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Brain Research, 437 (1987) 45-55 Elsevmr 45 BRE 13144 Intracellular study of rat substantia nigra pars reticulata neurons in an in vitro slice preparation: electrical membrane properties and response characteristics to subthalamic stimulation H. Nakanish;.*, H. Kita and S.T. Kitai Department of Anatomy and Neurobtology, The Umversity of Tennessee, Memphis, The Heahh Science Center, Memphis, TN38163 (U.S.A.) (Accepted 2 June 1987) Key words" Rat substantIa mg~a neuron; Shce preparation, Intracellulat recording; Membrane property; Subthalamonlgral input The electrical membrane properties of 5tl135tattlsa n.~,~ap~!rSreiiculata (SNR) neurons and the,r postsynapuc responses to si~.mulation of the subthalamic nucleus (STH) were studied in an m vitro slice preparation. SNR neurons were divided into two types based on their electrical membrane properties. Type-I neurons possessed (1 } spontaneous repetmve finngs, (2) short-duration action potentials, (3) less prominent spike accommodations, and (4) a strong delayed rectification during membrane depolarization Type-II neurons had (1) no spontaneous firings, (2) Iong-du_ratiQnaction potentials, (3) a prominent spike accommodation, (4) a relatively large post-active hyperpolarization, and (5) a less prominent delayed rectification. These membrane properties were very similar to those observed in substantm mgra pars compacta (SNC) neurons in slice preparations. Feawures coma~on to both ,ype~ of neurons include that (1) the input resistance was similar, (2) they showed an anomalous rectification during suoitg ~pcrp~lat~zatlous, and (3) they were capable of generating Ca potentials Intracellular responses of both types of SNK neurons to STH shmulatIon consisted of imtial short-duration monosynaptlc excitatory postsynaptic potentials (EPSPs) and a short-.durauon inhibitory $×~s~synapticpotential (IPSP) followed by a long-duration depolarization The IPSP was markedly suppressed by apphcatlon of blcucuihne methlodide and the polarity was reversed by lntracellular rejection of CI-. In the preparations obtained from internal capsule-transected rats, STH-mduced EPSPs had much longer duranons than those observed in the normal preparations, while the amphtude of IPSPs and succeeding smallamplitude long-duranon depolarizations was small. The results indicated that SNR contains two electrophysiologically different types of neurons, and that both types of neurons receive monosynaptic EPSPs from STH and IPSPs from areas rostral to STH INTRODUCTION The m a j o r afferents to the substantia nigra (SN) pars reticulata ( S N R ) originate f r o m the neostriaturn, the globus pallidus ( G P ) and the s u b t h ~ a m i c nucleus (STH). T h e neostriatum and S T H receive excitatory inputs f r o m the s e n s o r i m o t o r cortex 1'17'19'38. T h e r e f o r e , S N R receives cortically derived striatal input and cortically derived S T H input. Electrophysiological studies indicated that the predominant response of S N R neurons to neostriatal stimulation was inhibitory, while some excitatory re- sponses were also noted 29"39. The S T H afferents to S N R originate from neurons whose bifurcating axons also innervate G P and the entopeduncular nucleus (EP) 3"13'14"35. The S T H terminals form asymmetrical synapses on the dendrites of SN neurons 2"t5. Electrophysiological study to identify the functional significance of S T H inputs to S N R is limited to one study utilizing an extraceilular recording technique 9. T h e study indicated that S T H stimulation evoked an excitation of nigral neurons. O n the other hand, several reports suggested that S T H inputs to EP and G P are inhibitory and ~,-aminobutyric acid ( G A B A ) e r - * Present address: Department of Pharmacology. Faculty of Dentistry. Kyushu Umverslty. Fukuoka 812. Japan Correspondence S T Kital. Department of Anatomy and Neuroblology. The Univemty of Tennessee, Memphis. The Health Science Center, 875 Monroe Avenue. Memphis. TN 38163. U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Pubhshers B V. (Biomedical Dwision) 46 gic2s'30'31 These two opposing responses (excitatory vs. inhibitory) of STH inputs to EP/GP and SNR are somewhat puzzling since anatomical and electrophysiological studies clearly demonstrated that single STH neurons project both to EP/GP and SNR with their bifurcating axons. In order to examine the precise nature of STH inputs to SNR, we have recorded mtracellular responses of SNR neurons to stimulation of STH m an in vitro slice preparation. We considered that thts preparation is most appropriate for studying electrical membrane properties of SNR neurons since one can manipulate the external chemical enwronment of the neurons studied, and also one can make more precise measurements of membrane phenomena with stable recordings 18. The electrical membrane properties of the dopaminerglc cells in the SN pars compacta (SNC) have been examined in detail usmg an m vitro preparation and mtracellular recording techniques 16"2a. Since SNR contains both dopaminergic and GABAergic neurons 26, our aim was also to examine if SNR neurons can be distingmshed into two types on the basis of electrical membrane properties Our results indicate that SNR neurons can be classified into two types based on their electrical membrane characteristics. Both types of SNR neurons evoked monosynaptic excitatory postsynaptic potentials following stimulation of STH. The results suggest that cortically derived STH excitatory inputs and cortically derwed neostnatal inhibitory inputs are converging on SNR neurons. MATERIALS AND METHODS Male Sprague-Dawley rats (200-350 g) ~ere used. Immediately after decapitation, the brain was rapidly removed from the skull and trimmed tt a block. Parasaglttal shces (350-450/~m) containing the SN and the STH were cut usmg a Vibratome. Slices were preincubated in oxygenated Krebs solution for about 1 h at 35 °C before recording The recording chamber was constructed to allow Krebs solution (35 °C) to continuously flow on the bottom surface of the slice at a rate of 0.7-1.5 ml/mm, and warm and moist gas mixture (95% 0 2 - 5 % CO2) to flow over the surface of the shce 18. The Krebs solutlon was composed of (mM): NaCI 124, KCI 5 0, KH2PO4 1 24, NaHCO 3 26, CaCI 2 2.4, MgSO4 1.3 and glucose 10. Glass pipette.~ filled with 2 M potassium methylsulfate or 1.5 M potassium chloride were used for recording. Recording electrodes had DC resistance of 60-100 MfL Intraceilular recordings were obtained through a high-input impedance biological amplifier with an active bridge circuit which enabled the measurement of membrane potentials and the injection of intracellular constant current simultaneously. The output of the amplifier was fed into an oscilloscope. Electrical stimulation (intensity 5-30 V, duration 200/~s, 0.5 Hz) was applied through a bipolar electrode placed on the surface of the STH. The stimulating electrode was made by twisting a pair of 80-/~m-diameter nichrome wires, insulated except at the tip, with a tip separation of 200-400/tin. In some animals, the internal capsule (IC) at the level of the entopeduncular nucleus was transected using a Halazs knife, 6-10 days prior to the recording session, in order to eliminate afferent fibers to SNR originating rostral to STH. In some experiments, the Na channel blocker tetrodotoxin ( T r x , 10-s g/ml) and a K channel blocker tetraethylammonium chloride (TEA, 2-10 raM) were added to the superfusion medium. Cobalt chloride (2-4 raM) was used to block Ca conductance s. Bicuculline methiodine was dissolved in the superfusion medium with a concentration ranging from 50 to 100k~M to block GABAergic responses. RESULTS Intracellular recordings were obtained from 40 SNR neurons which had resting membrane potentials of more than 50 mV and generated action potentmls w~th an amphtude greater than 50 mV. Membrane properttes SNR neurons were divided into two types based on their electrical membrane properties One group of neurons which we named type-I (n = 26) had the following characferist~cs. They exhibited spontaneous repetmve firings with a rate of 1-40 spikes. The duration of the action potential was less than 2 ms. Injection of a depolarizing current pulse generated repetitwe firings from these neurons. Even though the finng frequency was highest (i.e., a maximum over 200 Hz) at the beginning of the current pulse, finngs with a regular interval were maintained throughout 47 A C 12om__ I ' 20 -0'4 je -0'2/,J / 02nA 04 e,,/e -20 eJ e -40 E / 200' H7 o *1,'tI B D 20 mV / 100" -f 1- / .,(,'/ ...................... o 0 0"3PA 0"6 Fig. 1. Input resistance and spike discharges of type-t neurons A membrane responses to mtraceilularly rejected hyper- and depolarrang currents of various intensities. In order to eliminate spontaneous finng, a hyperpolanzmg current of 0.06 nA was continuously injected m the neuron. B membrane responses to hyper- and depolarizing currents during appheauon of TIX (10-5 g/ml) to ehm,nate spikes Square waves at the bottom of oscdlographic records in tlus and all subsequent figures indicate the mtensmes of rejected clepolanzmg (upward square wave) and hyperpolanzmg (downward square wave) currents Cahbrauons m A also apply to B C currentvoltage relauon for a type-I neuron recorded m the shce superfuse¢ w~th TI'X-contaming solution Note membrane recuficauon m both hyper- and depolarizing directions D inleetion of a depolarizing current pulse m a continuously hyperpolanzed neuron produced repetmve finng followed by a small amplitude long-lasting hyperpolanzmg potentml after the termination of the current pulse E: relattons between the discharge frequency and the intensity of current pulses obtained from the lnterspike interval of first two spikes (1/tl) and last two spikes (l/t2) m the cell of D. the period of the current pulse (Fig. 1D). No large changes in the amplitude nor the duration of action potentials were observed even when relatively highfrequency (over 70 Hz) repetitive firing was generated by a strong current injection (Fig. 1D). A longlasting hyperpolarizing potential with a small amplitude (no more than 10 mV) was occasionally observed after termination of a large current pulse (Fig. 1D). SNR type neurons showed a large reduction of the input resistance (delayed rectification) during membrane depolarization (Fig. 1B,C). The m e m b r a n e properties of the other group of neurons, type-ll neurons (n = 14). ~ e r e very similar to those observed m SNC neurons in slice preparations 16-21. Type-ll neurons had no spontaneous firings. The duration of action potential was usually longer than 2 ms. InJection of moderate intensity depolarizing current pulse generated low-frequency (up to 10 Hz) repetitwe firings When a strong depolarizing current pulse was applied, the neurons showed a prominent sptke accommodation with a decrease in the spike amplitude and an increase in its duration, which finally led to a cessation of spike generation (Fig 2C). The membrane response after termination of large current pulse was a relatively large-amphtude (about 10 mV) long-lasting hyperpolanzation. The delayed rectification which was clearly observed in type-I neurons was less prominent in type-lI neurons Instead, type-ll neurons showed a clear skgn ot an act~ration of early K current as has been reported m SNC neurons 16 ~,8 / B w 0'3 -0 6 nA ./ // ./ -25 mV ./ .7 I • -50 1 Fig 2 Input res]s ce and sp~ke discharges of a type-II neuron A membrane responses to mtracellularly rejected hyper- and depolar~.zmgcurrents of various intensities B current-voltage relation for the type-II neuron C rejection of a ,trong depolarizing current palse produced only 4 spikes A strong spike accommodation can be seen m these responses The membrane potentml after the termination of the current pulse was a relatively large-amplitude long-lastingbyperpolanzmg potential. Calibrations in A also apply to C tance ot type-I and type-II neurons calculated from the slope of the current-voltage curve crossing at zero current point was very similar; the former Even though some membrane properties of two types of neurons were clearly different, there were features common to both of them. The input resis- BII, 20_v jj S0mse0 _T=-~-==t. . . . . . . . . . . . . . . . . . . . . . . . C D C ,,J J t _/--1 Fig 3 Slow depolarizing potentials evoked m a type-I neuron A injection of a depolarizing current pulse generated repeUttve firings and a small-amphtude membrane hyperpolanzatlon after the termmatlonofthecurrentt~td~, P, adepolartzmgcurrentpulsemjected dunng contmuous membrane hyperpolanzatlon triggered a slow depolanzir~g p,~tential ~utla.tmg the gt~rrem oulse C and D. responses to depolarizing current pulses during apphcauon of 10 mM TEA TEA mcre,~s,.d the duration of action potentials (C) and mcreased the amphtude and the duration of slow depolarizing potential (D) 49 ranged from 5(I to 150 MQ (n = 18, e . g , Fig 1C) and the latter ranged from 80 to 200 Mff2 (n = 10, e.g., Fig. 2B) The anomalous rectificatton of the cell membrane m response to strong hyperpolartzmg current injecttons was found in both types of neurons (Figs. 1C and 2B). Previous studies have clearly demonstrated that SNC neurons are capable of generating Ca potentials I~ 2~ We have also observed m this study that type-I neurons are also capable of generating Ca potentials. InJection of depolarizing current pulses in type-I neurons with a membrane potentml less negative than 60 mV generated repetitive f~rings (Figs. 1D, 3A). When the neuron was continuously hyperpolarized more negatively than 60 mV by a current injection, the depolarizing currer ~ pulse could generate repetitive firings followed by a long-duration slow depolarizing potential which outlasted the duration of the depolarizing pulse (Fig. 3B). Bath application of TEA (10 mM) resulted in the following changes in type-I neurons: (1) a decrease in or a termination of the spontaneous discharge, (2) an increase in the duration of action potentials generated by depolarizing current pulses (Fig. 3C), (3) an increase m the input membrane resistance, and (4) a marked augmentation in the duration and amplitude of the slow depolarizing potential which tnggered a number of action potentials (Fig. 3D). The type-I neurons were also able to trigger slow action potentmls in response to depolarizing current pulses when they were continuously hyperpolarized. The slow action potew~al had a duration of 30-100 ms and was accompanied by a burst of action potentials (Fig. 4A). A simdar burst response was observed at the offset of a strong hyperpolarizing current pulse (Ftgs. 1A and 4B). Superfusion of the Na conductance blocker TTX (10 -5 g/ml) abolished fast action potentials but preserved the slow depolarizing potential and the slow action po- O A nil 120 J 05nA 13 B q I E ] I Fig 4 Slow action p o t e n t i a l evoked by current pulses in a ~ype-I neuron A injection of a depolarizing current pulse in a continuously hyperpolarized neuron reduced a slow action potentml w~th spikes B a s~mdar slow action potential was generated after the offset of a hyperpolarizmg current pulse. C - E recordings ,during superfuslon with a medmm containing T r x (10-~ g/ml) L rejection of depolarizing current pulses generated a slow depolarizing potential (arrow) and a slow action potential D. a slow actton potenllal triggered at the offset of a hyperpolanzing currem pulse. E: addition of Co 2÷ to the superfusing medmm abolished slow action potenttal and slow depolarizing potential. 50 B A8 l~v o m ous depolarizing or hyperpolartz~ . . during injection o! co.nunu ,._a ,,,,tin short-duration EPSPs }' . . . . . . ded from a type [ ~euron _.,:~ STH stimmatton evott~,~ ....... I ,~'-"" . various -two components. " •The arrow on the top trace points to onset of IPSPs- B: • ca-~ mlecuv,, stlmulat ton "induced responses _-.nmos with stimulus tnten~m,-~ mg current appeared to ha,Je l'tg -~ o,,-. . . . . . S A fast sweep recur,, ~, slowshort.duration sweep recordings. These and IPSPs were followed by a iong-durauon depolanzauon The amplitude of the intual EPSPs and IPSPs. The EPSPs tmtial EPSPs powas decreased by depolarizing currents and increased by hyperpolanztng current mi ecti°ns' The amplitude of IPSPs was increased by depolarmng currents and decreased by hyperpolarizing current rejections. Bottom traces in A and B represent extraee|lular f~eld tent~als with a relatively large negative potenual often appeared to have two components because of an inflection on the nsmg phase (e g,, Figs 5A, 7A). tential (Figs. 4C,D). Both of the Tl?X-reststant poThe amplitude of the initial depolarization showed tentials were augmented by application of TEA (data graded changes upon increase in the stimulus intensinot shown) and abolished by an addition of Co2+ (3 ty, and tt was increased during iniecti°n of hyperporaM) to the TTX-contalmng medium (Fig. 4E) lanzing currents and decreased dunng depolarizing currents (Fig 5A,B) STH stimulation with various Postsyna?UCresponses evoked by STH stimulation intensities revealed that the latency of the earliest It was commonly observed that STH stimulation and second components ~howed shght and graded evoked relatively large (i.e,, up to 5 mV) negative shortening upon increase in the stimulus intensity field potentials with a latency of about 1 ms and a du(Fig 5A). The graded change of the latenctes was ration of about 1.5 ms. The latency of the negative considered to be due to an increase in the current potential was constant in spite of changes in stimulus spread at the stimulus site, since the change never exintensities or repeuUve stimulations of up to 500 Hz ceeded 0 5 ms. The shortest iatency of the earliest (data not shown). Intracellular responses of both depolarizing potential was 1.g ms (mean; range = types of SNR neurons to STH stimulation consisted 1.2-2.4 ms, n = 11) and that of the second compOof initial short duration (i.e. less than 10 ms) depolarnent was 4.1 ms (mean; range = 3 2-4 6 ms, n = 5). ization and a sl-~ortduration (i.e. less than 15 ms) hyThese observations indicated that the initial depolar perpolarizauon followed by a long duration (i.e. 20-40 ms) depolarization- The initial depolarization 51 jsmv m A C A D I lOmV 10 msec A Fig 6. Effects of blcuculhne and intracellular mlecnon of CIon the synaptlcresponses induced by STH stimulation. Recordrags m A-C are obtained from an SNR neuron. A: STH stimulation-induced EPSP~ followed 0y IPSPs. B bicuc.iline suppressed the IPSPs and increased the amphtude and the duration of the EPSPs C. extracellular field eotenuals D: the use of KCl-fdled recording electrode rapidly reversed the polanty of IPSPs in depolarizing direction. ,zing potentials induced by STH stimulation include two monosynaptic excitatory postsynaptic potentials (EPSPs) with different latencies. The amplitude of the short-duration hyperpolarizing response was increased during intrac¢llular injection of depolarizing current and decreased during hyperpolafizing current (Fig. 5B). These data indicated that the hyperpolarizing response is an lnh,bitory postsynaptic potential (IPSP). Precise latencies of IPSPs could not be determined since they were masked by the preceding EPSPs. The latency, however, was estimated to be 2-3 ms from the records obtained during injection of strong depolarizing current, which minimized the overlapping EPSPs (Fig. 5A, arrow on top traces). Fig. 6A shows responses of an SNR neuron following stimulation of STH. STH stimulation incuced EPSPs with or without spike followed by IPSPs. Application of bicuculline methiodide (100 mM) markedly suppressed STH-induced IPSPs and at the same time increased the amplitude and duration of EPSPs (Fig. 6B). In other experiments, the polarity of the IPSPs was rapidly reversed in depolarizing direction when KCl-fiiled recording electrodes were used. The duration of the depolarizing postsynaptic potentials, which include EPSPs and the reversed IPSPs, was more than 30 ms (Fig. 6D). As mentioned previously, STH stimulation induced a long-duration depolarizing potential which is preceded by short-duration EPSPs and IPSPs. The amplitude of the loo,g duration depolarization was less than 4 mV and only slightly altered by injections of depolarizing or hyperpolanzmg currents (Fig. 5B). Recordings from SNR neurons in the slices obtained from IC-transected rats are shown m Fig 7 In these preparations, STH-induced EPSPs had much longer durations than those observed in the normal preparations, while the amplitude of IPSPs and succeeding small-amplitude long-duration depolarizations was markedly reduced (i.e., compare Figs. 5B and 7B). As in the normal preparation, the latency of these EPSPs was fairly constant in spite of changes in the stimulus intensities (Fig. 7A,B), indicaring a monosynaptic nature of the responses. The negative field potential observed in the normal preparation was also almost totally abolished m these preparations (compare the bottom traces in Figs. 7A and 5A). These results would indicate that the negative field potentml recorded in SNR in the normal preparation was caused by an activation of the descending fibers passing through or by STH. DISCUSSION Electrical membrane properties It has been known that SNR contains both GABAergic and dopaminergic neurons. The GABAergic neurons which are main representatives of SNR pro- 52 A B ]smv 08 5mseo -OB8 -oB A A F~g 7 STH stimulation-reduced responses reeurded from Iype-I neurons m a shce preparation obtained from an IC-transected rat A: fast-sweep recordings w~th various stimulus intensities STH st~mulatmn evoked imtml shortoduratmn FPSPs w~thtwo components. B: slob-sweep recordings The IPSPs and the Iong-durauon depolanzatmn observed m the noLaal preparatmn were not prominent The negative field potentml (bottom tracesj observed m the normal preparatmn was also markedly reduced (compare to Fig 5) lect to the thalamus or the tectum and the dopaminergic neurons project to the neostriatum 26'2s'32. The present data clearly indicate that SNR neurons could be separated into two types based on their electrical membrane properties. Type-I neurons were characterized by having a spontaneous &schmge, a short duration action potential, an ability to generate sustained high-frequency discharges, and a strong delayed rectification. They are likely to be G A B A e r glc neurons since we encountered this type of neuron more frequently in SNR, and their short duration action potentials and ability to fire at high frequency are characteristic properties of SNR G A B A e r g l c neurons 7 Type-I1 neurons had no spontaneous actlvrues, a long-duration action potential, a strong spike accommodation and a prominent long-lasting post-active hyperpolarlzation which was reported to be caused by a Ca-dependent K conductance and an early K conductance 16 Type-II neurons also did not have strong delayed rectification. Since the mere- brane properties of type-II neurons are very similar to the neurons recorded from SNC in the slice preparation 16'21, it m~ght be reasonable to assume that they are dopaminergic neurons located in the SNR. The differences in the ionic conductance between the two types of neurons could be summarized as follows. Type-I neurons had a strong voltage-dependent K conductance which might be involved in the delayed rectification and short-duration action potential seen in this type of neuron. It has been recently proposed that a function of the striatonigral pathway might be a disinhibition of tonically firing nigrothalamic neurons 5. The strong voltage-dependent K conductance observed m type-I, presumably GABAergic, neurons might be the basic mechanism underlying the sustained high-frequency spike discharge, since they avoid the membrane potential to reach the Na inactivation level even when they receive tonic excitatory inputs such as from the subthalamic nucleus 15.24. Neither Ca-dependent K conduc- 53 tance nor early K conductance were prominent in this type of neuron. On the other hand, type-lI neurons had a strong Ca-dependent K conductance and early K conductance but a less prominent voltage-sensitive K conductance. In spite of these differences between the two types, there were some membrane characteristics common to both. They had a similar range of input resistance. The membranes of both types showed anomalous rectification when they were hyperpolarized by current injection. Both types of neurons were able to generate two Ca potentials (i.e., TTXresistant Ca-sensitive slow depolanzauons and slow action potentials) (Figs. 3 and 4, also see refs. 16,21). The Ca potentials were observed in SNR neurons only with a membrane potential more negative than 60 mV, and both responses were markedly augmented by application of TEA to the superfusing medium. These features were very similar to those observed from the neurons of the infenor olive 22, thalamus II, spinal cord 23, SNC 16°21 and S T H 24. The observations suggest that responses of SNR neurons to phasic synaptic inputs may differ depending on their level of tonic inputs (i.e. the 6aembrane potential level). Responses evoked by STH sumulation We have demonstrated that STH stimulation evoked a series of responses, monosynaptic EPSPs. IPSPs, followed by a long depolanzation, in both types of SNR neurons. The initml short-latency (1.2-2.4 ms) EPSPs were considered to be mediated by STH afferents since (1) STH projections to the SNR are well demonstrated by both anatomical-" ~4 15.35 and electrophysiological studies 3 13 (2) the latency of EPSPs coincides well with the conduction time of STH neurons to SNR t3 plus a synaptic delay. and (3) STH-mduced EPSPs were not affected by the chronic IC transection placed rostral to STH. A previous extracellular single-unit recording str~dy also suggested that the subthalamonigral pathway is excitatory 9. Recently, electron m~croscop~c analysis of STH axon terminals labeled with both mtracellular rejection of H R P and the anterograde neurotracer Phaseolus vulgaris leucoagglutmm revealed that they were making asymmetric synapt~c contacts with dendntlc shafts of SN neurons-" t5 The preclse origin of the second monosynapt~c EPSPs which had longer latencies (3.2-4.6 ms) than the initial EPSPs could not be determined at this time. It is most likely, however, that the EPSPs were not induced by afferents originating rostral to STH, since the chronic IC transection did not abolish the response. Simdarly, relatively long-latency (2.8-4.5 ms) monosynaptlc EPSPs were evoked in STH neurons in a slice preparation after stimulation of IC, and the EPSPs we,e not abolished by chronic IC transection at the level of the entopeduncular nucleus z4. These observations suggest that both STH and SNR receive common excitatory inputs originating from midbrain nuclei (e g., from the raphe nucleus 34, which has been shown to be excitatory to the neostnatum 27"37, and from the pedunculopontine tegmental nucleus32.33). STH stimulation also evoked IPSPs in SNR neurons. We consider that the IPSPs which follow monosynaptlc EPSPs must include Ci-mediated GABAergic inputs since application of bicuculline methiodide suppressed the IPSPs, and intracellular injection of CI- reversed their polarity in a depolarizing direction. Even though the IPSPs appeared to have a relatively short latency (2-3 ms), a precise measurement of the latency could not be made be, ause of the overlapping EPSPs. We consider that the IPSPs were evoked by stimulation of the striatonigral fibers and/or pallidonigral fibers since there was a large reduction in the amplitude of IPSPs after chronic transection of the IC. Both the stnatonigral and pallidonigral projections are known to be inhibitory and GABAerglcS.~0 13.25.29.30.36.39. In addition, an involvement of local inhibitory circuits mediated by axon collaterals or intrinsic neurons of SNR 4"12 could also not be dismissed in the generation of the IPSPs. These local circuits may be responsible for small IPSPs which persisted after chronic transection of IC. The chronic IC transection also resulted in a large increase in the amplitude and the duration of STH-induced EPSPs. These results would indicate that the striatonigral or palhdonigral inhibitory inputs are very effective in suppressing the subthalamic and other excitatory inputs to SNR neurons. It has been well demonstrated that the neostriatum and STH receive excitatory inputs from the sensorimotor cortex ~'~9"38. SNR receives inputs from both the neostriatum and the STH. Therefore, there are two parallel pathways between the sensorimotor cortex and SNR. The striatonigral input is known to be 54 inhibitory. The present study ind,cated that the STHntgral input ~s excitatory. From these previous and present observations, ~t ~s conceivable that SNR neurons summate cortically derived neostrmtal inhibitory inputs and cortically derived STH excitatory inputs. The observations also ~mply that STH input to SNR plays slgmficant roles in basal ganglia motor control functions. In sum, o-r present data indicate that there are two types of SNR neurons, having different electrical membrane characteristics. Our data also clearly indi- cated that both of these SNR neurons receive monosynaptic excitatory inputs from STH neurons and inhibitory inputs originating from areas rostral to the STH. ACKNOWLEDGEMENTS The authors express thanks to Dr. T. Kita for her helpful comments during the experiment. This study was supported by NIH Grants NS 20702 and NS 23886. REFERENCES 1 Buchwald, N . 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