizing pulses. The voltage sags and rebounds w e r e reminiscent of an I -Iike rectification H that would tend to limit (McCormick and Pape 1990) long hyperpolarizations. In type 1 neurons, we found that Cs application, a blocker of l , eliminated the sag a s + H well as the voltage dependence of the latency for the rebound 39 response. It is likely, then, that l contributes H to post-inhibitory rebound responses in type 1 neurons. 4.3 Classification of types 1 and 2 neurons In many aspects, types 1 and 2 neurons were similar. However, type 1 neurons had other outstanding features that invited a detailed examination of their electrical behavior: burst firing of two potentials; and, hyperpolarization. classification + As spikes a in conjunction long-lasting, discussed below, with these 1" relate to the activation functional indistinguishable states plateau criteria for of a p e r s i s t e n t neuronal of one class. (2) post-excitatory If it were possible to regulate this morphologically represent four (3) as "type N a conductance. these to (1) spontaneous firing; conductance, groups Our results, could however, suggest the separate classification of types 1 and 2 neurons. 4.4 Burst firing in type 1 neurons In these slice preparations, type 1 neurons fired spike bursts spontaneously. In response to neuron characteristically started depolarizing with current a brief burst injection, a discharge, 40 followed by a tonic pattern of single action potentials. The burst discharge consisted of 2-4 action potentials superimposed on a s l o w depolarizing hump. The application depolarization and eliminated of TTX blocked the action potentials. the slow A slow, Ca 2 + dependent depolarization was apparent, during blockade of both N a and K c u r r e n t s (cf. Figure 11). Under these conditions, + of N i 2 + application annihilated the slow depolarizing response to current injection variations which had a threshold of —40 mV. This may pulse reflect in a Ca -channel protein combination that binds to N i 2+ (cf. Zamponi et al. 1995) or a distribution + 2 + of Ca -channels in the 2+ dendrites, which are located remotely from a likely somatic site of recording. While such a transient Ca 2 + current may provide a minor contribution, the spike burst is largely attributable to the activation of persistent and transient N a conductances. + 4.5 Is the burst firing pattern of type 1 neurons representative of PrV neurons in vivo? In the early extracellular Smith (1960) observed that investigations of the PrV, D a r i a n - some neurons always started response to a stimulus with a spike burst but dismissed this their pattern 41 as evidence for possible cell damage. increase in the intensity In the in vivo studies, of electrical cutaneous (lip) stimulation did not change the interval between the two action potentials initial burst. burst to Much stronger appear at shorter occurred repetitively similar to the depolarizing further current we representative latencies; responsiveness of single rate. typel action the suggest in that vivo the and neurons in vitro striking evidence for the firing the potentials This behavior is very injected pulses of increasing amplitudes. of in the caused these spikes after at a greater firing comparison problematic, stimuli an Although data may similarities patterns with a be provide of PrV neurons in vivo. 4.6 The plateau potentials of type 1 neurons A distinguishing feature of type 1 neurons was an ability generate a plateau potential. In many respects, this similarities plateau (Llinas to the somatic and Sugimori latency on stimulus from the stimulus 1980a), amplitude; amplitude; potential including (1) of potential Purkinje has cells a dependence of (2) an independence of its (3) plateau oscillations, to its duration usually of 42 inactivating action potentials; a membrane potential amplitudes. cell of and, (4) a relatively ~-20 mV, despite the various Also, TTX-application plateau potentials. fixed plateau a t stimulus blocked both PrV and P u r k i n j e The Ca -dependent plateau potentials 2+ in cerebellar and spinal neurons (Hounsgaard and Kiehn 1989; Llinas and Sugimori 1980b), and the Na -dependent plateau + potentials in striatal and hippocampal neurons (Hoehn et al. 1993) are i n s e n s i t i v e to high TTX-concentrations. without significant concentrations The elimination alterations provides firm to action of plateau potentials potentials by low TTX evidence that their generation in P r V neurons requires the activation of a persistent N a current. + 4.6.1 Influence of cation currents Occasionally, depolarizing stimuli in neurons minimized under Ca 2 + Ca -activated currents + 1984), extracellular with K currents 2+ Sedlmeir normal elicited plateau conditions. potentials When w e Ca-free, Co -application, or blocked 2+ with such stimuli T E A application always (cf. Galvan and evoked plateau potentials. During the presumed, progressive reduction of these currents, was a gradual prolongation there of the plateau (cf. Figures 7 and 12). Therefore, Ca -dependent currents may contribute to the generation 2+ 43 and limit the duration of the plateau. conditions, an interaction Na + currents 2 + 2+ + would produce a depolarizing of a hyperpolarizing input. currents extracellular of C a , Ca -activated K , and p e r s i s t e n t spike burst in response to an excitatory interactive Under normal hump, causing a b r i e f stimulus or, on t e r m i n a t i o n In view of possible modulation of these (cf. Schwindt et al. 1992), modulate various aspects of plateau potentials mechanisms t h a t seem likely under physiological conditions (cf. Zheng and Gallagher 1995). In their investigations of Purkinje cells, Llinas and Sugimori (1980a) observed an increased plateau magnitude after conductances with Ba replacement 2+ of blocking K extracellular Ca + or 2 + intracellular application of high TEA-concentrations. We observed a reduced plateau conditions magnitude reduced extracellular by TTX. These findings in PrV neurons [Na ] or partial of blockade of N a conductances + directly under + support the possibility (Llinas and Sugimori 1980a) that the plateau may represent a balance of N a and + K + currents. 4.6.2 Repolarization of plateau potential What terminates the plateau in such an abrupt have observed that a reduction of C a 2 + influx manner? We causes a prolongation 44 of the plateau (Figures 6, 7). The application of TEA also gradually prolonged the total plateau time. Therefore, one explanation termination of the plateau involves activation of a K internal Ca . 2 + activates + conductance by Another is that the depolarization-initiated a similar, repolarizing K conductance. + for Na influx + Neither a c r i t i c a l intracellular [Ca ] nor [Na ], alone, can explain the decreased plateau 2+ duration when + Na influx + was gradually conditions where there is very little Ca reduced influx 2 + by TTX, under (cf. Figure 8). Oi reduction of the "persistent" N a influx, the K currents that balance + + with a persistent Na current to limit + mV, are sufficient justify the plateau potential to repolarize the neuron. a hypothesis that a combination current with These considerations of a voltage-dependent a Ca -dependent, or a Na -dependent, 2+ to ~ - 2 0 + K + K + current repolarizes the plateau. 4.7 Post-pulse Whatever hyperpolarization its exact (PPH) mechanism, the repolarization plateau potential presumably relates to the hyperpolarization occurred on termination of a depolarizing current of the which pulse (post-pulse hyperpolarization or PPH). The PPH was not likely a consequence of 45 the sole activation first of a voltage-dependent K conductance. In the + place, the TTX application, blocked the PPH in a voltage expected for the activation of the persistent N a current. voltage range where the TTX-sensitive Indeed, + blockade of the PPH required only low concentrations range of TTX. The PPH activated includes membrane depolarizations that were subthreshold for the generation of plateau potentials. We observed that the temporal sum (or area) of the P P H correlated roughly to the magnitude of the stimulus pulse or depolarization. On the other hand, we did not observe PPHs during TEA-application activation which is consistent the hypothesis that of a K conductance and not, electrogenic + produces the PPH in these neurons. potential the N a pumping, + In axons, the equivalent of the P P H (hyperpolarizing afterpotential) reversal with is affected by changes in the K and extracellular TEA application, + in a manner that is predictable for an activated K conductance, but not a f f e c t e d + by inhibitors of electrogenic N a pumping (Poulter et al. 1995). + We conclude that a "persistent" increase in internal [Na ] (rather than in + voltage) during the depolarization triggered the activation conductance, producing the P P H in type 1 neurons. of a K + 46 4.8 Functional considerations in type 1 neurons The early burst of a doublet during a depolarizing input in type 1 neurons implies that the onset of a natural stimulus would receive emphasis. The spike firing within over a wide range of input current rate of firing the burst usually was constant amplitudes. Hence, the in the burst cannot provide coding for the amplitude, unlike the subsequent firing of single action It would seems probable that spike bursts occur responses to natural excitatory (and post-inhibitory) the tonic firing pattern the nature (or modality) brief excitatory from rapidly bursts. receptors fiber inputs. postsynaptic potentials adapting threshold inputs whereas duration of of slowly sufficient If rapidly adapting afferents and On the one hand, activity may evoke only On the other, the extended activities tonic firing. potentials. (EPSPs) mediating mechano-receptors could cause depolarizations stimulus as would depend on the stimulus of afferent initial spike adapting duration were to provide input for to type 1 neurons, the latency to the spike burst may represent the only available code for the stimulus intensity, consideration in somatosensory physiology. an uncommon A more likely is that type 1 neurons have specialized response patterns dynamic aspects of mechanical stimuli. The larger scenario for the EPSPs a r i s i n g 47 from the faster the burst vibrissal latency and automatically frequency for vibratory follicle) strict afferents relationship deflections, inputs. for example, would decrease tune system Since the vibrissal are often directionally for the higher (and other sensitive, the hair relatively between the magnitude of hyperpolarization the latency to a rebound burst and response (Figure 3) would tend to emphasize certain aspects of stimulus dynamics. It had been shown (Schultz, Galbraith et al. 1976) that deflection of the sinus hair of the second type (St11) in the opposite to the optimal direction reduced background activity of both p r i m a r y afferent and cortical neurons to a zero-level. When the hair released a rebound response was evoked so that peri-stimulus-time-histogram own estimation (PSTH) was 3-5 its amplitude times bigger was in (our based on the Fig. 1C, Fig. 4A and Fig. 4B in the (Schultz, Galbraith et al. 1976) than background level. Although an effect of either change of the deflection speed of vibrissae angle or the change of the release to the neutral position on the rebound response magnitude had not been studied systematically in this work one can get some insight about it analyzing the data presented in the paper (Schultz, Galbraith et al. 1976). It is visible (see Fig. 1C, Fig. 4 48 A', Fig. 4 B \ Fig. 4 C ) in the (Schultz, Galbraith et al. 1976) that f a s t vibrissae return optimal deflection to the neutral position after higher angle non- evoked responses that were bigger in amplitude (impulses/bin in PSTH) and more sharply tuned in time. The fact that the same dependence can be observed at the two extreme levels of the sensory pathway leads us to assume that similar responses can be recorded in the secondary sensory neurons such as those located in the brainstem trigeminal assumption is correct complex, (unfortunately, work where this or similar including the PrV. If we could not find hyperpolarisation suddenly appears to be an important relation sensory to temporary the information background firing innervating vibrissae follicle can result in a reduction a single question was addressed) the ability the Type I PrV neurons to generate rebound response after processing. this of transient feature in For example, suppression in the primary afferent caused by non-optimal hair d e f l e c t i o n of a background excitatory transmitter release and thus in hyperpolarization of the postsynaptic site w h i c h in its turn can contribute to the whole neuron hyperpolarization. Then, transient increase of the primary afferent activity a neuron depolarization from the more hyperpolarized can lead to level of its 49 membrane potential. Such type of depolarization can evoke rebound response in the characteristics neuron so that would reflect its certain probability parameters and temporal of the incoming signal such as duration and depth of background activity reduction as well as the rate of its change. These parameters in their directly connected to the real-world turn are mechanical stimulation of the vibrissae. Based on the data obtained in the presented work one can hypothesize that the activation of an l -like current, which markedly H affects the latency-amplitude relationship (Figure 3), may represent an adaptation stimuli. for transmission An empirical of dynamic change in mechanical examination of these considerations would allow determination of the types of mechanoreceptive afferents that produce depolarization and hyperpolarization the nucleus principalis trigemini. in type 1 neurons of 50 5. R E F E R E N C E S BADER, C. R. A N D BERHEIM, L. AND BERTRAND, D. 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JONES, E. G . The Thalamus. New York, Plenum, KILLACKEY, H . P. A N D B E L F O R D , G . R. of p. 1985, 325-360. The formation of afferent patterns in the somatosensory cortex of the neonatal rat. J. Comp. Neurol. 1 8 3 : 285-303, Li, Y. 1979. Q., TAKADA, Identification neurons of M., MATSUZAKI, S., periaqueductal projecting to both the gray Res. 623: 267-277, Y. and dorsal trigeminal forebrain structures: a fluorescent in the rat. Brain SHINONAGA, sensory A N D MIZUNO, raphe nucleus complex retrograde double-labeling 1993. N. and study 53 LLINAS, R. Purkinje Electrophysiological A N D SUGIMORI, M . cell somata in mammalian properties cerebellar slices. of in v i t r o J. Physiol. Lond. 305: 171-195, 1980a. LLINAS, R. Purkinje Electrophysiological A N D SUGIMORI, M . ' cell dendrites properties of in v i t r o in mammalian cerebellar slices. J. Physiol. Lond. 305: 197-213, 1980b. MA, P. M . A N D WOOLSEY, T . vibrissae A. Cytoarchitectonic in the medullary trigeminal correlates of complex of the mouse. the Brain Res. 306: 374-379, 1984. MCCORMICK, D. activated A. cation AND P A P E , current H. C. Properties and its role of a h y p e r p o l a r i z a t i o n - in rhythmic oscillation in thalamic relay neurones. J. Physiol. Lond. 431: 291-318, 1990. MOUNTCASTLE, V. B. Central nervous mechanisms in mechanoreceptive sensibility. In: Handbook of Physiology. Sect. I, Vol. Ill, Physiological Society, Bethesda, M D , 1984, p.789-878. American 54 OLSZEWSKI, J . On the anatomical and functional trigeminal nucleus. J. Comp. Neurol. POULTER, M.O., dependent T., HASHIGUCHI, potassium Neuroscience 68: 487-495, RAMON Y C A J A L , S . 92: 401-413, AND PADJEN, conductance organization A. L. in of the 1950. Evidence for a s o d i u m frog myelinated axon. 1995. Histologie du Systeme Nerveux de I'Homme et des Vertebres. Madrid, Consejo Superior de Investigaciones Cientificas, 1910. S C H U L T Z , W., GALBRAITH, G. C , GOTTSCHLADT, comparison of primary afferent K. and cortical M., CREUTZFELDT, neurone activity sinus hair movements in the cat." Exp. Brain Res. SCHWINDT, P. C. AND CRILL, W. E. Properties O. 24: 365-381, of a persistent D. coding 1976 inward current in normal and TEA-injected motoneurons. J. Neurophysiol. 1700-1724, 1980. "A 43: 55 SCHWINDT, P . C , S P A I N , W . J . A N D C R I L L , W . excitability by a sodium-dependent neocortical neurons. J. Neurophysiol. E. Long-lasting potassium current of in cat 61: 233-244, 1989. SCHWINDT, P . C , S P A I N , W . J . AND CRILL, W . calcium chelation currents in cat E. Effects on voltage-dependent neocortical reduction and of intracellular calcium-dependent neurons. Neuroscience 47: 571-578, 1992. STROHMANN, B., rectifying properties SCHWARZ, D. of W. F. A N D PUIL, nucleus ovoidalis auditory thalamus. J. Neurophysiol. TORVIK, A. The ascending fibers nucleus: An experimental E. Mode of neurons firing in the and avian 71: 1351-1360, 1994. from the main trigeminal sensory study in the cat. Am. J. Anat. 100: 1-16, 1957. TRAVAGLI, R. A. AND GILLIS, R. A. Hyperpolarization-activated currents, l H and IK , in rat dorsal motor nucleus of the vagus neurons in vitro. J. IR Neurophysiol. 71: 1308-1317, 1994. 56 VAN DER L O O S , Neurosci. Barreloids in mouse somatosensoty thalamus. Lett. 2 : 1 - 6 , 1 9 7 6 , N., WILLIAMS, M . synaptic ZAHM, Differential D. S . AND JACQUIN, M . F. organization projections 453, H. of the principal and spinal foci trigeminal to the thalamus in the rat. Europ. J. Neurosci. T. A. A N D V A N DER Loos, H. The structural organization of l a y e r I V in the somatosensory region ( S I ) of the mouse cerebral Brain Res. 17: 205-242, Z A M P O N I , G . W . , BOURINET, distinct F. E., D U B E L , S . J . effects A N D GALLAGHER, dicarboxylic cortex. 1970. on neuronal ANDSNUTCH, calcium inhibition of activation-gating. Soc. Neurosci. ZHENG, 6: 4 2 9 - 1994. WOOLSEY, two and J . P. acid -induced (1S, burst 3R)-1 firing T. P. Nickel modulates channels: block and Abstr. 2 1 : 1 7 5 3 , 1 9 9 5 . -aminocyclopentane-1,3- is mediated by a native 57 pertussis toxin-sensitive metabotropic septal nucleus neurons. Neuroscience receptor at rat 68: 423-434, 1995. dorsolateral 58 6. APPENDIX 6.1 1. Data acquisition and processing program VMS In order to perform the experiments presented above, it necessary to design and write a new data acquisitionprogram for Instruments boards. the Macintosh data LabView Quadra 950 acquisition instrumentation and a n a l y s i s computer and DMA (direct development was and National memory access) environment was chosen for this purpose. LabVIEW includes is libraries specifically programs for are a general-purpose of functions and developmental data acquisition called and instrument tools that designed control. LabVIEW their appearance on a computer monitor and their operation imitate real- amplifier). Instruments system because world instruments Virtual programming (Vis) (e.g. oscilloscope, tape-recorder, The main advantage of using the virtual chart recorder, instrumentation approach, compared with conventional programming C, Pascal), is that every VI is a fully functional unit, similar stand-alone instrument, be separately combination with other which Vl's, can supplied used languages (e.g. commercially or to a or in written 59 independently. Thus, one can assemble an entire new instrumentation complex by simply connecting different laboratory. In addition, one can, in future, already working instruments without Vl's in an imaginary add new Vl's to a set of modification of existing V l ' s , or change parameters or functionality of the Vl's that are already at work. The VMS data acquisition and processing program used in the present work consists of seven main Vl's. These are: 1. A hardware setup; 2. The build protocol; 3. A play back module; 4. The chart recorder; 5. The protocol data acquisition module (DAQ); 6. A waveform generator; 7. A digital signal processing unit (DSP). A user can access each of these Vl's by clicking on the corresponding button in the main panel (Fig. A1) appearing on a computer monitor when the VMS program is loaded. The "Stop" button terminates the V M S . execution of 60 The VI "Hardware Setup" is used to change data a c q u i s i t i o n board settings board, and the such as the device number of the A-to-D number of input configuration used channels voltage for in the and output present and current project conversion channels. The t y p i c a l includes recordings two expandable input to maximum of 8 and one output channel (maximum 2) for current a or voltage command generation. The subpanel "External Gains" (Fig. A2) is used to feed information about external amplifier parameters (voltage gain, current gain and headstage gain) into the V M S program. The subpanel "Continuous DAQ" (Fig. 2A) defines parameters chart recorder VI (explained later). Figure A 2 Subpanel "Continuous DAQ" of a The VI "Build Protocol" is used to create a voltage or current command protocol. Every protocol can be a stimulus stimuli repeated in time. Three types of stimuli A pulse-type modulated example stimulus; sinewave); of the "Build 2. A chirp-type 3. Sinewave stimuli. Protocol" VI for or a set of are implemented: 1. stimulus (frequency Figure A3 shows a pulse-type an stimulus protocol generation. In the given example the protocol consists of the 10 voltage pulses (Fig. A3., right-hand slide dial) increasing in amplitude by 5 mV (Fig A3., right-hand column of controls). Each stimulus episodes (E-1, E-2, E-3, E-4, E-5; Fig. A3). Figure A 3 "Build Protocol" VI consists of 5 62 Stimuli with can be created visually a mouse and simply by marking parts of the dragging and placing the waveform corresponding cursor in a specific position on the display (e.g. Fig A3, E-1 cursor i s in the position x = 0.1 s and y= 0 mV). X- and Y-scaling on the display can be adjusted automatically or manually. The total number episodes available for a user is 7 in the visual mode of construction protocol and unlimited can be saved in in an alternative a file text and loaded stimulus mode. later for of Every use or modification. Figures A4 and A5 show the "Build Protocol" VI in the modes of the chirp-type and sinewave-type stimulus protocols respectively. $WM i $$:m -i liiiii Figure A 4 "Build Protocol" VI in the mode of the chirp-type stimulus generation i m 63 Figure A 5 "Build Protocol" VI in the modes of the sinewave-type stimulus generation The VI "Chart Recorder" is designed to substitute a standard chart recorder (Fig. A6). It is capable of continuously logging data to the disk without interruption of the data acquisition. In order to start data logging a user has to chose a file name for a record of the acquired information. The VI automatically minute, adding the current time to the file creates a new file every name specified by user (e.g. cell1_5-37 PM). The sampling rate for the data acquisition displayed time period are specified in the VI "Hardware Setup" the "Continuos DAQ." panel and in 64 Figure A 6 "Chart recorder" VI The front panel of the "Protocol DAQ" VI is shown in the Figure A7. This VI is used to conduct single-cell recording in discontinuous mode using pulse protocols (created with the "Build Protocol" VI) or arbitrary waveforms (created with the "Waveform generator" VI) or imported from a text file. 65 l i g f i i l input limits f 5 hlph limn | M ! low llfmt 1 bean rat? output limits m\ 509 0 UKHJEFOBM IsSTRUMtM PROTOCDl <*CQUI&iTIOS Figure A 7 "Protocol DAQ" VI A protocol "Protocol" or arbitrary waveform or "Waveform" can be loaded by pressing the buttons. Stimuli can be delivered manually (by pressing button "Next", Fig A 7 ) or automatically with a delay that is specified on the slide dial "Delay" (Fig. A 7 ) . Dials in the panels amplification respectively. "Input for limit" signal and "Output acquisition limit" and signal Acquired data are saved in a file The file name is shown in the window control on-board generation defined by the user. "Path to save". The buttons "Record" and "Approach" permit changing the voltage scale on the screen to a predefined value. In addition, both voltage and current 66 windows can be transferred into an auto-scale mode so that, independently of the input signal magnitude or duration, scaling i s maintained at an optimum. Figure A8 shows the front panel of the "Play back" VI. This VI is designed to provide easy access to the data obtained during experiment. A user can load a data file, like a video-tape into a video player, so that every experimental trial (frame) can be accessed sequentially in both forward and backward playback. To play back a l l frames from a file the user should press the "Total" button. , J, A A —•+• \ IBRD EJOCKli'HilQ F i g u r e A 8 "Play back" VI lOMulflJi!! 67 The button "Print" screens with file media (hard permits a transfer of both voltage and current name and location information drive) to the printer, creating from the storage a hardcopy of the displayed data. The main VI for data analysis is called " D S P " (fig. A9). F i g u r e A 9 "DSP" VI All data processing operations in this VI are performed on the data from either position voltage or current trace between two of these cursors can be changed and fixed cursors. at The selected positions with a mouse. As a segment of a trace is chosen, the user 68 can perform function. following Only implemented "Max/Min"): operations: 1. fit linear to and single date; 2. define data (button exponential maximum cursors are positioned "Fit") to a have been fits and minimum automatically to the (button maximum and minimum of the chosen trace segment; 3. calculate the average over the trace segment (button (button "dX/dt") using an arbitrary Filter data (button "lnt(mV*ms)"). "Filter"); "Mean"); 4. take derivative time window (dial "Window"); 6. take the An example of a linear fit shown, with the front panel of the "Fit" A11 shows front panel of the "dX/dt" temporal derivative the time integral 5. (button to a plateau potential is VI, in Figure A10. Figure VI with an example of the taken from the voltage trace segment between the cursor markings in the Figure A9. A user can transfer current trace directly (National Instrument a chosen data segment of the voltage to the HiQ numerical computation software) for on- or off-line or package analysis (fig. A 9 ; button "HiQ"). The data captured into a text file can be imported into any standard button spread-sheet "Capture", option or data processing "ASCII", not shown). program (fig A9; Finally any data segment can be captured and stored in binary format as an a r b i t r a r y 69 waveform to experiment be used as current(fig. A9; button or voltage-command "Capture", options during "Command", "Voltage" or "Current" (not shown). The V M S program is a new flexible tool designed for whole c e l l recording and data analysis. programs is, that new functions introducing errors in already The advantage can be added without existing modules. experiment control and data analysis, therefore, expansion in any direction neurophysiology. Figure A 10 "Fit" VI over of potential The alternative the risk of design of is ideally suited to development of the modern Figure A 11 " d X / d t " VI
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Firing properties and Na⁺-dependent plateau potentials of neurons in nucleus principalis trigemini of… Sandler, Vladislav Michael 1996
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