GABA and the nigrothalamic pathway

GABA and the Nigrothalamic D. W. STRAUGHAN, Department

of Pharmacology,

N. K. MACLEOD,

The School of Pharmacy.

Pathway

T. A. JAMES AND I. C. KILPATRICK 29-39 Bruns~tkk Squat-r, London WCIN IAX. England.

T. A. JAMES AND 1. C. KILPATRICK. GARA c///dthe, ,rig~,~tl~o/trrt~i~~ 1980.~In the rat, electrolytic or kainate lesions of one suhstantia nigra caused a small reduction in the levels of GABA, glutamic acid decarboxylase and glutamate in the ipsilateral medtal thalamus. Nigral stimulation gave similar extracellular synaptic responses in projection identified thalamic VM or PF neurones. These comprised a short latency inhibition (mean 5.1 msec) often following a brief excitation, and a longer latency inhibition (mean 22.3 msec) often following a more sustained excitatory burst. Iontophoretic bicuculline selectively blocked the elects of GABA, but not glycine or 5hydroxytryptamine. on these thalamic neurones, and also blocked both short and long latency synaptic inhibition. Intranigral injection of muscimol massively excited some PF thalamic neurones. The contralateral circling behaviour induced by intranigral muscimol was potentiated by intrathalamic microinjections of picrotoxin and attenuated by ethanolamine-0-sulphate. Thus, GABA appears to be the main inhibitory transmitter of the nigrothalamic pathway and for longer latency inhibition in the medial thalamus where it plays a key role in mediating some

STRAUGHAN,

D. W., N. K. MACLEOD,

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Thalamus

Nigrothalamic pathway

Electrophysiology

THE Substantia Nigra (SN) has a well defined system of dopamine neurones giving rise to the nigrostriatal pathway and is known to have an important role in motor behaviour in animals and extra-pyramidal disorders such as Parkinsonism. One of the major outputs from the striatum appears to project to the SN through the striatonigral pathway; y-aminobutyric acid (GABA) is a major inhibitory transmitter in this system as shown by neurochemical and physiopharmacological studies [7,9]. More recently attention has concentrated on the nigrothalamic pathway as a major outflow system from the SN and for the striatonigral pathway [6, 14,201. The cell bodies of this nigrothalamic pathway lie predominantly in the lateral and central regions of the zona reticulata of the SN and their axons project to the ventromedial nucleus (VM) and to a lesser extent to the Centromedianum-Parafascicular nuclear complex (CM-PF) of the ipsilateral thalamus in the rat [2,8] and to corresponding nuclei in other species, e.g. VM and Ventralis lateralis (VL) in the cat, VA and VL in the monkey. The nigrothalamic pathway provides a critical point for the control of motor behaviour and the regulation of posture; indeed, primate VL has been said to be an important relay component in a tremor circuit [ 161. This complements the knowledge that in Parkinsons disease sustained rhythmic bursts occur in the ventroposterior (VP) and VL nuclei of the thalamus [ 1,131 and that selective VL thalamic lesions relieve tremor symptoms. Further experimental studies on the nigrothalamic pathway and particularly the neurotransmitters involved would thus seem to be of value. The present paper reviews recent work on this pathway in our laboratory using neurochemical, neurophysiological, pharmacological and behavioural techniques. We are grateful to our colleagues M. S. Starr and A. Fletcher for their help.

Copyright

” 1980 ANKHO

International

GABA

TRANSMIITERS

IN THE NIGROTHALAMIC

PATHWAY

In initial neurochemical studies, changes in the levels of putative neurotransmitters in the thalamus were sought after lesions of the SN. Concentric bipolar electrodes (0.5 mm dia.) were placed in the right SN of male Wistar rats anaesthetised with halothane. Electrolytic lesions were made with DC currents of I mA for 10 seconds and the animals allowed to recover. Fourteen days later the animals were sacrificed and the brains removed. After confirming the site of the lesion, a block of medial thalamus (ca. 2 mm”)was removed from each side for assay. Amino acid contents were measured by microdansylation and glutamic acid decarboxylase (GAD), the enzyme synthesizing GABA by fluorometry. The results are summarised in Table 1 and show a small but statistically significant decrease by 19% in GABA and by 15% in glutamate with a 36?% decrease in GAD in the thalamus of the lesioned side compared with the control side. No significant changes in the free concentrations of alanine, glycine or aspartate were detected. The amino acid and GAD levels of the control side were similar to those seen in normal or sham operated rats (M. S. Starr - personal communication). In another set of experiments, the SN was lesioned chemically by microinjection of kainic acid (1 pg in 0.2 ~1). This also caused losses of thalamic GABA, glutamate and GAD which were similar to those seen with electrolytic lesions. Data on individual nuclei in rat thalamus are not available at present. Our GAD data confirm that from Di Chiara’s laboratory [6] where kainate or electrolytic nigral lesions gave a similar (about 33%) reduction in GAD in ventromedial but not ventrobasal thalamus. The simplest hypothesis is that the changes seen after SN lesions are compatible with, and result from, loss of GABA-

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TRANSMITTER AMINO ACIDS AND RELATED MEDIAL THALAMUS FOLLOWING SUBSTANTIA

ENZYMES IN NIGRA LESIONS

Mean percentage change of unlesioned side at 7 days

Glutamate GABA GAD *~<0.05

Electrolytic

Kainate

- 14.7% - 19.4vr” -36.7%

~11.9P ~ 15.3W -33.7s*

probability in paired Students r-test.

ergic nigrothalamic neurones. The small size of the effects seen might reflect (1) the sampling procedure and dilution by admixture of areas not directly affected by the lesion, and/or (2) a very minor contribution of the nigrothalamic pathway to thalamic GABA. Transynaptic changes or disuse atrophy might also cause changes in transmitter levels in the thalamus, although this is unlikely. The loss of thalamic glutamate after nigral lesions might reflect loss of actual nigrothalamic glutamate neurones or damage to corticothalamic axons passing through the SN (see later). Damage to adjacent excitatory tracts by chronic electrolytic and kainate lesions is now a distinct possibility when there are doubts about the selectivity of kainate for cell bodies (Nieuollon personal communication). The next series of experiments involved a combination of electrophysiological and iontophoretic techniques on projection and position-identified thalarnic neurones in the urethane anaesthetised rat. Extracellular recordings were made in the thalamus through a single barrel 3 M NaCl-filled glass micropipette glued to a conventional 7 barrel glass micropipette containing aqueous solutions of a variety of neurotransmitters and their antagonists. Bipolar stainless steel stimulating electrodes (tip dia. 0.5 mm) were placed in the ipsilateral internal capsule, caudate nucleus, frontal cortex, and the contralateral brachium conjunctivum. The stereotaxic co-ordinates were based on the atlas of Kiinig and Klippel [ 151. Conventional iontophoretic techniques with an operational amplifier current pump circuit and current neutralisation were employed as in previous studies from our laboratory. One barrel contained the dye pontamine sky blue which was ejected iontophoretically to mark the location of cells of interest for subsequent histology. The main technique used to identify individual neurones physiologically during an experiment, involved antidromic stimulation and collision. This is illustrated in Fig. 1. With SN stimulation, inhibition is the predominant synaptic response seen in the VM and PF thalamic nuclei. In 61 neurones this inhibition occurred with a short latency (mean=5.1 + 0.5 msec), and mean duration of 67 msec. In many of these neurones the inhibition was preceded by a brief excitation. This is shown in Fig. 2 (lower record) from which it will be appreciated that in the absence of the early excitation the true latency of inhibition might be even shorter. In the remaining 55 neurones, the inhibition was of much longer latency ranging between 15 and 50 msec (mean 22.3 msec) and mean duration 89 msec-one such typical neurone is shown in Fig. 3. In many of these neurones, the long latency inhibition was preceded by a sustained excitatory burst which occurred at a mean latency of about 8 msec

Imv IOms FIG. 1. Oscilloscope photograph (single sweep) of extracellular recording from a rat PF thalamic neurone. In record A the sweep is triggered by a spontaneous action potential, which is also used to trigger after a delay of 2 msec a pair of stimuli (5 msec apart) to the caudate nucleus. Both caudate stimuli give rise to antidromic action potentials in the thalamic neurone-the latency from the stimulus artifact (shown by 0) being about I msec. In record B the paired stimuli are delivered closer to the spontaneous potential (after about 1.5 msec) and because of collision the first stimulus now fails to evoke an antidromic action potential.

after nigral stimulation and persisted for about 20 msec. We believe that these short responses evoked in the rat thalamus by SN stimulation are very likely to be mono-synaptic. Thus, reports on 73 nigrothalamic neurones in rat brain [ 1l] give a latency range for antidromic activation of l-5 msec which corresponds closely to values obtained in a smaller sample of rat VM neurones [3]. It is also relevant to note the short latency of about 6 msec for orthodromic inhibition in the nigrothalamic pathway of the conscious cat [4]. The long latency synaptic inhibitions recorded in the rat thalamus after SN stimulation in the present experiments could well be polysynaptic and involve an inhibitory thalamic intemeurone as well as a longer pathway through other nuclei, perhaps activated orthodromically. In the present experiments, the majority of early synaptic excitations in the rat thalamus evoked by SN stimulation

GABA AND THE NIGROTHALAMIC

A

PATHWAY

l

2ms

20ms

B FIG. 2. Extracellular recordings from two PF thalamic neurones (20 superimposed sweeps). For the cell shown in A, a single stimulus to the substantia nigra (SN) at 0 produces a short latency excitation (shown on a fast time base in the top record) followed by a short latency, substantial period of inhibition (middle record). For a different cell shown in B, a single stimulus to the SN produces inhibition at a latency of 15 msec.

were considered to arise indirectly by activation of corticofugal fibres. Thus, capsular stimulation produced an almost identical excitation in the thalamus but with an even shorter latency. The crucial experiments to confirm this would require decortication, as in the cat, where this removed the short latency nigrothalamic excitation in the thalamus [4]. No clear difference in the SN evoked responses has emerged yet between rat VM or PF neurones. Many of the neurones studied here in both VM and PF could be driven antidromically from the caudate nucleus, whilst the VM neurones were driven in many cases by antidromic stimulation of the cortex or internal capsule, In cat experiments [4], the predominantly inhibitory responses to nigral stimulation were seen in both non-relay and relay neurones of the VL thalamus as well as in the VM and intralaminar nuclei. The

9

duration of these inhibitions in cat thalamus at 30 to 70 msec corresponds well to those seen in the present study in the rat. These present results accord well then with the known projections of thalamic neurones. The next experiments sought to characterise the inhibitory transmitter of the nigrothalamic pathway through pharmacological studies. All the 29 VM and PF thalamic neurones tested were inhibited by iontophoretic applications of GABA, glycine and, when tested, by S-hydroxytryptamine applied for periods up to IO set with ejecting currents up to 55 nA. The typical record in Fig. 3 shows in the centre panel that iontophoretic bicuculline blocks GABA responses selectively, so that the neuronal depression induced by glycine or S-hydroxytryptamine is unaffected. In other experiments, iontophoretic strychnine, which is known to be a potent and selective glycine antagonist in other regions, readily and selectively blocked the effects of iontophoretic glycine but not GABA. These antagonists were then tested against synaptic responses evoked in the thalamus by SN stimulation. In 27 out of the 31 thalamic neurones tested, iontophoretic bicuculline blocked both short and long latency synaptic inhibition, as in Fig. 3, though somewhat Iarger currents were required compared with those to block iontophoretic GABA-as with caudate-evoked inhibition of SN neurones [7]. Strychnine was effective in blocking synaptic inhibition in only one of the 35 cells examined. These bicuculline data provide powerful supporting evidence for GABA being an important inhibitory transmitter in the nigrothalami~ pathway. Taken together with our earlier data on latency, we suggest that GABA might be both released monosynaptically-from projection-type GABA neurones with cell bodies in the SN to give short latency inhibition, and perhaps indirectly-through activation of GABAreleasing thalamic interneurones to give short or long latency inhibition. Though, at present, we have been unable to detect neurones with the characteristics of inhibitoryintemeurones, they are known to be hard to detect in other brain areas. With regard to GABA-mediated control of the nigrothalamic pathway, it should be noted that the majority of projection-identified nigrothalamic and reticulata neurones in rat brain were inhibited by caudate stimulation and the Iong axon GABA-ergic striatonigral pathway [3,7]. However, it is worth noting that normally GABA-ergic neurones from other sites might also impinge on nigrothalamic neurones. Thus, substantial lesions in the main descending striatonigral GABA pathway at the level of the crus cerebri did not prevent the distinctive circling behaviours induced by the placement in the crru&l SN of a variety of drugs potentiating or blocking endogenous GABA-mediated synaptic transmission [ 12,191. These latter studies did not utilise direct agonists as these might act on “spare” extrasynaptic receptors as well as on functional synaptic GABA receptors and so give misleading information as to the actual role of GABA in the nigra. However, the effects of indirect agonists or potentiators are replicated by direct GABA agonists. Thus, several groups have shown that a single microinjection of muscimol at critical sites in the rat SN causes a distinction pattern of contralateral turning mediated essentially independently of the dopamine system [17,18]. It seemed of interest to confirm the role of the nigrothalamic pathway in mediating this muscimol-induced circling behaviour and determine the types of thalamic neurone principally involved in the rat. In recent experiments in our laboratory, a single microinjection into the SN

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L msec. FIG. 3. The teft hand panel shows superimposed oscihoscope pboto~phs of the firing of a medial thalamic neurone. The top record shows synaptic inhibition evoked by SN stimulation in the control period. In the middle record, the inhibition has been attenuated by the iontophoretic application of bicuculhne (20 nA; 5 min.). The right hand panel shows analogue ratemeter records from the same neurone. The top record shows the reduction in firing rate induced by brief iontophoretic applications of GABA, glycine and 5-HT. In the middle record the effects of GABA can be seen to be blocked seiectively by iontophoretic bicucuhine. In both panels the bottom records show recovery of synaptic inhibition and GABA responses respectively after stopping the application of bicueuhine.

25 d+gitoI mte meter

FIG. 4. Ratemeter (top record) and fiim record of extracellular action potentials (rn~dd~e) of a PF thalamic neurone with concurrent electrocorticogram (bottom trace). The injection of muscimoI400 ng in 0.2 pi into one SN at t produces after a defay of 3 min a profound increase in the rate of spontaneous firing. A prior injection of artificial CSF into the SN had failed to alter the firing of this thalamic neurone.

GABA AND THE NIGROTHALAMIC

II

PATHWAY

of muscimol (4 to 400 ng in 0.2 ~1) but not procaine or artificial CSF, initiated tight dose-dependent contralateral circling. This effect was reduced by 65% by chronic electrolytic lesions (0.6 mA 20 set) of the ipsilateral medial thalamus. This reinforces the importance of the nigrothalamic pathway in mediating the circling behaviour produced by pharmacological manipulations within the nigrostriatal system. Indeed, recent work [ 141 has shown that dopamine-mediated circling behaviour in rats is modulated by lesions of the ventromedial thalamic nucleus. Our muscimol microinjection experiments were then repeated in 14 anaesthetised rats whilst recording the activity of individual thalamic neurones. Two clear patterns of response were seen-typically, injections of muscimol, but not artificial CSF, into the ipsilateral SN produced a massive (about 20-fold) increase in the discharge rate of 8 PF thalamic neurones, some driven antidromically from the caudate nucleus. This is illustrated in Fig. 4. However, in four thalamic neurones, mainly in the VM and not driven antidromically from the caudate, intranigral muscimol only produced a small (about 3-fold) increase in the rate of spontaneous firing. It is worth noting that no change in firing rate was seen in the few cases when the muscimol was injected outside the SN or when the recording electrode was in thalamic nuclei other than VM or PF. Further evidence for the role of thalamic GABA in mediating responses involving the nigrothalamic pathway

comes from recent behavioural experiments in which the typical effects of intranigral muscimol were blocked by the prior (l-2 days) intrathalamic injection of ethanolamine-Osulphate (40 pg in 0.2 PI), an irreversible inhibitor of GABA transaminase [lo]. In addition, the contralateral circling behaviour induced by intranigral muscimol was potentiated in 11 rats by intrathalamic microinjections of the GABA antagonist picrotoxin (0.4 pg in 0.2 ~15 min previously). Thus, it seems that GABA mediates opposing patterns of motor behaviour in the SN and thalamus. In support of this view, when muscimol was injected into the medial thalamus in 18 rats it gave a characteristic dose-related but short-lived ipsilateral turning which was broadly resistant to the systemic administration of haloperidol, a dopamine receptor antagonist. Clearly, a major problem to be resolved now is the role of the GABA systems in the SN and thalamus, and the nigrothalamic pathway, in the normal control of motor behaviour. Though unilateral manipulations of the GABA system in experimental animals produce useful models of tuming behaviour, these may well be unphysiological. In this connection it is interesting to note that when chronic recordings are made from reticulata neurones in the SN of unanaesthetised monkey no consistent changes of firing rate were seen to accompany normal movement (W. Schultz; personal communication).

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