Sensitivity of identified medial hypothalamic neurons to GABA, glycine and related amino acids; influence of bicuculline, picrotoxin and strychnine on synaptic inhibition

Brain Research, 209 (1981) 145-158 © Elsevier/N0rth-H011andBiomedical Press

145

SENSITIVITY OF I D E N T I F I E D M E D I A L H Y P O T H A L A M I C N E U R O N S TO GABA, G L Y C I N E A N D R E L A T E D A M I N O ACIDS; I N F L U E N C E OF BICUCULLINE, P I C R O T O X I N A N D S T R Y C H N I N E ON SYNAPTIC INHIBITION

H. W. BLUME*, Q. J. PITTMAN** and L. P. RENAUD*** Division of Neurology, Montreal General Hospital and McGill University, 1650 Cedar Avenue, Montreal, Qud. H3G 1A4 (Canada)

(Accepted August 28th, 1980) Key words: medial hypothalamus -- amino acids - - microiontophoresis -- synaptic inhibition

SUMMARY Medial hypothalamic neurons in pentobarbital anesthetized rats were identified by location and response to electrical stimulation of the amygdala, medial preoptic area, midbrain periaqueductal gray and median eminence. Cells were then examined for their sensitivity to microiontophoretic applications of GABA, glycine and 3 related amino acids, i.e. fl-guanidinopropionic acid, dt-aminovaleric acid and fl-alanine. Application of all agents decreased the spontaneous and glutamate or aspartate evoked activity of the majority of neurons in all identified categories. The majority of neurons were more sensitive to fl-guanidinopropionate, ~aminovalerate and GABA than to glycine and fl-alanine. Bicuculline and picrotoxin produced a selective and reversible antagonism of depressions evoked by GABA and GABA-Iike amino acids whereas strychnine produced a selective and reversible antagonism of depressions evoked by glycine and fl-alanine. BicucuUine and picrotoxin, but not strychnine, were observed to diminish synaptic inhibition evoked by electrical stimulation of several sites when these agents were administered by microiontophoresis and by i.v. injections in convulsive doses. These observations suggest that many medial hypothalamic neurons have both * Present address: Beth Israel Hospital, Dept. of Neurosurgery, 330 Brookline Avenue, Boston, Mass. 02215, U.S.A. ** Present address: University of Calgary, Faculty of Medicine, Division of Pharmacology and Therapeutics, Calgary, Alberta, T2N IN4, Canada. *** To whom correspondence should be addressed.

146 GABA and glycine receptors but that GABA may have the more important role as a neurotransmitter common to afferent or recurrent inhibitory hypothalamic pathways.

INTRODUCTION Inhibition is a prominent feature of the response evoked among many medial hypothalamic neurons following stimulation of their afferent or efferent connections1, ag,24,25,a2,36. The medial hypothalamus contains several substances that may function as inhibitory neurotransmitters in these pathways3,10. Previous studies utilizing microiontophoretic techniques have demonstrated that the activity of unidentified medial hypothalamic neurons may be depressed during the application of monocarboxylic amino acidsS,Xl,29. In order to further assess the amino acids as inhibitory neurotransmitters among intrinsic hypothalamic connections, the present in vivo investigation compared the responses of medial hypothalamic neurons identified by location and synaptic connections to the microiontophoretic application of GABA, glycine and related amino acids, and examined the influence of antagonists on both the amino acid responses and on synaptic inhibition evoked from various stimulation sites. Our observations indicate that medial hypothalamic neurons are generally more sensitive to GABA than to glycine and that GABA but not glycine antagonists may diminish postsynaptic inhibition on some medial hypothalamic neurons. A portion of these results has been briefly reporteda3, 34. MATERIALSAND METHODS

Preparation Experiments were performed on male Sprague-Dawley rats initially anesthetized with i.p. pentobarbital (50 mg/kg) followed by supplementary i.v. doses (3-5 mg) every 2-4 h. The heart rate was monitored continuously and the temperature maintained at 37.5 °C throughout each experiment. The hypothalamus was exposed via a transpharyngeal approach3L

Stimulation Bipolar nichrome electrodes (o.d. 230/~m, tip separation 0.5 mm, insulated except for the terminal 0.5 ram) were stereotaxically positioned in two or more of the following sites: lateral septum, the amygdala (cortical, medial, basolateral or basomedial nuclei), medial preoptic area and periaqueductal gray or adjacent reticular formation. These electrodes were connected to isolated stimulation units that delivered monophasic current pulses (0.05 msec, 0.1-0.8 mA). Terminals of 'tuberoinfundibular' neurons were activated with monophasic current pulses (0.05 msec, 0.15-1.85 mA) applied to a concentric bipolar electrode positioned on the median eminencea2. Evoked unitary potentials arising from these stimuli were classified as antidromic, orthodromic 'excitatory' or orthodromic 'inhibitory' according to previously defined criteriaaL

147

Recording Extracellular action potentials were recorded through 2 M NaCl-filled glass micropipettes (impedance 5-8M f~) connected to a preamplifier and conventional system amplifier with variable band pass. A variable voltage gate was utilized to select action potentials for various spike train analyses (time interval and poststimulus latency discharge probability, and time frequency analysis) using a PDP 11/40 computer.

Localization The position of each microelectrode penetration was carefully measured with reference to midline, the posterior border of the optic chiasm, and the ventral surface of the brain. Electrode tips were broken and left in place during perfusion with 10 formalin. All electrode tracts were reconstructed on the basis of the histological location of the last electrode penetration observed in 40 #m thionin-stained sections. Positions of stimulation electrodes were also verified histologically. Data on cell locations, latencies, type of response to stimulation and to pharmacological agents were entered into a computer file; selected characteristics could then be subjected to statistical analysis, or the locations of particular neurons could be visualized on a set of coronal planes through the hypothalamus and preoptic area.

Microiontophoresis Seven barrel micropipettes with a total tip diameter of 5-7/~m, filled 12-24 h prior to recording, contained the following solutions: sodium L-glutamate (0.2 M, pH 7.0); sodium L-aspartate (0.2 M, pH 7.0); y-aminobutyric acid (0.2 M, pH 4.0); glycine, (0.2 M, pH 4.0); fl-alanine (0.2 M, pH 3.5); 6-aminovaleric acid (0.2 M, pH 4.0); fl-guanidinopropionic acid (0.2 M, pH 4.,0); bicuculline hydrochloride (5 mM in 165 mM sodium chloride, pH 3.5); picrotoxin (5 mM in 165 mM sodium chloride, pH 7.5); strychnine sulfate (5 mM in 165 mM sodium chloride, pH 5.5); sodium chloride (0.2 M, pH 7.0) for use as a current control. Each multibarrel micropipette was then rigidly attached to a single recording electrode whose tip protruded approximately 10-15/zm from the tip of the drug-containing micropipette. Individual drug barrels were connected through chlorided silver wires to constant current sources. In order to mininfize leakage of active substances when not in use, a 10-12 nA current opposite in polarity to that of the ejection current was applied to all drug-containing channels. RESULTS

Neuronal identification Data were obtained from 104 medial hypothalamic neurons distributed within the anterior hypothalamic-periventricular area (9 cells), arcuate (4 cells), ventromedial (85 cells) and dorsomedial nuclei (6 cells). Table I classifies these neurons according to their initial response (antidromic, orthodromic excitation or inhibition, no response) to 1 Hz electrical stimulation. All neurons were tested for a response to stimulation in two or more areas. Since the latencies and type of responses obtained by stimulation in

148 TABLE I

Classification of neurons according to initial response to 1 Hz electrical stimulation The column on the left refers to the stimulation sites used in these experiments and characterizes all medial hypothalamic neurons according to their initial response to single 1 Hz stimulation in these areas. A, antidromic; E, orthodromic excitation; I, orthodromic inhibition; NR, non-responsive. The numbers refer to cells in each of these stimulus tested categories further subdivided according to the change in their spontaneous and glutamate- and/or aspartate-evoked action potential frequency during microiontophoretic applications of 5 amino acids (I', increase; ~,, decrease; ~ , no change).

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w e r e c o m b i n e d . T a b l e I a l s o s u m m a r i z e s t h e effects o f m i c r o i o n t o p h o r e t i c a p p l i c a t i o n of GABA, glycine, and related amino acids on spontaneous

(28 cells) o r g l u t a m a t e

a n d / o r a s p a r t a t e e v o k e d a c t i v i t y (76 cells).

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f r e q u e n c y o f all s p o n t a n e o u s l y a c t i v e n e u r o n s a n d t h e m a j o r i t y o f g l u t a m a t e a n d / o r aspartate activated neurons in each area of the medial hypothalamus. Two patterns o f r e s p o n s e w e r e o b s e r v e d . A n f i n o r i t y (8 ~ ) o f n e u r o n s w e r e d e p r e s s e d b y G A B A

149 applied with low currents (i.e. 10-15 nA) but required high currents (i.e. 60-120 nA) in order to produce complete suppression of their spontaneous or evoked activity; a 50 reduction in excitability was achieved with 52.5 ± 4.2 nA tmea, ± S.E.M.). The majority of GABA-sensitive neurons were depressed at much lower ejection currents and a difference of less than 10 nA usually separated the threshold current from that required to produce a complete suppression of activity (Fig. 1); for these neurons a 5 0 ~ reduction in excitability was achieved with 14.3 ± 1.1 nA. These differences in sensitivity seemed unrelated to the micropipettes utilized since the two patterns were recorded from adjacent neurons tested in the same penetration. Glycine-sensitive neurons

Microiontophoretic applications of glycine were associated with a reduction in spontaneous (10 cells) or glutamate- and/or aspartate-evoked activity (52 cells) of the majority (84 ~ ) of tested neurons. Glycine-responsive cells were found throughout the medial hypothalamus in each of the stimulation tested categories listed in Table I. For 10 ~o of neurons comparable depressant responses that were rapid both in onset and in recovery were obtained with equal or lower currents for glycine than for GABA. However, most neurons appeared less sensitive to glycine than to GABA since lower ejection currents were required for the GABA associated responses (Fig. 1). In studies on 41 neurons a 50% reduction in excitability could be achieved with 13.3 ± 1.9 nA for GABA compared with 25.1 ± 2.3 nA for glycine. As indicated earlier, these data were derived largely from tests on ventromedial nucleus neurons. However, cells in other hypothalamic locations did not display GABA -4

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Fig. 1. Oscilloscope records from a glutamate activated (15 nA) neuron in the ventromedial nucleus illustrate comparative depressant actions of GABA and glycine. On the left, the depressant action of G A B A is present following a reduction (--4 nA) or removal (0 nA) of the GABA-retaining current (maximum - - 1 0 nA). On the right, glycine-evoked responses require higher iontophoretic currents over a greater range to achieve a comparable effect.

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1 rain Fig. 2. Ratemeter records from a glutamate activated (22 nA) neuron of the ventromedial nucleus illustrate comparative depressant actions of GABA, 6-aminovalerate (VAL) and /3-guanidinopropionate (BGP). During microiontophoretic application of picrotoxin (20 nA) there was a gradual but readily reversible reduction in the efficacy of all 3 test agents. A control application of sodium ions (40 nA; not illustrated) had no effect on cellular excitability. significantly different results in terms of their relative G A B A and glycine sensitivities, and therefore the data were pooled from all areas. Furthermore, there appeared to be no correlation between G A B A and glycine sensitivities and synaptic connectivity of neurons in different locations. Most neurons were responsive to stimulation of more than one site. Actions of related amino acids In accordance with their actions on spinal cord neurons and interactions with G A B A and glycine antagonists, fl-guanidinoproprionic acid and (~-aminovaleric acid have been classified as 'GABA-like' amino acids, whereas fl-alanine has been classified as a 'glycine-like' amino acid% In view of the apparent difference in sensitivity of medial hypothalamic neurons to GABA and glycine, their actions were compared with the actions of these related amino acids. Using currents that provided for a 50 % reduction in excitability, it was noted that consistently lower currents were required for fl-guanidinoproprionate (13.5 ± 1.4 hA) than for GABA (18.6 ± 1.2 hA) in studies on 13 neurons (Fig. 2). 6-Aminovalerate was occasionally more effective as a depressant agent than G A B A (Fig. 2), but this difference was not significant when the mean effective currents for a-aminovalerate (15.8 ± 1.4 nA) and G A B A (18.5 ~ 1.9 nA) were compared on 13 neurons. On the other hand, consistently lower ejection currents were required for G A B A (13.1 4- 2.4 nA) than for either fl-alanine (19.9 ± 2.6 nA) or glycine (28.9 ~ 2.9 nA) during studies on 30 neurons. These results suggest that the order of potency for these amino acids on medial hypothalamic neurons is fl-guanidinoproprionate > 6-aminovalerate GABA > fl-alanine > glycine.

151 Picrotoxin, bicuculline and strychnine Picrotoxin and bicuculline are considered as G A B A antagonists whereas strychnine is considered as a glycine antagonistT, 20. Influence on amino acid responses. W h e n applied with currents o f 100-200 hA, both bicuculline and picrotoxin reversibly antagonized G A B A - e v o k e d depression o n all of 20 neurons tested within 30 sec whereas several minutes were required to observe a m a x i m u m effect o f these agents when applied with lower current levels (Figs. 2 and 3). The lowest effective current level noted for picrotoxin action was 20 n A (6 cells); bicuculline's effect as a G A B A antagonist was seldom evident with currents under 80 n A (8 cells) probably due to the comparatively lower solubility and/or transport n u m b e r o f bicuculline. Applications o f picrotoxin at 20-40 n A and bicuculline at

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1 tin Fig. 3. Sequence of polygraph ratemeter records obtained from a glutamate activated (20 nA) ventromedial nucleus neuron illustrate the effects of bicuculline and strychnine on GABA- and glycineevoked responses. In the upper sequence, bicuculline antagonized the GABA response and partially altered the glycine-evoked response; however, the effect was much more pronounced on GABA-evoked responses. Note the brisk recovery. In the lower sequence, strychnine application was associated with a complete suppression of the control glycine-evoked response, and recovery occurred only after several minutes. Note also the partial interference with the GABA-evoked responses, hut the relatively rapid recovery compared with that for glycine.

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Fig. 4. Ratemeter records from a periventricular neuron comparing the dose-dependent depressant actions of GABA, fl-alanineand glycine.During a relatively brief application of strychnine (20 nA) depressions of all 3 agents were antagonized. Note, however, that the response to GABA returned several minutes earlier than that to glycineor fl-alanine. 80-100 nA were also associated with a gradual and reversible antagonism of the depressions evoked by GABA, fl-guanidinoproprionate and 6-aminovalerate with no appreciable change in the glycine or fl-alanine evoked responses (Fig. 2). When picrotoxin and bicuculline were applied with higher ejection currents, control flalanine and glycine evoked depressions were also altered (Fig. 3), although to a lesser extent than for GABA and the other amino acids. Since recovery of control responses to glycine and fl-alanine preceded recovery for the other amino acids, it would appear that these antagonists are more specifically directed towards the actions of GABA and GABA-Iike amino acids. Applications of strychnine with currents of 10-14 nA partially and preferentially antagonized glycine and fl-alanine actions reversibly with little or no effect on GABA, fl-guanidinoproprionate or t~-aminovalerate actions. However, when strychnine was applied for varying periods of time with currents of 20-40 nA, depressions evoked by glycine, GABA and the other amino acids were all diminished; recovery to GABA, flguanidinoproprionate and 6-aminovalerate actions returned abruptly and several minutes prior to those evoked by glycine and fl-alanine (Figs. 3 and 4). Influence on synaptic inhibition. In view of picrotoxin and bicuculline's selectivity for GABA receptors, and strychnine's selectivity for glycine receptors, it was considered that these convulsants might be useful tools to assess the roles of GABA and glycine as inhibitory synaptic transmitters in the medial hypothalamus. In the first series of experiments, bicuculline, picrotoxin and strychnine were applied by microiontophoresis to hypothalamic neurons that displayed inhibition either as the initial response to a stimulus (4 cells) or following an initial excitatory response (21 cells). When bicuculline or picrotoxin were applied to 16 cells with currents under 100

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155 nA, no notable change was observed in synaptic inhibition. However, when bicuculline and picrotoxin were applied to 19 cells with currents of 160-300 nA, i.e. clearly sufficient to abolish any GABA-induced depressions (Fig. 5), 70% of neurons displayed a reversible prolongation and increase in magnitude of any stimulus evoked excitatory responses and a decrease (but not abolition) in the magnitude and duration of all synaptic inhibitions (Fig. 5). During the microiontophoretic application of picrotoxin to two neurons initially excited by lateral septum and amygdala stimulation, we observed a marked increase in both the intensity and duration of the initial synaptic response (Fig. 6). At this time, the silent period that followed the initial excitation in the control poststimulus histogram was no longer evident. Coincidentally there was an increase in the number of short interspike intervals, and an increase in spontaneous activity (often seen during application of all 3 convulsants). These changes reversed slowly after termination of the picrot0xin applications. In contrast, none of the 25 neurons tested with microiontophoretic applications of strychnine with currents up to 100 nA displayed any change in their synaptic responses. In a second series of experiments, convulsants were administered intravenously to pentobarbital anesthetized animals paralyzed with curare and ventilated artificially. Eight neurons were tested with each convulsant. With half of the cells, inhibition was the initial response to stimulation; with the other half, inhibition was observed to follow an initial excitatory response. When picrotoxin, bicuculline or strychnine were administered in subconvulsant doses, we observed no change in either the duration or the magnitude of synaptic inhibition. Only when picrotoxin and bicuculline, but not strychnine, were administered in doses sufficient to produce convulsions, was there some decrease in duration (but never complete abolition) of any synaptic inhibition. Unfortunately, it was not possible to record from the same neuron for more than 20-30 min and therefore verify the reversibility of these alterations in synaptic response patterns. DISCUSSION The amino acids G A B A and glycine have been identified as one class of probable neurotransmitters at chemical inhibitory synapses in vertebrate central nervous

Fig. 6. On the left, the upper 'control' poststimulus histogram from a neuron in the dorsomedial hypothalamic nucleus illustrates an initial excitation, with subsequent depression and late excitatory 'rebound' following single stimulation of the lateral septum (LS) and medial amygdaloid nucleus (A). During the application of picrotoxin (200 nA), note the dramatic increase in the magnitude and duration of the stimulus-evoked excitation observed in the middle histogram on the left. Recovery occurred very slowly, and was still not complete 50 min after cessation of the picrotoxin application (lowest record on the left). The centre column of time interval histograms (TIH) illustrates a marked decrease in the mean interspike interval associated with the picrotoxin application. The column of poststimulus histograms on the right represents a similar sequence of data obtained before, during and following an application of bicuculline (200 nA, middle histogram). Bicuculline had only a marginal influence on the synaptic activity patterns, tending to diminish the magnitude of the silent interval following the initial stimulus-evoked excitation.

156 systems 7,17,26. In the brain stem and spinal cord, many neurons respond to glycine and GABA, and our observations indicate that neurons in several hypothalamic nuclei are also depressed by both GABA and glycine (cf. refs. 8 and 11). These data would suggest that medial hypothalamic neurons, irrespective of their location and synaptic connections have receptors for both amino acids, and are generally more sensitive to GABA than to glycine. The relative specificity of strychnine as a glycine antagonist and of picrotoxin and bicuculline as GABA antagonists prompted the use of these convulsants to attempt differentiation between glycine and GABA mediated synaptic inhibitions on neurons sensitive to both amino acids. Studies in the brain stem and other supraspinal structures already suggest that many of the glycine receptors on these neurons are probably extrajunctional and not likely to be involved in synaptic inhibition, since the latter is unaffected by strychnine administered systemically or electrophoretically2,4,5,15,1s,27, ~1. While glycine may have some (as yet undetermined) role as a hypothalamic inhibitory neurotransmitter, strychnine has no detectable effect on synaptic inhibition in this region. On the other hand, data from the present in vivo studies (cf. refs. 26, 36, 41) and from recent in vitro studies 12 illustrate that the GABA antagonists picrotoxin and bicuculline do modify the time course and magnitude of synaptic inhibition in the medial hypothalamus, favoring the proposal that GABA may function as one of the inhibitory neurotransmitters in this region. Furthermore, since there is a similarity in both the magnitude and the duration of synaptic inhibitions evoked among medial hypothalamic neurons by stimulation at several sites, and in the response of these inhibitions to GABA antagonists, GABA may be a neurotransmitter that is common to several afferent or recurrent inhibitory pathways. A somewhat disappointing aspect of the present investigation was our inability to demonstrate that GABA antagonists could consistently and completely abolish synaptic inhibition in the medial hypothalamus (cf. ref. 26). In view of the multiplicity of potential neurotransmitters available to medial hypothalamic neurons3, ~0 and their potential to respond to many of these agents when applied exogenouslyll,z0,2a,26,~9, 33, this observation may not be surprising, but rather reflect that synaptic inhibition among many medial hypothalamic neurons is only partially mediated by monocarboxylic amino acids. Recent evidence indicates that GABA has a functional role in the medial hypothalamus. Both GABA and glutamate decarboxylase (GAD), a useful marker for GABA-containing neurons, are present in high levels within the hypothalamus~S,3L Hypothalamic slices display evidence of a high affinity GABA-uptake system 13 and part of this uptake appears to be concentrated within neuronal structures 21. GABA can be released by electrical stimulation from preparations of hypothalamic synaptosomes 9 suggesting that GABA is located within neuronal endings. GABA has been implicated in several hypothalamic functions, including feeding16 and neuroendocrine mechanisms, i.e. control of ACTH 14,22, gonadotropin 80 and prolactin release2S,30,35, 87,40. These data in combination with the electrophysiologic observations described above support the proposal that GABA has a neurotransmitter role in the medial hypothalamus.

157 ACKNOWLEDGEMENTS

The authors thank the Medical Research Council of Canada and Conseil de la recherche en sant6 du Qu6bec for support. They also wish to thank William Ellis for expert technical assistance, Mrs. Gwen Landrigan for typographical assistance and Mr. Serge Lafontaine for assistance with the computer analysis. We are grateful to Dr. M. Manske (University of Waterloo, Canada) for generous supplies of bicuculline hydrochloride.

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