29/39 Brunswick Square, London WC1N lAX (Received 26 February

J. Physiol. (1976), 259, pp. 825-849 825With 1 plate and 5 text-figure8Printed in Great Britain

CAUDATE STIMULATION AND SUBSTANTIA NIGRAACTIVITY IN THE RAT

BY A. DRAY, T. J. GONYE AND N. R. OAKLEYFrom the Department of Pharmacology,

The School of Pharmacy, University of London,29/39 Brunswick Square, London WC1N lAX

(Received 26 February 1976)

SUMMARY

1. The responses of spontaneously active single neurones in the sub-stantia nigra and overlying mesencephalic reticular formation have beenanalysed during the electrical stimulation of the ipsilateral caudatenucleus. Experiments were performed in rats anaesthetized with urethaneor pentobarbitone. All recordings were made extracellularly with multi-barrelled glass micropipsttes which were also used to test neuronalresponsiveness to electrophoretically administered substances. Themicropipette tip position was marked and the distribution of neuronesstudied has been analysed.

2. Single shock stimulation of the caudate nucleus inhibited neuronalactivity in the substantia nigra (270/320 cells: mean latency 5-4 msec)and in the mesencephalic reticular formation (62/72 cells: mean latency16-6 msec). However, these effects were often accompanied by periods ofexcitation. In pentobarbitone anaesthetized animals the latency andduration of these substantia nigra inhibitions was increased.

3. Compared with the zona reticulate, fewer neurones in the zona com-pacta of the substantia nigra responded to caudate stimulation in bothurethane or pentobarbitone anaesthetized animals.

4. The activity of most cells was depressed by electrophoreticallyadministered GABA or glycine and increased by acetylcholine or gluta-mate. Neurones of the mesencephalic reticular formation were less sensitiveto GABA and glycine than substantia nigra neurones. Within the sub-stantia nigra, both zona compacta and zona reticulate neurones weremore sensitive to GABA than to glycine. Over-all, glutamate was a morepotent excitant than acetylcholine (ACh).

5. Electrophoreticbicuculline methochloride (BMC) consistently reduced"ABA but not glycine depression of substantia nigra neurones. Approxi-mately twice as much BMC was required to reduce the endogenous

A. DRAY, T. J. GONYE AND N. R. OAKLEY

inhibition of the same substantia nigra neurones and the amplitude of theconcomitantly evoked positive field potential as was required to abolishexogenous GABA responses. Some evoked substantia nigra inhibitionswere resistant to BMC.

6. Electrophoretic strychnine consistently reduced glycine but notGABA depression of substantia nigra neurones, and did not modifycaudate evoked inhibition of these neurones or the accompanying fieldpotential.

7. The results support the concept of a slowly conducting caudato-nigral pathway which has both facilitatory and inhibitory components.The inhibitory pathway uses GABA as the neurotransmitter. The identityof the possible excitatory transmitter is unknown. The monosynapticnature of this pathway is uncertain and the possible contribution of otherbicuculline insensitive nigral inhibitory processes is discussed.

INTRODUCTION

There is convincing anatomical and histochemical evidence for theexistence of efferent projections from the striatum to the ipsilateral sub-stantia nigra (Voneida, 1960; Szabo, 1962, 1967, 1970; Kemp, 1970;Grofova & Rinvik, 1970; Kim, Bak, Hassler & Okada, 1971; Hajdu,Hassler & Bak, 1973; Usunoff, Hassler, Wagner & Bak, 1974; Grofova,1975).The substantia nigra contains large concentrations of y-aminobutyric

acid (GABA) (Fahn & Cot6, 1968; Okada, Nitsch-Hassler, Kim, Bak &Hassler, 1971; Kim & Hassler, 1975), its synthesizing enzyme, gluta-mate decarboxylase (GAD) (McGeer, McGeer, Wada & Jung, 1971;Fonnum, Grofova, Rinvik, Storm-Mathisen & Walberg, 1974; Kataoka,Bak, Hassler, Kim & Wagner, 1974) and its degrading enzyme GABA-transaminase (Salvador & Albers, 1959; Kataoka et al. 1974). Both theuptake of GABA into nerve terminals in the substantia nigra (Hattori,McGeer, Fibiger & McGeer, 1973) and its release from slices of substantianigra (Hokfelt, Jonsson & Ljungdahl, 1970; Okada & Hassler, 1973)have been demonstrated. Furthermore, electrophysiological studies haveshown that the activity of neurones in the substantia nigra may be re-duced by electrical stimulation of the ipsilateral caudate nucleus (Gross-man, 1966; Frigyesi & Purpura, 1967; Feltz, 1971; Goswell & Sedgwick,1971; Yoshida & Precht, 1971; Crossman, Walker & Woodruff, 1973;Feger & Ohye, 1975) and also by GABA applied by micro-electrophoresis(Feltz, 1971; Crossman et al. 1973). In addition, both the caudate evokeddepression of substantia nigra neurones and that produced by GABAmay be reduced by picrotoxin (Precht & Yoshida, 1971; Crossman et al.

826

SYNAPTIC ACTIVITY IN SUBSTANTIA NIGRA

1973) and on this evidence it has been suggested that the striato-nigralpathway is inhibitory and utilizes GABA as the neurotransmitter.

However, some of these electrophysiological studies reveal the activityevoked in the substantia nigra to be more complex than simple inhibition.Thus, the possibility of an additional excitatory input to the substantianigra has been raised by Frigyesi & Purpura (1967) who showed thatstimulation of the caudate nucleus induced an e.p.s.p.-i.p.s.p. sequence insubstantia nigra neurones and both Feltz (1971) and Feger & Ohye (1975)have demonstrated an excitation of substantia nigra neurones whichpreceded inhibition. The lack of such responses in other studies has beensuggested to be a result of barbiturate anaesthesia (Feger & Ohye, 1975).In an attempt to clarify this and to amplify previous studies made in rats(Grossman et al. 1973) we have made a more comprehensive electrophysio-logical and pharmacological investigation of neuronal responses in the ratsubstantia nigra to stimulation of the caudate nucleus during urethane orpentobarbitone anaesthesia. Additionally, since the substantia nigramay be sub-divided into two distinct areas the zona compact and zonareticulate (Rinvik & Grofova, 1970; Gulley & Wood, 1971; Schwyn &Fox, 1974) with apparently differing pharmacology (Aghajanian & Bun-ney, 1974) it seemed worth while to explore this and to define the char-acteristics of the possible striatal input to these regions.Some preliminary results of this study have been communicated to the

Physiological Society (Dray & Gonye, 1975).

METHODS

These experiments were performed on male albino rats (230-270 g) lightly anaes-thetized by i.P. injections of either urethane (1.2-1-4 gfkg) or pentobarbitone(Nembutal, 30-40 mg/kg). The depth of anaesthesia was judged by testing for theloss of corneal and limb-withdrawal reflexes. The electrocorticogram was not con-sidered to be a reliable index since the pattern of activity produced by these agentsdiffers markedly.

Since both the caudate nucleus and the ipsilateral substantia nigra were ap-proached dorsally the overlying cortex was initially exposed and covered withwarm agar. Pulsations of the brain and cerebral oedema were minimized by allowingcerebrospinal fluid to leak from a cisternal puncture. The electrocorticogram wasroutinely recorded from two stainless-steel screws placed on the surface of the cortexand the animal's temperature was maintained at 36-37° C throughout.A bipolar stainless-steel stimulating electrode (Rhodes Medical Instruments)

(tip separation 0*25 mm) with either a coaxial or bipolar configuration, insulated towithin 0-25 mm of the tip, was advanced through the cortex and positioned stereo-taxically in the caudate nucleus, using co-ordinates obtained from the rat stereotaxicatlases of Fivkova & Marsala (1967) or Konig & Klippel (1963). Subsequent histo-logical examination revealed that the electrode tip was in the medial portion of thecaudate nucleus; stereotaxic co-ordinates L 2-0-3-0; A 8 0-9 0; D 1-0-1 5 (Konig &Klippel, 1963).

827

A. DRAY, T, J. GONYE AND N. R. OAKLEYPulses generated from an isolated constant voltage stimulator (Hivotronic) were

delivered to the stimulating electrode at periods determined by a gated-generator(Hivotronic). The stimulus current was monitored on an oscilloscope by measuringthe voltage drop across an 100 Q resistor. Stimulation parameters were chosen to bejust supra-threshold for evoking activity in the substantia nigra, but at the sametime producing minimal effects on the electrocorticogram. Short trains of high fre-quency stimulation (4100 Hz. 10 msec train) appeared not to modify either the typeof responses observed in the substantia nigra nor their duration or intensity. Thecaudate stimulation used in this study had the following parameters; single square-wave pulses of 20-180 flA, duration 0-2-0-6 msec at 1-2 Hz.

Action potentials were recorded extracellularly from single neurones in the sub-stantia nigra or in the overlying mid-brain reticular formation, within an areadelineated by the co-ordinates L 2-0-3-0; A 1-0-3-0; D 0-6-2-6 (atlas of Konig &Klippel, 1963). Seven barrel glass micropipettes (tip diam. 6-8 /tm) were used for thispurpose and to apply drugs close to neurones by electrophoresis. Micropipettes werefilled with aqueous solutions either by centrifugation or by a glass fibre method(Tasaki, Tsukahara, Ito, Wayner & Wu, 1968) immediately before use. The centrerecording barrel contained 3 M-NaCl and another containing 1 M-NaCl was used tobalance the net current at the electrode tip to zero or to expel Na+ in order to evalu-ate the effects of iontophoretic current. The other barrels were filled with drugs atthe following concentration and pH: GABA, 0-2 M, pH 3-5 (Sigma); glycine, 0-2 m,pH 3-5 (B.D.H.); strychnine sulphate, 5 mm, pH 3-5 (Hopkin and Williams); (+ )-bicuculline methochloride, 5 mm, pH 3-5 (synthesized by Dr J. F. Collins); acetyl-choline chloride, 0-5 M, pH 4*5 (B.D.H.); Na glutamate, 0-2 M, pH 8-5-9-0 (B.D.H.).

Extracellular spikes were amplified and displayed on an oscilloscope. Actionpotentials were voltage-gated, converted to pulses of constant amplitude and dura-tion (Courtice, 1975) and these were displayed with a pen recorder as a continuousrecord. The gated pulses were also fed on-line into a computer (PDP-12) whichgenerated post-stimulus time histograms of cell responses during stimulation.Evoked potentials were also recorded through the micropipette and were eithersummed on a storage oscilloscope or averaged on the PDP- 12 or on a BIOMAC 1000computer. Measurements of the latency and duration of neuronal responses weremade either from photographed (Medelec) records of post-stimulus time histogramsobtained from summing 100-200 consecutive stimuli or from photographed recordsof oscilloscope traces.

Spontaneously firing cells in both the zona compacta or zona reticulate of thesubstantia nigra and in the overlying mesencephalic reticular formation weretested for their responsiveness to microiontophoretic applications of GABA, glycine,acetylcholine or glutamate and for their behaviour during electrical stimulation of theipsilateral caudate nucleus. Cells which were considered to be within the substantianigra, and which responded to both GABA and glycine as well as to caudate stimu-lation were tested with either bicuculline methochloride (BMC) or strychnineapplied from an adjacent barrel of the micropipette.The position of the micropipette tip was marked (P1. 1) by ejecting pontamine

sky blue dye (2-5 #uA for 5-10 min) from one barrel of the electrode (Hellon, 1971)after recording from a neurone that had responded to caudate stimulation or onwhich a pharmacological study had been completed. These positions were verifiedhistologically after each experiment by examining serial sections of brain which hadbeen cut on a freezing microtome and stained with a myelin or Nissel stain. Thebrain was fixed after an experiment by injecting formol-saline directly into the leftventricle after first clamping the descending aorta and severing the jugular veins.Similar perfusions with formol-saline containing potassium ferricyanide (2-5 %)

828

SYNAPTIC ACTIVITY IN SUBSTANTIA NIGRA 82and its subsequent reaction with iron deposited by the prior passage of a brief cur-rent (0.1-2 mA for 10 sec) facilitated the precise localization of the stimulatingelectrode tip (P1. 1). Statistical analysis of results was performed using Student'st test.

RESULTS

A total of 646 spontaneously active cells were tested for their responseto stimulation of the caudate nucleus. Of these 320 were localized in thesubstantia nigra and 132 in the overlying mesencephalic reticular formationin urethane anaesthetized animals. A further 116 substantia nigra cellsand seventy-eight mesencephalic cells were tested in pentobarbitone-anaesthetized animals.The distribution of spontaneously active cells corresponded closely

to the anatomical structures encountered during the descent of therecording micro-electrode. Thus cells were found in the mesencephalic

FOR 3

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Text-fig. 1. Diagrammatic representation of three transverse sectionsthrough the substantia nigra. Representative sample of the position,revealed by dye-marking, of n~eurones inhibited (dots) or unaffected(crosses) by caudate stimulation are shown in the different anatomicalareas. Micro-electrode penetrations were made through the vertical plane.No cells were encountered in the medial lemniscus (LM). Neurones, in-hibited by caudate stimulation were located mainly in the substantia nigra,zona reticulate (SNR) and also in the zona compacta (outlined by the inter-rupted line immediately above SNR). Further details are given in the text.r, red nucleus, CC, cerebral peduncle; FOR, mesencephalic reticularformation (Konig & Klippel, 1963).

829

830A. DRAY, T. J. CON YE AND N. R. OAKLEYreticular formation and in the substantia nigra areas, but few in the areacorresponding to the medial lemniscus (Text-fig. 1). Although caudatestimulation evoked activity in cells in the substantia nigra and in themesencephalic reticular formation, both the latency of response and tosome extent the types of responses were different in the respective areas.Pontamine blue dye marking of responsive cells showed an apparentrandom distribution in the mesencephalic reticular formation whereas inthe substantia nigra they appeared to be confined in general to the medialportion and were on the whole more readily observed (see later results)in the zona reticulate (Text-fig. 1). However, cells responding with onepattern of response or another were encountered in all areas of thesubstantia nigra.

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Text-fig. 2. Effects of electrical stimulation of the caudate nucleus on theactivity of neurones in the ipsilateral substantia nigra. The top left figureshows an a.c. record (ten superimposed sweeps) of inhibition of spontaneousneuronal activity after caudate stimulation. The period of inhibition wasaccompanied by a focal positive field potential (ten sweeps, d.c.), and theafter-excitation by a negative field potential. Below (left) is a computergenerated post-stimulus time histogram (PSTH) summning the eventsfrom the same neurone after 200 consecutive stimuli (sweep duration250 msec). The position of the stimulus is indicated by the arrow andcalibration bars are given in the Figure. On the right is an example ofmore complex activity evoked in a different substantia nigra neuroneduring caudate stimulation. Inhibition was interrupted by a brief periodof excitation (top trace, ten sweeps). These events were accompanied by apositive field potential which coincided with the initial inhibitory pauseand a negative field potential during the period of excitation. The PSTHbelow (sweep 500 msec) represents the summation of 200 consecutivestimuli and shows the events clearly.

830


Responses in the substantia nigra(a) Urethane anaesthesia

Inhibition of spontaneous activity after caudate stimulation was themost common response observed in substantia nigra neurones (275/320)(Text-figs. 2 and 3). In the majority of these neurones (192 cells) thereduction of spontaneous activity occurred after a similar and relativelyconstant latency (mean 5-4 + 0-2 msec) (Text-fig. 4). In other neurones(83/275) inhibition was preceded by a period of excitation (Text-figs.2 and 3) and sometimes was followed by an after-excitation (81/275)(Text-figs. 2 and 3) which occasionally showed periods of clear reverbera-tion (Text-fig. 3). The initial period of excitation was usually brief (mean

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Text-fig. 3. Post-stimulus time histograms (200 sweeps) of evoked activityin substantia nigra neurons. A, simple inhibition; B, excitation precedinginhibition and marked rebound excitation; C, clear post-inhibitory re-verberation; DA excitation preceding inhibition with a peak of excitationduring the inhibitory pause. The position of stimulus is indicated by thearrow. Sweep duration is 250 msec, but 500 msec in C.

12-2 + 1-2 msec) and occurred with a similar latency (5.9 + 0 4 msec) tothe evoked inhibition described above. These initial excitations usuallyconsisted of a burst of action potentials which did not always faithfullyfollow 2 Hz stimulation or higher frequency stimulation at 100 Hz. Morerarely a brief excitation (9/192) occurred during an inhibitory pause(Text-fig. 3).

831

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A. DRA Y, T. J. GONYE AND N. R. OAKLEY

The duration of evoked inhibition was variable (range 8-380 msec)and had a mean period of 65-4 + 8-5 msec when it occurred alone, but wassignificantly shorter (mean 34-8 + 5-2 msec) when followed by an after-excitation. The duration of the inhibition which was preceded by aninitial excitation was not significantly different (mean 74-8 + 10-1 msec)from that of inhibition alone.A small number of neurones (13/320) were simply excited by caudate

stimulation. These excitations occurred after a similar latency (mean5-2 + 0-8 msec) to the excitations preceding an inhibition. However, theduration (21-6 + 1-7 msec) of the former was significantly longer. Bothinhibition or excitation of a neurone was induced concomitantly with thesame threshold of stimulation and the direction of neuronal responsesrarely changed during repeated testing. The effects described above havebeen summarized in Table 1.

Location of the micro-electrode tip with pontamine-blue dye revealedthat fifty-six of the cells studied were in the zona compacta and 264were in the zona reticulate. Responses of all types previously describedwere found in both areas (Table 2) and neurones located in one or otherarea could not be distinguished on the basis of latency or duration of theevoked effect. However, it was notable that proportionally fewer cells(37/56 = 66 %) in the zona compacta were affected by caudate stimulationcompared with the zona reticulate (251/264 = 950/)

(b) Pentobarbitone anaesthesiaQualitatively similar responses of substantia nigra neurones were

observed during pentobarbitone anaesthesia as those seen during urethaneanaesthesia (Table 1). Thus inhibition of spontaneous activity was thecommonest response (104/116 cells). This occurred alone (46/104) or wasaccompanied by an after-excitation (23/104). The latency of onset of in-hibition was more variable (Text-fig. 4) and overall was significantly longer(mean 8-9 + 0-7 msec; P < 0-001) than that observed during urethaneanaesthesia. In contrast to the observations under urethane, the durationof inhibition alone (mean 79-7 + 9-7 msec) was similar to that which wasfollowed by after-excitation (mean 89-6 + 2-1 msec). Only one cell showeda reverberatory excitation/inhibition following the inhibitory pause.No cells responded by excitation alone.The proportion of cells (35/104) in which excitation preceded inhibition,

was similar to that observed during urethane anaesthesia. Moreover, thelatency of onset (mean 6-3 + 0-4 msec) and duration of the initial excita-tion (10-6 + 1-4 msec) as well as the duration of the inhibitory pause(96.0 + 14-8 msec) were not significantly different from that measuredduring urethane anaesthesia.

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A. DRA Y, T. J. CONYE AND N. R. OAKLEY

Of the cells studied, twenty-three were in the zona compacta and therest (ninety-three) in the zona reticulate. Responses of each type describedwere observed in both areas and these have been summarized in Table 2.However, as before, proportionally fewer cells responded in the zonacompacta (16/23 = 69-6%) compared with the zona reticulate (88/93 =94-7%).

TABLE 2. Responses of neurones in the zona compacta (SNO) or zona reticulate(SNR) of the substantia nigra during stimulation of the caudate nucleus. Thenumbers refer to the number of neurones

Inhibition ExcitationExcitation Inhibition followed by before Not

only only excitation inhibition affected

Urethane SNC 6 12 10 9 19SNR 7 99 71 74 13

Pentobarbitone SNC 0 9 2 5 7SNR 0 37 21 30 5

Responses in the mesencephalic reticular formationNeurones in the mesencephalic reticular formation, like those in the

substantia nigra exhibited complex patterns of activity after caudatestimulation. Over-all qualitatively similar responses were observed, butthere were marked differences in time course and in the proportions ofneurones responding. The effects have been summarized in Table 3.

Urethane anaesthesiaCompared with the substantia nigra (90 %), fewer cells in the mesen-

cephalic reticular formation (55%) responded to caudate stimulation.However, of the responsive cells (72/132), inhibition was the predominanteffect (62/72) and this was observed to occur alone (thirty-five cells) orwas followed by an after-excitation (eleven cells) which was sometimesreverberatory (3/11 cells). The latency of inhibition was variable and wassignificantly longer than in the substantia nigra (16.6 + 2*2 msec) (Text-fig. 4). However, similar differences were observed in the duration of in-hibition. This was significantly shorter (28-8 + 8-7 msec) when accompaniedby after-excitation than when occurring alone (82.6 + 8-2 msec).

In other cells (16/62) inhibition was preceded by an excitation whichwas brief (19.9 + 3.5 msec) but clearly occurred with shorter latency(8.3 + 1.1 msec) than the inhibition of mesencephalic reticular formationneurones. However, the duration of the inhibition preceded by initialexcitation was similar (78-2 + 160 msec) to inhibition alone (Table 3).

834

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Text-fig. 4. Latency histograms of inhibition of substantial nigra (SN) andmesencephalic reticular formation (MRF) neurones after stimulation of theipsilateral caudate nucleus. The number of cells has been plotted againstlatency in msec. Mean latency is indicated by the arrows. The latency ofinhibition of SN neurones during urethane (mean 5-4 msec) and pento-barbitone (mean 8-6 msec) anaesthesia was significantly different. Therewas a wide scatter in the latency of inhibition of MRF neurones duringboth urethane (mean 16-6 msec) and pentobarbitone (mean 21-3 msec)anaesthesia.

Pentobarbitone anaesthesiaOver-all there was a notable difference in the proportion of responses

(92%) compared with that during urethane (55 %) anaesthesia (Table 3).This difference may be due to sampling differences resulted from record-ing from heterogeneous neuronal populations during micro-electrodepenetrations through 'reticular' areas. Nevertheless the predominantresponse observed was inhibition of cell firing (71/78). This occurred alone(42/71); with an after-excitation (13/71) or was preceded by initial excita-tion (16/71). The latency of onset and duration of the effects observedwere broadly similar to those during urethane anaesthesia. However,similar to the observations in the substantia nigra during pentobarbitoneanaesthesia, the duration of inhibition followed by excitation was longer

836


(145.8 + 48-1 msec) than that during urethane anaesthesia. In additionfewer simple excitations (one cell) were observed and no cells showedreverberatory sequences following a period of inhibition.

Neuronal firing rateOverall there was a significant difference (P < 0-01) between neuronal

firing rate in the substantia nigra areas and the mesencephalic reticularformation (19-0 + 1-4 Hz). Further neuronal firing rate in the zona com-pacta (mean 11 6 + 0 9 Hz) was significantly slower (P < 0-001) than inthe zona reticulate (23.6 + 1 2 Hz) of the substantia nigra. Action poten-tials of cells in the zona compacta were generally smaller (100 fV) thanin the zona reticulate (100-400 ,tV) and often showed irregular bursts ofactivity though no detailed analysis of this was undertaken. The dischargerate of cells in the zona reticulate were generally regular. The differencesin discharge rate, firing pattern and spike size were not however consideredto be sufficient for the routine identification of individual zona compactaor zona reticulate neurones. It was difficult to identify zona compactacells electrophysiologically, since none were observed to be antidromicallyactivated during possible stimulation of their axon projections in thestriatum. In the final analysis histological localization of dye marks hadto be relied upon for identification of the precise substantia nigra areastudied.

There was little correlation between nigral cell firing rate and thedirection or pattern of response to caudate stimulation (Table 1). In addi-tion cell firing did not appear to be significantly affected during pento-barbitone anaesthesia.

Evoked field-potentialsDuring caudate stimulation evoked field-potentials, representing local

current movements, were recorded through the micro-electrode in thesubstantia nigra. These generally coincided with the activity evokedfrom a single cell and were positive-going during a period of cell inhibition(Text-fig. 2) and negative-going during a period of cell activation (Text-fig. 2). The amplitude of the evoked-wave was usually related to theintensity of caudate stimulation. Positive field-potentials (100-500,V)were always recorded when the electrode-tip was situated in or close to thesubstantia nigra, but similar smaller fields (50-200 ,V) could be recordedless consistently in the mesencephalic reticular formation.

Effects of electrophoretic applications of putative neurotransmittersThe responses of spontaneously active cells located in the mesencephalic

reticular formation, zona compacta or zona reticulate of the substantia

837

A. DRAY, T. J. GONYE AND N. R. OAKLEYnigra were tested with electrophoretic applications of either GABA, gly-cine, ACh or glutamate in urethane anaesthetized animals. The activity ofmost cells was depressed by GABA (345/372) and by glycine (239/252)whereas ACh (69/81) and glutamate (84/88) excited most cells. Theeffects of these compounds have been summarized in Table 4. Neuronal

TABLE 4. Responses of spontaneously active cells in the mesencephalic reticularformation and substantia nigra (zona compacta (SNC) and zona reticulate (SNR)to electrophoretically applied GABA, glycine, ACh or glutamate. The figures in thenumerator column refer to the number of cells responding and those in the denomina-tor column to the number of cells tested. The mean expelling current required toproduce 50-100 % change in the firing rate of all the cells studied is also indicated

GABA Glycine ACh Glutamate

MRF No. of cells 134 147 99 104 20 23 23 29Current (nA) 11991 101 3 25-2 11'2

SNC No. of cells 54 58 34 38 17 18 19 19Current (nA) 52-5 73-1 24*7 9-4

SNR No. of cells 157 167 106 110 42 50 52 54Current (nA) 32-3 53-1 31-3 8-6

responses were considered to be genuine since they were not mimickedby iontophoretic applications of Na+ and changes in neuronal firing of50-100% could be reproduced consistently (three trials) during a 30-60sec application of an agonist at an appropriate current. In addition themagnitude of the electrophoretic current required to produce closely com-parable responses during an identical period of ejection was used as ameasure of cell sensitivity to the agonist.

Over-all, there were no differences in the proportion of neurones re-sponding to one or other of the compounds in each region studied. Therewere, however, differences in the electrophoretic currents required toelicit a comparable response. Cells in the mesencephalic reticular forma-tion appeared to be significantly (P < 0-01) less sensitive to GABA andglycine than those in the substantia nigra (Table 4). This was found forthe responses over-all as well as for responses to these compounds ex-pelled from the same electrode during individual penetrations through themesencephalic reticular formation and substantia nigra. The mean GABAexpelling current required to depress mesencephalic reticular formation cellactivity was 119-1 nA compared with 42-4 nA for nigral cells (P < 0-001).The mean glycine current was 101-3 nA in the mesencephalic reticularformation compared with 63-1 nA in the substantia nigra (P < 0.01).

Within the substantia nigra, GABA (mean 42-4 nA) was significantly(P < 0-02) more potent than glycine (mean 63-1 nA). In addition, zona

838


compacta neurones were overall less sensitive to GABA or glycine thanzona reticulate neurones.

Neuronal sensitivity in the mesencephalic reticular formation to ACh(mean 25-2 nA) or glutamate (mean 11-2 nA) was not over-all significantlydifferent from that in the substantia nigra (mean ACh current, 28-0 nA:mean glutamate current, 9-0 nA). Neither were there any differences inthe sensitivity of zona compacta compared with zona reticulate neurones(see Table 4) to these excitants. However, it was evident that in each areaglutamate was consistently the more potent excitant.

Effects of antagonistsNeurones in the substantia nigra which were both inhibited by caudate

stimulation in a consistent and reproducible manner and which werealso depressed by GABA and glycine were tested with electrophoreticapplications of either bicuculline methochloride (10-50 nA for periods of1-5-20 min) or strychnine (2-20 nA for 1-18 min). The effect of each anta-gonist was assessed on the caudate evoked inhibition of substantia nigracells only after a selective effect had been demonstrated on the responsesto electrophoretic GABA or glycine.

Bicuculline methochloride (BMC) significantly reduced or abolishedthe depressant effects of GABA (sixty-five cells) (Text-fig. 5) withoutaffecting depression by glycine (sixty-five cells) when these agonists wereapplied alternately, with a constant time cycle and with a constant currentto the same cell. The mean BMC expelling charge (current x time) re-quired to produce such selective effects was 6-8 x 10-6 C.BMC also generally reduced both the period of endogenous inhibition

(56/65 cells) and the amplitude of the concomitantly recorded positivefield-potential (59/65) (Text-fig. 5). However, to demonstrate this ap-proximately twice the expelling charge ofBMC was required (12.1 x 10-6 C)compared with that required to selectively antagonize exogenouslyapplied GABA. Often the positive field-potential was completely sup-pressed at times when the duration of neuronal inhibition was unaffected(twenty-two cells). It was notable that although evoked inhibition couldbe reduced by BMC it was exceedingly difficult to abolish. In fact in nineof the sixty-five substantia nigra cells tested caudate evoked inhibitionwas unaltered by BMC despite exhaustive testing (Table 5). No change inthe latency of an inhibitory response was observed during BMC admini-stration, when the inhibitory period had been partially reduced. BMC,however, often produced an increase in the intensity and duration of re-bound excitation which followed an inhibitory pause, but whether thiswas the result of a direct excitatory action or indirectly due to attenuationof the preceding inhibition is uncertain.

839


Although BMC often increased the spontaneous firing rate of cells.this action did not appear to be responsible for selective GABA antago-nism and the effects were temporally unrelated (Text-fig. 5). In additiorit was considered that the increase in firing rate was not responsible foireducing the period of endogenous inhibition, since this could only bcminimally effected by elevating neuronal firing with a continuous application of ACh or glutamate.

Micro-iontophoresis PSTH Evoked potential

20 Caudate stim.1o n r control

10 15iLJmin T500,4V

BMCI~<>ctr~ . 2 nA 10 min

Bicuculine methochlorideZ20 nA JitSS.

Recovery (2 min.

50 msec

17 min

1. ~~~~~~~~~~~ LText-fig. 5. Effects of electrophoretic administration of bicuculline metho-chloride (BMC) on the inhibitory responses of a spontaneously firing sub-stantia nigra neurone to GABA and glycine; and on evoked inhibitionproduced by caudate stimulation. On the left, the continuous rate-meterrecord (spikes per second against minutes; the scale on each trace is thesame) shows reproducible depression of firing produced by consecutiveelectrophoretic applications of GABA (1O nA) and glycine (15 nA). Thecentre Figure shows the PSTH from the same neurone (200 sweeps; sweepduration 500 msec). Inhibition was preceded by brief excitation andfollowed by rebound excitation. On the right, the evoked extracellular fieldpotential comprises' a positive phase corresponding with the period ofneuronal inhibition and a negative phase corresponding with reboundexcitation. During the continuous administration of BMC (20 nA) theresponse to GABA was selectively abolished before any significant increasein background firing. No significant change in evoked inhibition wasobserved at this time but was reduced after 10 min of BMC application.The positive phase of the evoked potential was also reduced. Recovery ofthe responsiveness to GABA (bottom trace) occurred approximately 4-5min after terminating the BMC expulsion. Evoked neuronal inhibition andthe evoked positive potential recovered some 12-5 min later.

840


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841


Strychnine was tested on evoked inhibition in the nigra (thirty-threecells) after a selective antagonism of the depression produced by electro-phoretically applied glycine had been demonstrated (mean expellingcharge 3-7 x 10-6 C. Neither the period of evoked inhibition (31/33) northe amplitude of the positive field-potential (29/33) were usually affectedby strychnine when it was applied in amounts (7-8 x 10-6 C) considerablyin excess of those required to selectively reduce the effects of exogenouslyapplied glycine (Table 5). On the few occasions that evoked inhibition(2/33) and the evoked positive wave form (4/33) were reduced, it wasfound that the effects of electrophoretically applied GABA had also beenreduced (Table 5).

DISCUSSION

In the present experiments the activity evoked in the substantianigra by stimulation of the ipsilateral caudate nucleus was complex. Re-duction of spontaneous activity was the most common effect observedand this is in agreement with previous studies in the cat (Frigyesi &Purpura, 1967; Feltz, 1971; Goswell & Sedgwick, 1971; Yoshida & Precht,1971; Feger & Ohye, 1975) and rat (Crossman et al. 1973).

Inhibition occurred with a relatively constant latency (5.4 msec).In the cat Yoshida & Precht (1971) have reported that substantia nigraneurones were monosynaptically inhibited by caudate stimulation andcalculated the conduction velocity of these fibres to be 0*9-1*0 m sec-1.The present observations in rats indicate a similar conduction velocitythough it is not certain that this represents a monosynaptic pathway.That some inhibitions were of longer latency might indicate the activationof polysynaptic pathways possibly via the globus pallidus (McGeer et at.1971; Hattori et al. 1973) or within the substantia nigra itself (Dray &Straughan, 1976; see also later discussion). In addition, the prolongationof the inhibitory latency during pentobarbitone anaesthesia is compatiblewith a possible contribution from polysynaptic pathways.The duration of inhibition was variable and was sometimes followed

by a rebound excitation or a period of rhythmic excitation/inhibition.Similar responses have been observed in other studies (Frigyesi & Pur-pura, 1967; Feltz, 1971; Goswell & Sedgwick, 1971; Yoshida & Precht,1971; Feger & Ohye, 1975) and may result from an activation of intra-nigral feed-back circuitry. A feed-back circuit may be provided either byinterneurones similar to anatomically identified small-axon neurones(Taber, 1961; Gulley & Wood, 1971; Schwyn & Fox, 1974) or by axoncollaterals from nigral neurones which terminate in the substantia nigraitself (Rinvik & Grofova, 1970; Schwyn & Fox, 1974). When such reboundeffects occurred the initial inhibitory period was significantly shorter.

842


Brief excitation, preceding inhibition was also observed. This occurredwith similar constant latency to evoked inhibition and may have resultedfrom the activation of excitatory striato-nigral fibres. Although similarresponses have previously been observed (Grossman, 1966; Frigyesi &Purpura, 1967; Feltz, 1971; Feger & Ohye, 1975), the lack of such re-sponses in other studies was suggested to be a result of barbiturate anaes-thesia (Feger & Ohye, 1975). In the present experiments these responseswere elicited with the same threshold of stimulus as neuronal inhibitionand moreover both excitation or inhibition could be evoked by smallstimulus currents (20 /%A). It therefore seemed unlikely that excitationresulted from spread of the stimulating current to structures outside thecaudate. This conclusion is supported by the observations of Feger &Ohye (1975) in unanaesthetized monkeys. The experiments with pento-barbitone failed to show any over-all changes in the occurrence of initialexcitation preceding inhibition although fewer neurones showing reboundexcitation were encountered. In addition pentobarbitone significantlyprolonged the period of evoked inhibition (see also Nicoll, Eccles Oshima &Rubia, 1975). The intense nature ofthe initial excitation of substantia nigraneurones may render it less likely to be suppressed by the dose ofanaestheticused. The possibility is therefore raised that the striato-nigral pathway mayafter all haveboth excitatoryand inhibitory components. Indeed anatomicalstudies (Grofova & Rinvik, 1970; Kemp & Powell, 1971) suggest that thereare two types of long axon projections from the caudate to the substantianigra. Moreover, the nerve terminals of these projections appear to containanatomically different vesicles (Grofova & Rinvik, 1970), indicating thepossibility that each projection may release a different neurotransmitter.Of particular interest was the distribution of neurones studied within

the substantia nigra, revealed by dye-marking. Over-all, and in agreementwith other studies (Aghajanian & Bunney, 1974; Rebec & Groves, 1975;Tulloch, 1975), neurones in the zona compacta discharged more slowlyand more irregularly than those in the zona reticulate. It was apparent,however, that considerable bias had occurred in sampling neurones in thesubstantia nigra. Approximately five cells were studied in the zonareticulate for every one in the zona compacta. This may have been due topreferential recording from larger cells in the zona reticulate (Gulley &Wood, 1971; Grofova & Rinvik, 1970). However, similar types of responsewere observed, during caudate stimulation, in the zona compacta as in thezona reticulate, but over-all fewer cells in the zona compacta were in-hibited. Similar observations were made during pentobarbitone anaesthe-sia. The fact that fewer terminals from striatal projections end in the zonacompact than in the zona reticulate (Rinvik & Grofova, 1970; Grofova &Rinvik, 1970) might explain the present observations.

843

A. DRAY, T. J. GONYE AND N. R. OAKLEYThe extracellular action potentials recorded from zona compacta and

zona reticulate neurones were generally small (100 1sV), but larger actionpotentials (400 1V) could be recorded from some zona reticulate neuroneswith the same micro-electrode during the same penetration. Although themagnitude of extracellularly recorded spikes might merely reflect cellgeometry rather than cell size, these observations support the anatomicalevidence for a large neurone found exclusively in the zona reticulate (Gul-ley & Wood, 1971) which may constitute a nigro-thalamic projection(Carpenter & Strominger, 1967; Gulley & Wood, 1971; Rinvik, 1975).Neurones in the mesencephalic reticular formation also showed similar

patterns of response to caudate stimulation as those in the substantianigra. However, the effects usually occurred with longer and more variablelatency, suggesting their mediation through polysynaptic pathways. Thesignificance of the caudate evoked activity in neurones in the mesen-cephalic reticular formation is not clear and may represent contributionsfrom both direct and indirect caudate inputs.Evoked inhibition of substantia nigra neurones was accompanied by a

focal positive field potential which corresponded to that reported in catsby Yoshida & Precht (1971) and which was taken to represent the out-ward flow of inhibitory post-synaptic current (Precht & Yoshida, 1971).The amplitude of this field potential was reduced by picrotoxin but not bystrychnine (Precht & Yoshida, 1971) and it was therefore suggested to be aresult of endogenously released GABA. The present observations generallysupport this conclusion since local administration of BMC, another GABAantagonist (Hill, Simmonds & Straughan, 1973; Dray, 1975) significantlyreduced the amplitude of this positive wave, whereas strychnine did not.However, the evoked inhibition of neuronal firing, tested after a selectivereduction of exogenous GABA responses had been demonstrated was notalways readily suppressed by bicuculline methochloride. These observa-tions may be explained in one or other of the following ways; that evokedinhibition was more intense and more prolonged than the effects ofexogenously applied GABA and would require more antagonist to suppressit, or a totally effective concentration of antagonist cannot be achievedat distant inhibitory dendritic terminals, or that endogenously activatedreceptors are not identical to those activated by exogenously appliedGABA, or an inhibitory neurotransmitter other than GABA is re-leased within the substantia nigra and its action is resistant to bicucullinemethochloride. The latter explanation appears possible in view of theinitial excitations evoked in some substantia nigra neurones and the factthat the substantia nigra contains other putative inhibitory neurotrans-mitters notably dopamine and 5-hydroxytryptamine (see Dray & Straug-han, 1976) whose actions should not be affected by bicuculline (Dray, 1975).

844


Most neurones in the substantia nigra and in the mesencephalic reticularformation were inhibited by electrophoretic administration of GABA.However, substantia nigra neurones particularly those in the zonareticulate were more easily affected, a difference which may reflect thedistribution of GABA releasing boutons and hence receptive sites in thesubstantia nigra (Rinvik & Grofova, 1970). Glycine also depressed theactivity of mesencephalic reticular formation and substantia nigra neuronesand a similar differential spectrum of neuronal sensitivity was observedas that with GABA. It was notable, however, the substantia nigra neuroneswere in general less sensitive to glycine than to GABA. The fact that thesubstantia nigra contains significant amounts of glycine (Dray & Straug-han, 1976), that it may readily depress neurones in this region and thatthis depression is readily suppressed by strychnine raises the possibilityof a transmitter function for glycine. Though these data provide circum-stantial evidence for transmitter identity and evidence for similarities inthe receptors for glycine in the substantia nigra compared with otherbrain areas, further evidence concerning its distribution and release fromnerve terminals in the substantia nigra must be forthcoming before atransmitter role can be seriously considered.Most cells in the regions studied could be excited by ACh and glutamate.

Though there appeared to be no regional differences in neuronal sensitivityto these agents, glutamate was consistently the more powerful agonist.Putative transmitter substances have previously been shown to affectneuronal activity in this region, though differences in sensitivity of differ-ent neuronal populations have not been routinely explored. However,Aghajanian & Bunney (1973) have reported that zona compacta and zonareticulate neurones were depressed by GABA as were undefined substantianigra neurones in other studies (Feltz, 1971; Crossman et al. 1973; Cross-man, Walker & Woodruff, 1974). In addition glycine readily depressedsubstantia nigra neurones (Crossman et al. 1974) and glutamate excitedneurones in both regions of the substantia nigra (Aghajanian & Bunney,1973). ACh also consistently excited rat substantia nigra neurones (Cross-man et al. 1974) especially in the zona reticulate (Aghajanian & Bunney,1974). On the other hand in the cat, ACh and glycine were ineffective inthis region (Feltz, 1971) suggesting possible species variations.The observations from the present study lend support for a slowly con-

ducting caudate-nigral pathway which has both facilitatory and inhibitorycomponents. That GABA is the inhibitory transmitter is also supported,but the possible participation of other inhibitory neurotransmitters shouldbe considered. The identity of the excitatory transmitter in this pathwayis unknown, but clearly the effectiveness of ACh and glutamate makesthese substances possible candidates for such a role.

845

A. DRAY, T. J. GONYE AND N. R. OAKLEY

Studies involving discrete lesions in the substantia nigra (Dray, Oakley& Simmonds, 1975; Dray, Fowler, Oakley, Simmonds & Tanner, 1975)suggest that both subdivisions of this nucleus are important in con-trolling motor behaviour. However, though it appears that the zona reti-culata rather than the zona compacta receives much of the sensory inputto this nucleus and is involved in its integration (see Schwyn & Fox,1974; Dray & Straughan, 1976), the significance of the differences in firingrate, evoked activity and pharmacology of these regions, described inthis report, cannot be readily explained. Further anatomical and physio-logical studies on more precisely identified substantia nigra neurones arerequired to elucidate their contributions to the activity of this region.

We wish to thank Professor D. W. Straughan for his helpful criticism of thismanuscript. This work was supported by an M.R.C. Programme Grant to D.W.S.

REFERENCES

AGHAJANIAN, G. K. & BUNNEY, B. S. (1973). Central dopaminergic neurons:neurophysiological identification and responses to drugs. In Frontier8 in Catechol-amine Research, ed. SNYDER, S. H. & USDIN, E., pp. 643-648. Long Island City,N.Y.: Pergamon.

AGHAJANIAN, G. K. & BUNNEY, B. S. (1974). Dopaminergic and non-dopaminergicneurons of the substantia nigra: differential responses to putative transmitters.J. Pharmacol. (Fr.). 5 suppl. 1, 56-57.

CARPENTER, M. B. & STROMINGER, N. L. (1967). Efferent fibres of the subthalamicnucleus in the monkey. A comparison of the efferent projection of the sub-thalamic nucleus, substantia nigra, and globus pallidus. Am. J. Anat. 121, 41-72.

CoURTIcE, C. J. (1975). Action potential selection by amplitude and width.J. Phyaiol. 246, 18-19P

CRossMAN, A. R., WALKER, R. J. & WOODRUFF, G. N. (1973). Picrotoxin antago-nism of gamma-aminobutyric acid inhibitory responses and synaptic inhibitionin the rat substantia nigra. Br. J. Pharmac. Chemother. 49, 696-698.

CRossMAN, A. R., WALKER, R. J. & WOODRUFF, G. N. (1974). Pharmacologicalstudies on single neurones in the substantia nigra of the rat. Br. J. Pharmac.Chemother. 51, 137-138P.

DRAY, A. (1975). Comparison of bicuculline methochloride with bicuculline andpicrotoxin as antagonists of amino acid and monoamine depression of neurones inthe rat brain stem. Neuropharmacology 14, 887-891.

DRAY, A., FOWLER, L. J., OAKLEY, N. R., SIMMONDS, M. A. & TANNER, T. (1975).Comparison of circling behaviour following unilateral inhibition of GABA-transaminase or discrete electrolytic lesioning in the rat substantia nigra. Br. J.Pharmacy. Chemother. 55, 288P.

DRAY, A. & GoNqE, T. J. (1975). Effects of caudate stimulation and micro-iontophoretically applied substances on neurones in the rat substantia nigra.J. Phy8iol. 246, 88-89P.

DRAY, A., OAKLEY, N. R. & SIMMONDS, M. A. (1975). Rotational behaviour follow-ing inhibition of GABA metabolism unilaterally in the rat substantia nigra.J. Pharm. Pharmac. 27, 627-629.

DRAY, A. & STRAUGHAN, D. W. (1976). Synaptic mechanisms in the substantia nigra.J. Pharm. Pharrmac. 28. 400-405.

846

SYNAPTIC ACTIVITY IN SUBSTANTIA NIGRAFARN, S. & COT*, L. J. (1968). Regional distribution of gamma-aminobutyric acid(GABA) in brains of the Rhesus monkey. J. Neurochem. 15, 209-213.

FEGER, J. & OHYE, C. (1975). The unitary activity of the substantia nigra followingstimulation of the striatum in the awake monkey. Brain Re8. 89, 155-159.

FELTZ, P. (1971). Gamma-aminobutyric acid and a caudato-nigral inhibition. Can.Jnl Phy8iol. & Pharmacol. 48, 1113-1115.

FIFKOVA, E. & MARSALA, J. (1967). Electrophysiological Methods in BiologicalResearch, pp. 653-731. New York: Academic Press.

FONNUM, F., GROFOVA, I., RINVIK, E., STORM-MATHISEN, J. & WALBERG, F. (1974).Origin and distribution of glutamate decarboxylase in substantia nigra of the cat.Brain Res. 71, 77-92.

FRIGYESI, T. & PURPURA, D. P. (1967). Electrophysiological analysis of reciprocalcaudato-nigral relations. Brain Res. 6, 440-456.

GoswELL, M. J. & SEDGWICK, E. M. (1971). Inhibition in the substantia nigrafollowing stimulation of the caudate nucleus. J. Physiol. 218, 84-85P.

GROFOVA, I. (1975). The identification of striatal and pallidal neurons projecting tosubstantia nigra. An experimental study by means of retrograde axonal transportof horseradish peroxidase. Brain Re8. 91, 286-291.

GROFOVA, I. & RINVIK, E. (1970). An experimental electron microscopic study onthe striatonigral projection in the cat. Exp. Brain Res. 11, 249-262.

GROSSMAN, R. G. (1966). Mechanisms of generation of potentials produced in theaubstantia nigra following stimulation of the neostriatum. Neurology, Minneap.16, 320.

GULLEY, R. L. & WOOD, R. L. (1971). The fine structure of the neurons in the ratsubstantia nigra. Tis8ue & Cell 3, 675-690.

HAJDU, F., HASSLER, R. & BAK, I. J. (1973). Electron microscopic study of thesubstantia nigra and the stria-nigral projection in the rat. Z. Zellforsch. mikrosk.Anat. 146, 207-221.

HATTORI, T., McGEER, P. L., FIBIGER, H. C. & McGEER, E. G. (1973). On the sourceof GABA-containing terminals in the substantia nigra. Electron microscopicautoradiographic and biochemical studies. Brain Res. 54, 103-114.

HELLON, R. F. (1971). The marking of electrode tip positions in nervous tissue.J. Physiol. 214, 12P.

HiLL, R. G., SIMMONDS, M. A. & STRAUGHAN, D. W. (1973). A comparative study ofsome convulsant substances as y-aminobutyric acid antagonists in the felinecerebral cortex. Br. J. Pharmac. Chemother. 49, 37-51.

HOKFELT, T., JoNssoN, G. & LJUNGDAHL, A. (1970). Regional uptake and sub-cellular localization of [3H]-gamma-aminobutyric acid (GABA) in rat brainslices. Life Sci. Oxford 9, 203-212.

KATAOKA, K., BAK, I. J., HASSLER, R., KIM, J. S. & WAGNER, A. (1974). L-gluta-mate decarboxylase and choline acetyltransferase activity in the substantia nigraand the striatum after surgical interruption of the strio-nigral fibers of thebaboon. Exp. Brain Res. 19, 217-227.

KEmp, J. M. (1970). The termination of strio-pallidal and strionigral fibers. BrainRe8. 17, 125-138.

KEMP, J. M. & POWELL, T. P. S. (1971). The structure of the caudate nucleus of thecat: light and electron microscopy. Phil. Trans. R. Soc. 262, 383-401.

KIM, J. S., BAK, I. J., HASSLER, R. & OKADA, Y. (1971). Role ofy-aminobutyric acid(GABA) in the extrapyramidal motor system. 2. Some evidence for the existenceof a type of GABA-rich strio-nigral neurons. Exp. Brain Res. 14, 95-104.

KIM, J. S. & HASSLER, R. (1975). Effects of acute haloperidol on the gamma-aminobutyric acid system in rat striatuin and substantia nigra. Brain Res. 88,150-153.

847

848 A. DRA Y, T. J. GONYE AND N. R. OAKLEYKONIG, J. F. R. & KLIPPEL, R. A. (1963). The Rat Brain: A Stereotaxic Atla8

of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins:Baltimore.

MCGEER, P. L., MCGEER, E. G., WADA, J. A. & JuNG, E. (1971). Effect of globuspallidus lesions and Parkinson's disease on brain glutamic acid decarboxylase.Brain Re8. 32, 425-431.

NICOLL, R. A., ECCLES, J. C., OSHIMA, T. & RUBIA, F. (1975). Prolongation ofhippocampal inhibitory postsynaptic potentials by barbiturates. Nature, Lond.258, 625-627.

OKADA, Y. & HASSLER, R. (1973). Uptake and release of y-arninobutyric acid(GABA) in slices of substantia nigra of rat. Brain Re8. 49, 214-217.

OKADA, Y., NITSCH-HASSLER, C., KIM, J. S., BAK, I. J. & HASSLER, R. (1971).Role of gamma-aminobutyric acid (GABA) in the extrapyramidal motor system.1. Regional distribution of GABA in rabbit, rat, guinea pig and baboon CNS.Exp. Brain Re8. 13, 514-518.

PRECHT, W. & YOSHIDA, M. (1971). Blockage of caudate-evoked inhibition of neu-rons in the substantia nigra by picrotoxin. Brain Re8. 32, 229-233.

REBEC, G. V. & GROVES, P. M. (1975). Apparent feedback from the caudate nucleusto the substantia nigra following amphetamine administration. Neuropharma-cology 14, 275-282.

RINVIK, E. (1975). Demonstration of nigrothalamic connections in the cat by retro-grade axonal transport of horseradish peroxidase. Brain Re8. 90, 313-318.

RINVIK, E. & GROFOVA, I. (1970). Observations on the fine structure of the sub-stantia nigra in the cat. Exp. Brain Ree. 11, 229-248.

SALVADOR, R. A. & ALBERS, R. W. (1959). The distribution of glutamic-y-amino-butyric transaminase in the nervous system of the rhesus monkey. J. biol. Chem.234, 922-925.

SCHWYN, R. C. & Fox, C. A. (1974). The primate substantia nigra: A Golgi andelectron microscopic study. J. Hirnforech. 15, 95-126.

SZABO, J. (1962). Topical distribution of the striatal efferents in the monkey.Expl Neurol. 5, 21-36.

SZABO, J. (1967). The efferent projections of the putamen in the monkey. ExplNeurol. 19, 463-476.

SZABO, J. (1970). Projections from the body of the caudate nucleus in the rhesusmonkey. Expl Neurol. 27, 1-15.

TABER, E. (1961). The cytoarchitecture of the brain stem of the cat. 1. Brain stemnuclei of cat. J. camp. Neurol. 116, 27-69.

TASAKI, K., TSUKAHARA, Y., ITO, S., WAYNER, M. J. & Wu, W. Y. (1968). A simple,direct and rapid method for filling microelectrodes. Physiol. & Behav. 3, 1009-1010.

TULLOCH, I. F. (1975). Dopaminergic cells - responses to olfactory input. J. Physiol.248, 47P.

USUNOFF, K. G., HASSLER, R., WAGNER, A. & BAK, I. J. (1974). The efferent con-nections of the head of the caudate nucleus in the cat: an experimental morpho-logical study with special reference to a projection to the raphe nuclei. Brain Re8.74, 143-148.

VONEIDA, T. J. (1960). An experimental study of the course and destination of fibersarising in the head of the caudate nucleus in the cat and the monkey. J. comp.Neurol. 115, 75-87.

YOSHIDA, M. & PRECHT, W. (1971). Monosynaptic inhibition of neurons of thesubstantia nigra by caudato-nigral fibres. Brain Re8. 32, 225-228.

The Journal of Physiology, Vol. 259, No. 3 Plate 1

A. DRAY, T. J. GOYNE AND N. R. OAKLEY

I

1.

...... I

,"'iv.

(Facing p. 849)

SYNAPTIC ACTIVITY IN SUBSTANTIA NIGRA 849

EXPLANATION OF PLATE

Transverse histological sections through the rat brain showing (top) the positionof the stimulating electrode tip (arrow) in the caudate nucleus, (bottom) the positionof the micropipette tip in the substantia nigra (SN). The size of the dye spot (arrow)has been exaggerated to facilitate illustration. ML, medial lemniscus; CC, cerebralpeduncle.

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29/39 Brunswick Square, London WC1N lAX (Received 26 February