Progrrn

in Ncurohioloy~.

1977. Vol. 9, pp. 123-145.

GABA-RECEPTORS

Pergamon

Press. Prmted in Great

IN THE VERTEBRATE SYSTEM F. V.

“Ram&

Britain

NERVOUS

DEFEUDIS

y CajaP’ Center and Autonomous Madrid, Spain

Medical

School,

Contents 1. Introduction 2. Physiologic-pharmacologic studies 2.1. Introduction 2.2. Picrotoxin and bicuculline 2.2.1. Picrotoxin 2.2.2. Bicuculline 2.3. Other possible GABA-antagonists 2.4. Structure/activity relationships 2.5. Studies with tissue slices and cultures 2.6. Possible “activators” of GABA-receptors 2.7. GABA and presynaptic inhibition 2.8. GABA-receptors of sympathetic ganglia 3. “Binding” of GABA 3.1. Introduction 3.2. Bicuculline-sensitive GABA binding 3.3. Other agents that might act on GABA-receptors 3.4. Regional variations in GABA binding 3.5. Effects of maturation and environment on GABA 3.5.1. Maturation 3.5.2. Environment 4. Concluding remarks Acknowledgements References

binding

123 124 124 125 125 125 126 126 127 128 128 129 129 129 130 134 134 136 136 137 138 138 138

1. Introduction

Four o-amino acids (GABA, glycine, taurine, fi-alanine) have been proposed as inhibitory neurotransmitters in the vertebrate CNS (e.g. Curtis and Johnston, 1974a; Krnjeviti, 1974; DeFeudis, 1975a, 1977; DeFeudis and Martin de1 Rio, 1977). Of these, only GABA is rather uniquely localized to the vertebrate CNS, though it is present in high concentration in the peripheral nervous systems of various invertebrates (e.g. Kravitz et al., 1963; Otsuka et al., 1967). Although GABA is not a substrate for protein synthesis, it is heavily involved in general metabolism, it is a constituent of certain small peptides (e.g. y-aminobutyrylhistidine; a-(y-aminobutyryl)-lysine; GABA-l-methylhistidine; see Kanazawa et al., 1965; Nakajima et al., 1969; Baxter, 1970; van Regemorter et al., 1972; Fig. l), and it can stimulate cerebral protein synthesis in vitro (e.g., Campbell et al., 1966; Tewari and Baxter, 1969; Kelly and Luttges, 1972; Baxter, 1976). GABA has fulfilled all established criteria for characterizing central neurotransmitters, and appears to be a major postsynaptic inhibitory transmitter in the vertebrate CNS (see e.g., Curtis and Johnston, 1974a; Krnjevib, 1974; DeFeudis, 1975a, 1977; reviews in Roberts et al., 1976). GABA might also serve as a postsynaptic excitatory transmitter in some structures (e.g., Flock and Lam, 1974) and may be involved in presynaptic inhibition (see Section 2.7). Recent studies have implicated the central GABA system in many vertebrate behaviors and in certain neurologic disorders, especially those related to extrapyramidal, mesolimbit and vestibular areas of the CNS (e.g., Perry et al., 1973; McGeer et al., 1973; Urquhart et al., 1975; Cools, 1975; McGeer and McGeer, 1975; Pycock and Horton, 1976; Cools and Janssen, I976; Roffler-Tarlov and Tarlov, 1976; Pycock et al., 1976; Hornykiewicz et al., 1976), and in convulsive mechanisms (e.g., Tapia et al., 1969; Love& 123

JP.N. 9,3-

A

F. V. DEFEUUIS

4

Acetyl-CoA

i Malate

I

TCA CYCLE

I”’ ,I

I-Hydroxybutyrate

Homocarnosine

(f-f3utyroloctom)

Homoanserine d-

T’-D~amlnobutyrate

Homopantothemc S-Adenosylmethiomne

/-Amlnobutyrylchollne

FIG. 1. Proven and suggested pathways of GABA metabolism. (Modified and reproduced with permission from Baxter, 1970).

1971; Bradford, 1976; Tower, 1976). Other studies have revealed that “GABA-ergic” mechanisms might also be involved in the neural regulation of secretion of certain pituitary hormones (e.g., luteinizing hormone, prolactin; vasopressin, ACTH; Ondo, 1974; Makara and Stark, 1974; Ondo and Pass, 1976; Feldberg and Silva, 1977; Pass and Ondo, 1977) and in the mechanisms of ethanol (e.g., Hakkinen and Kulonen, 1967, 1976; Rawat, 1974; Sytinsky et al., 1975; Chan, 1977) and barbiturate (e.g., Sutton and Simmonds, 1974; Tzeng and Ho, 1977) actions and tolerance. Therefore, for both basic and applied reasons, it seems imperative to study vertebrate GABA-receptors. This review will focus on recent physiologic-pharmacologic and biochemical studies that have been aimed at elucidating GABA-receptors. Several recent reviews are related to this subject (Curtis and Johnston, 1974a; Krnjevit, 1974; DeFeudis, 1975a, 1977; Johnston, 1976a, b; Olsen, 1976; Peck et al.. 1976; Young et ul., 1976).

2. Physiologic-Pharmacologic

Studies

2.1. INTRODUCTION

After the potent depressant effects of GABA on central neurones had been shown (Curtis and Watkins. 1960a, b), further studies led Krnjevii: and Phillis (1963) to suggest that GABA could be a naturally-occurring inhibitory transmitter. Since then, much evidence has accumulated in support of this proposal.

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125

2.2. PICROTOXIN AND BICUCULLINE Picrotoxin and bicuculline block the neuronal depressant actions of GABA (e.g., Galindo, 1969; Obata and Highstein, 1970; Nicoll, 1970; Engberg and Thaller, 1970; Curtis et al., 1970a, b, Bruggencate and Engberg, 1971; Duggan and McLennan, 1971; Hill et al., 1973a; Altmann et al., 1976), and strychnine blocks predominantly those neuronal depressions produced by “glycine-like” amino acids (e.g., Curtis et al., 1968, 1969, 1971a; Tebecis, 1970). These agents also antagonize synaptically-mediated postsynaptic inhibitions (e.g., Kellerth and Szumski, 1966; Curtis et al., 1970a, b; Ito et ul., 1970; Nicoll, 1970; Obata and Highstein, 1970; Obata et al., 1970; Altmann et al., 1976), and, in the case of picrotoxin and bicuculline, presynaptic inhibitions (e.g., Curtis et al., 1971b;. Davidoff, 1972a, b) in the vertebrate CNS. Hence, these agents have been used extensively to examine inhibitory processes of the vertebrate CNS (Fig. 2). Bicuculline-sensitive, strychnine-insensitive synaptic inhibitions occur in most CNS regions, whereas strychnine-sensitive, bicuculline-insensitive inhibitions are more restricted to spinal and brain stem regions. PNI

(1

c

N’CH3 0 2 :I

2

Plcrotoxlnin

O 04

Bicuculline

0.X _o’

* _

‘C

\C/c\C/N”3 GABA

Strychnine

0\\

A

,+NH; -0 Glycine

FIG. 2. Structures of agonist-antagonist pairs, GABA and bicuculline or picrotoxinin. glycine and strychnine. (Modified and reproduced with permission from Johnston, 1976a.)

2.2.1. Picrotoxin Galindo (1969) first demonstrated a picrotoxin-sensitive, GABA-mediated inhibition on neurones of the feline cuneate nucleus. Picrotoxin also blocks stellate and basket cell inhibitions of Purkinje cells, Golgi cell inhibition of granule cells (Bisti et al., 1971; Woodward et al., 1971), and granule cell inhibitions of mitral cells in the olfactory bulb (Nicoll, 1971), all of which appear to be mediated by GABA. GABA action and neurally-evoked inhibitions at several other central sites are also blocked by picrotoxin (see above, and refs in Krnjevib, 1974; DeFeudis, 1975a). However, picrotoxin does not appear to block specifically the neuronal depressant action of GABA in feline cerebral cortex (Krnjevii: et ul., 1966) or in rat caudate nucleus (Bernardi et al., 1976). The report of Davidoff and Aprison (1969), that iontophoresed picrotoxin consistently blocked the depressant action of glycine on spinal interneurones, has never been confirmed (e.g., Curtis et al., 1969; Engberg and Thaller, 1970; Felpel, 1972). The usefulness of picrotoxin is limited by its low aqueous solubility, by its lack of specificity as a GABA-antagonist, and by its direct excitatory action on neuronal membranes. The actions of picrotoxin-analogues have been recently reviewed (Curtis and Johnston, 1974b; Johnston, 1976b). 2.2.2. Bicuculline Curtis and colleagues (1970a, b) first demonstrated a GABA-antagonizing bicuculline on certain neurones of the mammalian CNS. Since then, many

action of studies on

126

F. V. DEFFUDIS

the actions of this substance have been conducted (see reviews by Curtis and Johnston, 1974a; Krnjevik, 1974; DeFeudis, 1975a, 1977: Davidson, 1976; Johnston, 1976b). Like picrotoxin, bicuculline does not appear to produce effective and specific blockades of GABA-induced depressions or synaptic inhibitions in the mammalian cerebral cortex and caudate nucleus, and it may directly excite neuronal membranes (e.g., Straughan et al., 1971; Krnjevii, 1974; Bernardi et (II., 1976). Bicuculline is poorly soluble in aqueous media and is hydrolyzed to its less active derivative, bicucine, under physiological conditions (e.g., Olsen et al., 1975). Several effects of bicuculline-like compounds have been observed that are not related to a GABA-receptor action; e.g., its inhibition of acetylcholinesterase (Svenneby and Roberts. 1973); its potentiation of acetylcholine action (Miller and McLennan, 1974); its direct effects on axonal (Freeman, 1973) and muscle membrane conductances (Shank et 111..1974).

2.3. OTHER POSSIBLE GABA-ANTA(;ONISTS Benzylpenicillin (i.v.) antagonized bicuculline-sensitive prolonged inhibition, but not strychnine-sensitive direct inhibition in spinal cord, and its iontophoretic application blocked GABA- but not glycine-induced spinal depressions (Davidoff, 1972a, c; Curtis rt al., 1972). The acetylcholine-receptor antagonist. rl-tubocurarine, is a rather potent GABA-antagonist in the feline cerebral cortex and cuneate nucleus, but it also affects the action of glycine (Hill et ul., 1972, 1973a. b). Tetramethylenedisulphotetramine (TMDST), upon systemic administration, antagonized prolonged spinal inhibition (Curtis and Johnston, 1974a). Since TMDST antagonized the actions of both GABA and glycine in parallel on neurones of the rat cuneate nucleus. midbrain reticular formation, and medulla (Collins et al.. 1975; Dray, 1975a), this agent could interact with an ionophore that is common to GABA and glycine (Dray. 1975a). TMDST is of limited usefulness since it is practically insoluble in aqueous solutions (Kerr rt ul., 1976). Cuaniol reduced the depressant effect of GABA, relative to that of glycine, on feline spinal neurones (Curtis and Johnston, 1974b), and caprolactams blocked positive field potentials elicited in rabbit substantia nigra by strio-pallidal stimulation (Kerr et al., 1976). Although none of these compounds appear to be better GABA-antagonists than picrotoxin or bicuculline, it should be noted that both these agents can affect the actions of some other neuronal depressants (e.g., glycine, taurine, imidazoleacetic acid, 5-HT) in the CNS (e.g., Curtis et ~rl., 1971b; Tebecis ct ~1.. 1971 : Straughan and Watson. 1972; Curtis and Johnston, 1974a: Krnjevik, 1974; DeFeudis. 1975a).

2.4. STRUCTURE/ACTIVITY RELATIONSHIPS On rat central neurones (-)bicuculline-methochloride was ineffective as a GABAantagonist though it produced neuronal excitation similar to that of the (+)isomer. which is used in iontophoretic studies (Collins and Hill, 1974). (+)Bicuculline-methiodide antagonized both the action of iontophoretically-applied GABA and synaptic inhibition in the feline cuneate nucleus, whereas the (-)isomer possessed neither action (Hill rt al., 1974). Dray (1975b) showed that bicuculline-methochloride was a more potent and more selective GABA-antagonist than bicuculline or picrotoxin on cells of the medullapons of rats, and that all three compounds reduced or abolished imidazoleacetic acidinduced depressions before those of GABA, more potently antagonized imidazoleacetic acid than GABA, and did not significantly affect norepinephrineor 5-HT-induced depressions. Imidazoleacetic acid could interact with GABA-receptors (Curtis rt ul., 1971b; Haas et al., 1973), or the more potent antagonism of the action of imidazoleacetic acid by picrotoxin and bicuculline might signify that its receptor differs from the GABAreceptor (Dray, 1975b). The observed difference in potency between bicuculline and bicuculline-methochloride was perhaps caused mainly by a difference in aqueous solubility.

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Both GABA and bicuculline are flexible molecules; hence the problems of the “active conformation(s)” of GABA have been approached indirectly by studying GABAanalogues of restricted conformations. The pitfalls of using this approach have been discussed by Johnston (1976b). Five compounds have been found to act as GABAagonists on bicuculline-sensitive, strychnine-insensitive inhibitions on feline spinal neurones, the most potent being trans-3-aminocyclopentane-l-carboxylic acid (about twice as potent as GABA) and the least potent being 4-aminotetrolic acid (about 2&50x as potent as GABA). As these compounds possess three isosteric atoms corresponding to the charged atoms of the GABA zwitterion, an “extended” conformation of GABA might act on bicuculline-sensitive receptors (see Johnston, 1976b). In other in uiuo iontophoretic (Krogsgaard-Larsen et al., 1975) and in vitro studies (Krogsgaard-Larsen and Johnston, 1975) muscimol (3-hydroxy-5-aminomethylisoxazole) and its derivatives were used to separate receptor- from uptake-binding activities, since muscimol, though exerting a potent “GABA-like” depressant action (Johnston et al., 1968), is a weak inhibitor of GABA uptake (Johnston, 1971). Results revealed that GABA interacts with its receptor and uptake system in different conformations (see also Segal et al., 1973). Bicuculline-insensitive GABA-receptors may also exist in the vertebrate CNS (see Ryall. 1975; Johnston, 1976b). Iontophoretically-applied cis-4-aminocrotonic acid, tram-2- and trans-3-aminocyclohexane carboxylic acid produced bicuculline-insensitive, strychnine-insensitive depressions of feline spinal neurones (Johnston et a/., 1975; Johnston, 1976b). Hence, GABA might act with bicuculline-sensitive receptors in an “extended” conformation (see above) and with bicuculline-insensitive receptors in a “folded” conformation, since cis-4-aminocrotonic acid is a folded analogue of GABA. Iontophoretically-applied Lioresal (Balcofen, or /?-(p-chlorophenyl)-GABA), a lipophilic substance, also exerted a bicuculline-insensitive depressant action on central neurones (Davies and Watkins, 1974; Curtis et al., 1974a). 2.5. STUDIESWITH TISSUESLICESAND CULTURES In superfused slices of guinea-pig cerebellar cortex, GABA decreased the frequency of spontaneous action potentials (Okamoto and Quastel, 1973). Both picrotoxin and bicuculline reduced GABA-induced depression of spontaneous activity in high-Clmedium and abolished GABA-induced excitation in low-Cl_ medium (Okamoto et al., 1976). Bicuculline also blocked the depressant effects of fi-alanine, taurine and glycine on these cells in high-Cl- medium, but not their excitatory effects in low-Cl_ medium. These results are in accord with the belief that all four of these amino acids cause inhibition by increasing Cl- (and perhaps K+) permeability of neuronal membranes (see Curtis and Johnston, 1974a; Krnjevik, 1974; DeFeudis, 1975a). All four of these w-amino acids depress spontaneous firing of neurones of the feline cerebellum in situ (e.g., Kawamura and Provini, 1970), and studies on cat cerebellum have revealed that both GABA and glutamate-cr-decarboxylase (GAD) are specifically localized in Purkinje axon terminals (Fonnum et al., 1970; Otsuka et al., 1971; Fonnum and Walberg, 1973). In further studies, Okamoto and Quastel (1976) showed that three molecules of GABA combined with the GABA-receptor site to produce inhibition or excitation in this preparation and that one molecule of picrotoxin combined with its receptor site. This result with GABA is in accord with the proposal of Brookes and Werman (1973) that three molecules of GABA are required to activate its receptor in flexor tibialis muscle fibers of the locust. Other studies on Purkinje cells of rat explant cultures (Gahwiler, 1975) provided evidence that GABA, bicuculline and picrotoxin all act by influencing synaptic receptors. Bicuculline (10m8 M) and picrotoxin (10e5 M) antagonized the inhibitory effect of GABA (10d5 M), whereas strychnine (10-7-10-5 M) generally had no such eff$t. Further studies by Gahwiler (1976) revealed that benzodiazepines are very potent GABA-antagonists in this same preparation, the inhibition of spontaneous activity of Purkinje cells by 1O-5 M GABA being antagonized by diazepam (EC,, = 2.4 x lo-” M) and by chlordiazepoxide (EC,, E 1.5 x 10m9 M); all drugs were bath-applied. Also, bath-

F.

12x

V. D~Feurxs

applied diazepam (lo-“ M) partially blocked the depressant action of iontophoreticallyapplied GABA. A reversal of GABA blockade in spinal cord explants has also been produced by bicuculline and picrotoxin (Grain, 1974).

2.6. PCSSIBLE“ACTIVATORS” OF GABA-RECEPTORS

Of the three actions of pentobarbital &at have been observed in the spinal cord, those on pre- (see below) and postsynaptic inhibitions could involve an uctivation of GABA-receptors. Nicoll (1975a) showed that pentobarbital ( 1O-4 M. or more) hyperpolarized frog motoneurones by an action that was sensitive to reversible blockade by picrotoxin and bicuculline. but not by strychnine, and that at 2 x lo-” ~-10~~ M, it increased the amplitude and duration of the GABA response. Pentobarbital (30mg/kg, i.?.) also hyperpolarized feline hippocampal neurones and markedly prolonged their IPSPs by a direct, dose-dependent action on inhibitory synapses (Nicoll et al., 1975), findings which supported the view. based on earlier studies (Nicoll. 1972). that barbiturates prolong synaptic inhibitions. Barbiturates could produce such an effect by prolonging the release of inhibitory transmitter, by directly affecting the GABA conductance mechanism, or by retarding the removal of GABA from postsynaptic receptor sites (see Nicoll et a/., 1975). It has also been suggested that benzodiazepines might stimulate GABA-receptors indirectly by causing a release of GABA from cells of the cerebellum (Costa et al.. 1975; Haefely et al., 1975). However, studies by Steiner and Felix (1976) on vestibular and cerebellar neurones of cats and rats did not support this notion, since GABA-mediated inhibition was clearly antagonized by these drugs (see also Gahwiler, 1976; Section 2.5). Studies with crude CNS receptor preparations have also linked benzodiazepine action to glycine (Young et al.. 1974; Snyder, 1975). However, critical iontophoretic tests of this hypothesis by Dray and Straughan (1976) on spontaneously-active cells of the medullae of urethane-anaesthetized rats revealed an involvement of benzodiazepines with GABA- rather than with glycine-mediated processes. 2.7. GABA

AND PRESYNAPTICINHIBITION

Eccles et al. (1963) proposed that GABA might be a presynaptic inhibitory transmitter since its topical application was shown to depolarize dorsal roots. Further evidence now supports an involvement of GABA in primary afferent depolarization (PAD) and presynaptic inhibition. especially in the amphibian spinal cord (e.g., Barker and Nicoll, 1973; Davidoff et cd., 1973; Davidoff and Adair, 1974: Barker et a/., 1975a, b; Constanti and Nistri. 1976; Davidson, 1976; Obata, 1976; Gmelin. 1976), but a role for GABA as the mediator of this process remains uncertain (see e.g.. Curtis et a/., 1971b; Krnjevib, 1974; McLennan, 1976). Both picrotoxin and bicuculline block PAD. long duration inhibitions, and the effects of GABA on primary afferent terminals (e.g.. Tebecis and Phillis. 1969; Curtis rt al., 1971b; Davidson and Southwick, 1971; Barker and Nicoll, 1972; Davidoff, 1972a, b; Levy and Anderson, 1972; Levy. 1975; Barker or trl.. 1975a). Also, experimentally-produced reductions of spinal GABA content decreased PAD (Banna and Jabbur, 1971; Bell and Anderson, 1972: Miyata and Otsuka, 1972), whereas inhibition of GABA-c(oxoglutarate-transaminase (GABA-T: the GABA-catabolizing enzyme) increased PAD and facilitated presynaptic inhibition (Davidoff et trl.. 1973). However, since at least three different types of neutral amino acid receptors might exist on primary afferent terminals of frog spinal cord (a “GABA-like” receptor, a “taurinelfl-alanine” receptor, and and a “glycine-like” receptor; Barker et NI., 1975a, b) and since curare. nicotine atropine can antagonize responses elicited by these amino acids on primary afferents (Nicoll, 1975d), it remains difficult to define a specific role for GABA in PAD. Barbiturates exert presynaptic actions on vertebrate CNS neurones (e.g. Loyning et al.. 1964; Nicoll, 1975b). In isolated frog spinal cord, pentobarbital depolarized primary

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afferents and greatly prolonged presynaptic inhibition (Nicoll, 1975b) by an action that was reversibly blocked by picrotoxin and bicuculline, but not by glycine-antagonists or Mg*+ and which, therefore, appeared to involve an activation of GABA-receptors. These findings confirmed earlier observations that anaesthetics can prolong presynaptic inhibition (e.g., Eccles and Malcolm, 1946; Schmidt, 1963). Costa et al. (1975) have suggested that benzodiazepines might act by facilitating GABA-mediated presynaptic inhibition. In support of this proposal, v&y low doses of benzodiazepines facilitated PAD in the spinal cord (e.g., Schmidt, 1963; Schmidt et al., 1967), and the enhanced presynaptic inhibition can be reduced by picrotoxin (Stratten and Barnes, 1971) and bicuculline (Pole et al., 1974). However, the recently demonstrated GABA-antagonizing actions of benzodiazepines should be considered before accepting this view (see Section 2.5). Lioresal might activate presynaptic inhibition (e.g., Burke et al., 1971; Knutsson et al., 1973). However, since it produced a depression of central neurones that was resistant to both bicuculline and strychnine (Curtis et al., 1974a; Davies and Watkins, 1974), and hyperpolarized primary afferent terminals of frog spinal cord (Davidoff and Sears, 1974; Nistri, 1975), this agent does not appear to enhance GABA action during presynaptic inhibition.

2.8. GABA-RECEPTORSOF SYMPATHETIC GANGLIA GABA can depolarize cat superior cervical, inferior mesenteric, dorsal root, and nodose ganglia in situ (e.g., DeGroat, 1970, 1972; DeGroat et al., 1971, 1972), rat superior cervical ganglion in vitro (e.g., Adams and Brown, 1973; Bowery and Brown, 1974; Bowery et al., 1976b), and can increase the release of catecholamines from isolated bovine adrenal medulla, perhaps by depolarizing chromaffin cells (Sangiah et al., 1974). In isolated, superior cervical ganglia of the rat, half-maximal depolarization was elicited by about 1.3 x lo-’ M GABA, and was antagonized by bicuculline; EDGE= 1.4 x lo-’ M (Bowery and Brown, 1974). Conformationally-restricted GABA-analogues, (4-aminotetrolic acid, trans-4-aminocrotonic acid, imidazoleacetic acid) elicited depolarizations similar to that of GABA, and their actions were prevented by bicuculline, methylbicuculline and TMDST (Bowery et al., 1975a; Bowery and Jones, 1976). y-Aminobutyrylcholine also exerted “GABA-like” activity on sympathetic ganglia (Bowery and Brown, 1972). Some convulsant bicyclic organophosphorus esters can antagonize GABA-induced depolarizations of rat superior cervical ganglion (Bowery et ul., 1976a).

3. “Binding” of GABA 3.1. INTRODUCTION These studies are being aimed at separating the “binding” of GABA into components related to its uptake (inactivation) and receptor-interaction; the receptor component will be emphasized here. “Binding” of GABA and other functionally-active amino acids to uptake sites of subcellular particles of the CNS occurs by Na+-dependent energy-independent, non-enzymatic mechanisms (e.g., Sano and Roberts, 1963; Elliott et al., 1965; DeFeudis, 1973a, b). This “binding” mechanism for GABA might subserve a “carrier-mediated” process for its transfer and accumulation by subcellular particles, and could be involved in the inactivation of synaptically-released GABA (e.g., Varon et al., 1965; Weinstein et al., 1965; Kuriyama et al., 1968% b). Changes in neither the constitution of cerebral particulate fractions nor in their rates of sedimentation can account for the increase in GABA “binding” produced by Na+ (DeFeudis, 1973b). Kuriyama et al. (1968a) showed that some GABA “binding” occurred to a synaptic vesicle fraction of mouse brain in the presence of Na+, and other evidence indicates that GABA might be stored in vesicles of some CNS nerve terminals (Hattori et al., 1973). However, Rassin (1972) found that

130

F.V. DEFEUDIS

even though substantial amounts of GABA, as well as taurine, aspartate, glutamate and glycine, were bound to synaptic vesicle fractions of guinea-pig cortex, these vesicular amino acids were not released by hypo-osmotic conditions (see also DeBelleroche and Bradford, 1973a). Thus, “transmitter” amino acids may not be stored in synaptic vesicles, as is acetylcholine. A criticism which might be applied to all of these studies with subcellular preparations is that “gliosomes”, which may be present in such preparations, may bind and/or accumulate GABA and other amino acids (see e.g., Henn et al., 1976). However. a recent comparison of the binding of P-alanine (a presumed marker for glial GABA uptake; Schon and Kelly, 1975) and GABA to synaptosome-enriched fractions of three regions of rat brain revealed that Na+-dependent GABA binding is not likely to be associated with glial elements (Somoza et ul., 1977). In the following sections, studies on the binding of GABA performed both in the presence and absence of Na+ will be discussed, though most GABA binding occurs to uptake sites in the presence of Na+. 3.2. BICUCULLINE-SENSITIVE GABA

BINDING

In these studies either synaptosome-enriched fractions or membrane fractions prepared by osmotically shocking such fractions have been employed. That properties of GABAreceptors can be studied in these preparations seems likely since synaptosomes generally contain postsynaptic thickenings and postsynaptic membranes (see DeRobertis et al., 1967; review by DeBelleroche and Bradford, 1973b). In the initial studies with bicuculline, an attempt was made to separate the binding of GABA to postsynaptic receptors from that associated with Na+-dependent GABA uptake by pre-incubating a synaptosomal fraction of rat cerebellar cortex with 5 x lo_4111 chlorpromazine (CPZ) for 10min at 4”C, followed by incubations in the presence of CPZ plus various concentrations of 3H-GABA, with or without a lOOO-fold excess of unlabelled GABA (Peck et al., 1973; see also Peck et cl/., 1976). CPZ-insensitive (“specific”) 3H-GABA uptake by the particles (lCr159;; of total GABA uptake) was saturable and was inhibited by bicuculline but not by L-diaminobutyrate, picrotoxin, penicillin, or glycine. “Specific” 3H-GABA b’m d’mg to a synaptic membrane fraction of rat cerebellar cortex in the absence of added Naf, was also saturable (K,, ? 2.3 x 10e5 M) and competitively inhibited by bicuculline (Ki z 5.7 x 10s5 M). A solubilized GABAbinding component was isolated which had a K,, 2 2.3 x lo-” M and a B,,;,, ? 3.4 picomoles GABA/pg protein; bicuculline also competitively inhibited this binding with a Ki 2 5.6 x lo-’ M (Peck et al., 1976). Experiments with degradative enzymes indicated that this GABA-binding component was either a lipoprotein or a protein dependent on lipid for its structural integrity and/or binding activity (see also Sano and Roberts. 1963). Zukin et al. (1974) examined GABA binding to a synaptic membrane fraction of rat brain incubated at 4°C for 5 min in the absence of added Na+. “Specific” 3H-GABA binding, obtained by subtracting from the total bound radioactivity the amount not displaced by ,high concentrations of bicuculline (lop4 M) or GABA (10e3 M),was saturable (K, 2 1 x 10m7M),and was half-maximally displaced by about 5 x 10m6M bicuculline, and maximally displaced by about 1 x 10M4M bicuculline (i.e., bicuculline had about 1/50th the affinity of GABA for these sites). The potencies of various amino acids at inhibiting “specific” GABA binding paralleled their potencies as neuronal depressants, but differed markedly from their relative affinities for synaptosomal GABA uptake (see also Enna and Snyder, 1975). Naf-dependent GABA binding was hardly affected by bicuculline, imidazoleacetic acid and 3-aminopropanesulfonic acid, agents which exerted considerable effects on Na+-independent binding. It was concluded that the postsynaptic GABA-receptor is best shown in the absence of added Na+ using frozen/thawed synaptic membrane fractions. Scatchard plots of GABA binding to synaptic membrane fractions of rat brain indicated single populations of binding sites for

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131

both Na+-dependent and Na+-independent mechanisms (but see below), the KB for Na+-dependent GABA binding being about 1.2 x lOA M and that for Na+-independent binding being about 4 x 10e7 M; B,,, was about 30 picomoles/mg protein for Na+dependent binding and about 0.7 picomole/mg protein for Na+-independent binding (Enna and Snyder, 1975). For Na+-dependent GABA binding, the rate constant for association was 6 x lop7 Kimin-‘, association was complete after about 100 s, halfmaximal binding occurred at about 30 s, and the rate of dissociation of bound 3H-GABA had a half-life of about 1.9 min; rate constants for the association and dissocuatioh of Naf-independent GABA binding were so rapid that they could not be measured (Enna and Snyder, 1975). In further studies, Enna and Snyder (1976) examined Na+-independent GABA (8 x 10p9~) binding to synaptic membrane fractions prepared from frozen bovine and sheep retinae, alone or in the presence of lop3 M GABA, 10e4 M bicuculline, or other drugs. Half-maximal binding occurred at about 1 x 10e7 M GABA, and a Scatchard plot revealed two distinct components, a “high-affinity” site with K, z 1.8 x 10e8 M’ and B,,, E 0.6 picomole/mg protein and a “low-affinity” site with K, z 2.2 x 10e7 M and B,,, s 1.6 picomole/mg protein (cf. DeFeudis and Somoza, 1977; below). The single binding site observed in the earlier study of Enna and Snyder (1975) corresponded to the low-affinity site of this latter study. It was suggested that treatment of the tissue with Triton X-100 facilitated the separation of these components. Bicuculline inhibited this binding to bovine retina, the (+) isomer (the physiologically effective GABA-antagonist; see Johnston, 1976a) being more effective than the (-)isomer. As had been shown previously with rat CNS preparations (Zukin et al., 1974) picrotoxin exerted a negligible effect on GABA binding to retina. Hill plots yielded coefficients close to unity for both bicuculline and GABA (see also Enna and Snyder, 1975; DeFeudis and Somoza, 1977). The density of “high-affinity” GABA receptors of retina, about 1 picomole/mg protein, was similar to those found for rat whole brain in the absence of Na+ (Enna and Synder, 1975) and for synaptosomal fractions of rat cerebral cortex in the presence of Na+ (DeFeudis et al., 1975; DeFeudis and Somoza, 1977; see below). Fiszer de Plazas and DeRobertis (1975) showed that r4C-GABA binding to a fraction enriched in nerve-ending membranes, in the presence of Na+, occurred with a K, E 3 x 10m5M and a B,,,, G 0.4 picomole GABA/@g protein, and was inhibited by about 60% by CPZ (5 x 10e4~) and by about 45% by bicuculline (4 x 10m4M). An hydrophobic protein fraction prepared from these membranes bound r4C-GABA, in the absence of Na+, with a K, g 3 x 10m5M and a B,,, 2 10.5 picomoles GABA/pg protein; bicuculline competitively inhibited this binding (Ki % 2.7 x 10m4 M). Binding curves for both membrane and protein fractions indicated the presence of a single type of GABA binding site (cf Giambalvo and Rosenberg, 1976; see below). This GABA binding site was partially separated from a site involved in the binding of L-glutamate, and differed kinetically and chromatographically from a GABA binding site isolated from crustacean muscle (DeRobertis and Fiszer de Plazas, 1974). The above-mentioned studies can lead one to believe that the binding of GABA to its receptor and the action of bicuculline on this binding are shown best in the absence of added Naf, since the Naf-dependent binding of GABA is prevented by this condition. However, a bicuculline-sensitive component of GABA binding has also been demonstrated under more physiological conditions (e.g., Peck et al., 1973; DeFeudis et al., 1975; Fiszer de Plazas and DeRobertis, 1975). The binding of 3H-GABA (4.8 x lo-’ M) to a synaptosome-enriched fraction of rat cerebral cortex in an isosmotic medium containing physiological concentrations of Na+ and other ions at 0°C was significantly decreased by bicuculline methiodide (BMI) at 10-7-10-3 M (DeFeudis et al., 1975; Fig. 3). The ECUS of BMI (about 1.7 x 10m8 M) was about one-fifth as great as the K, of GABA (8 x lOmEM) for interaction with the postulated “GABA-recepto?. Further kinetic analyses of such data have revealed that “double-” or “tripleaffinity” mechanisms appeared to be involved in this binding of GABA, that “highestaffinity” GABA binding occurred with a K, z 6 x lo-’ M and a B,,,, 2 2 nanomoles

13’

F. V. DEFEUDIS

GABA/g pellet, and that BMI inhibited this binding with an ECU,, 2 7 x 10-s M (DeFeudis and Somoza, 1977). The observed K,, value is similar to the suggested K,, value for GABA action under in uivo conditions (Curtis and Johnston, 1974a; Curtis et al., 1974b) and is lower than the KDs estimated for Na+-independent GABA binding to membrane fractions of rat brain (3.7 x 10d7 M; Enna and Snyder, 1975) and hamster cerebral cortex (5.6 x lo-’ M; Simantov et al., 1976). This K,, value (6 x lo-’ M) agrees best with that recently obtained by Enna and Snyder (1976) for the “high-affinity” Na+independent binding of GABA to bovine retina (1.8 x lo-* M; see above). This “highaffinity” binding site was perhaps not observed in the studies of Enna and Snyder (1975) and Simantov et al. (1976) since their method for correcting the data tends to obscure this component (see DeFeudis and Somoza, 1977). The EC,,S of 1.7 x 10m8 M (DeFeudis et al., 1975) and 7 x lo-’ M (DeFeudis and Somoza. 1977) for BMI displacement of GABA binding are at least an order of magnitude lower than the EC~~S estimated for bicuculline action on GABA binding to rat cerebral or retina membrane fractions in the absence of added Na+ (Zukin et al., 1974; Enna and Snyder, 1976), and are in accord with values predicted from in ciuo iontophoretic experiments (Curtis et al., 1974b; Johnston, 1976b). Thus, the action of bicuculline on GABA-receptors might depend upon Naf to some extent. Hill coefficients close to unity (DeFeudis and Somoza, 1977) agreed with those found by Enna and Snyder (1975) for GABA binding both in the presence and absence of Na+. However, recently Giambalvo and Rosenberg (1976) have found that 14C-GABA binding to junctional complexes of rat cerebellum, at 25°C in the presence of Na+, proceeded with a Hill coefficient of about 2.2, indicating positive cooperativity (i.e., that the GABA-receptor might be oligomeric, containing multiple binding sites per molecule). This binding was inhibited by methyl-bicuculline, picrotoxinin and imidazoleacetic acid, whereas curare, strychnine and ~-2, 4-diaminobutyrate were ineffective (see also Giambalvo, 1975). These latter results are in accord with the proposal of Takeuchi and Takeuchi (1969) that two molecules of GABA are required to activate the GABAreceptor of the crayfish neuromuscular junction. Other recent studies have indicated that three molecules of GABA are required to activate its receptor in slices of guinea-pig cerebellar cortex and in locust muscle (Brookes and Werman, 1973; Okamoto and Quastel, 1976; see Section 2.5). Giambalvo and Rosenberg (1976) also showed that phospholipids competed with GABA for binding sites, and that phospholipase C increased GABA binding by 260%. They calculated that the density of GABA binding sites was about a value which compares well with estimates of the densities of 27,000 molecules/pm2, a-toxin and a-bungarotoxin binding sites made from studies on electric tissue (Miledi et al., 1971; Cohen and Changeux, 1975). Neither Na+ nor Ca2+ appeared to be required for the GABA binding process. A “triple-affinity” mechanism (three populations of binding sites) for GABA binding in the presence of Na+ had been shown previously with data that had been corrected by using sucrose distribution ratios (DeFeudis and Schiff, 1974, 1975; Fig. 4). More recently, “triple-affinity” mechanisms have also been demonstrated for the binding of GABA to a membrane fraction of rat cerebral cortex in the presence of Na‘ (Estrada, 1977), and for the binding of aspartate to an hydrophobic protein fraction of rat cerebral cortex in the absence of Na+ (Fiszer de Plazas and DeRobertis, 1976). In the presence of Na+, BMI (10-j M) displaced about l-2 picomole GABA/mg protein, or about 50-100 picomole GABA/g original wet weight of tissue (DeFeudis et al., 1975; DeFeudis and Somoza, 1977), a value which agreed with estimates of GABAreceptor density made with synaptic membrane fractions of rat (e.g., Enna and Snyder, 1975), hamster (Simantov et al., 1976) and chick (Enna et al., 1976) brain, and bovine retina (Enna and Snyder, 1976) in the absence of added Naf. This BMI-displaceable component of GABA binding represents only about 1/40,00Oth of the total GABA content of cerebral cortex or about l/lOOOth of the total amount of GABA than can be bound to synaptosome-enriched fractions in the presence of a physiological concentration of Na’.

133

GABA-RECEPTORS

Concentration

of

added

GABA

(Ml

FIG. 3. Effect of bicuculline methiodide (BMI) on the binding of GABA to a synaptosomeenriched fraction of rat cerebral cortex in the presence of physiological concentrations of Na+ and other ions. Note that at low concentrations of GABA, BMI can displace about 2&30% of the bound GABA. (Unpublished graph; data in DeFeudis et al., 1975.)

These studies, together with others (see above), have revealed that “GABA-receptors” can be studied in the presence of the potent Na+-dependent binding mechanism for GABA (which is likely related to its uptake by neurones and glia), and that the GABAreceptor (as revealed by BMI) might possess a greater .affinity than the uptake system for GABA. The close quantitative similarity of the BMI-sensitive GABA binding comreceptor (as revealed by BMI) might possess a greater affinity than the upptake system for GABA. The close quantitative similarity of the BMI-sensitive GABA binding component shown in the presence of Na+ and the bicuculline-sensitive GABA binding that occurred in the absence of Na+ suggests that the same process(es) is involved. Thus, the GABA-receptor appears to remain intact after CNS particles have been subjected to freezing/thawing and the absence of Na+ (see also Giambalvo and Rosenberg, 1976). However, though some properties of GABA-receptors might be shown in Na+-free medium, one cannot avoid considering that the properties of the physiological process, receptor-interaction, should depend upon a normal physiological environment. Other studies conducted with a crude mitochondrial fraction of mouse brain revealed an uptake (“binding”) of 14C-GABA at 0°C that was saturable; K, z 2.8 x 10e5 M (Olsen et al., 1975). This process was strictly Na+-dependent in synaptosomal fractions and synaptosomal membranes from whole brain, cerebral and cerebellar cortices. Chlorpromazine and imipramine (both at 5 x 10e4 M) inhibited GABA uptake non-competitively by as much as 100%; picrotoxin (3 x lop4 M) was ineffective, whereas 3 x 10e4 M of bicuculline-like compounds did inhibit the process. Most recently, Lloyd et al. (1977) have shown that Na+-independent binding of 3H-GABA to membranes prepared from human cerebellar cortex occurred with a KB z 3.4 x 10e7 M (see also Enna et al., 1976) and that this binding was inhibited by bicuculline; EC,, z 2.2 x 10m5 M. GABA binding to membranes of substantia nigra, but not to other regions studied, was lower in Parkinsonian patients. The above studies revealed apparent dissociation constants for “high-affinity” GABA binding of about 6 x 10m8-3 x lo-’ M and that about 2 x 10-‘-4 x lo_4111 bicuculline-like substances were required for half-maximal displacement of the bicucullinesensitive component of bound GABA. However, it should be noted that, like picrotoxin, bicuculline not act directly on GABA-receptors. As revealed by their non-competitive inhibition of GABA responses in invertebrates, both agents may interact with GABAregulated ionophores (Takeuchi and Takeuchi, 1969; Takeuchi and Onodera, 1972).

F. V. DEFEUDIS

13-l

3.3. OTHER AGENTS THAT MIGHT ACT ON GABA-RECEPTORS Of the other possible GABA-antagonists, picrotoxin usually had no effect on Na+-independent GABA binding. whereas d-tubocurarine did inhibit this process; IC,, ? 3.8 x 10m5 M (Zukin rt al., 1974; Enna and Snyder. 1975, 1976; see above). Also, d-tubocurarine, though having only about l/lOOth the potency of GABA for inhibiting Na’-independent GABA binding, possessed over 200 times more affinity for Naf-independent, than for Na+-dependent GABA binding (Zukin et ul., 1974; Enna and Snyder, 1975). Strychnine decreased Na+-independent GABA binding (Zukin et al., 1974), and also decreased the binding of both GABA and glycine to synaptosome-enriched fractions of mouse and cat brain in the presence of Na+, perhaps by an effect on the transport mechanisms for these amino acids (DeFeudis et [I/.. 1976a, 1977). Bicuculline might also inhibit the binding of both glycine and GABA (G. Svenneby and E. Roberts; personal communication to Olsen, 1976). These findings strengthen the views that binding studies do not necessarily provide results that reflect irl L?L’Odrug actions, and that bicuculline and strychnine are not highly specific agents.

3.4. REGIONAL VARIATIONS IN GABA

BINDING

Regional variations in GABA “binding” might be correlated with variations in its endogenous content, receptor density, and/or iontophoretic potency. Although “receptor” binding of GABA appears to represent only about l/lOOOth of total GABA-binding which occurs in the presence of Na+, this can account for a much greater proportion at low concentrations of GABA (see above and Fig. 3); hence, both Na+-dependent and Na+-independent binding will be discussed here. The first study on regional dependency of Na+-dependent GABA binding revealed, in tissues homogenized at 0°C in an isosmotic sucrose solution containing 40m~ NaCl plus 14C-GABA, that this binding occurred to a greater extent to “crude nuclear” fractions prepared from rat cerebral cortex and hippocampus than to those of whole cerebellum or pons-medulla (DeFeudis and Black, 1973). The binding of 3H-GABA and 14C-glycine to subcellular particles of rat cerebral cortex and spinal cord was examined

~~~~~~~~’ I 3

B,n-moles

amino

75 100 acid in P,/g,tissue

150°

FIG.4. Ho&tee plots for the binding of GABA and glycine to a synaptosome-enriched fraction of rat cerebral cortex at OT, 10 min. in the presence of an isosmotic sucrose solution containing systems. (Re-drawn 32mM NaCI. K,, and B,,,,,, values are indicated for these “triple-affinity” from DeFeudis and Schiff. 1975. with permission from Erprrimrnral Neurology.)

GABA-RECEPTORS

135

both by the above-mentioned method and by a method with which “synaptosomal-mitochondrial” (P2) fractions were first isolated in the absence of added salt (to prevent “clumping”; see Gray and Whittaker, 1962) and then exposed to the labelled amino acids in the presence of 32 mM NaCl (DeFeudis, 1973~). “Regional dependency” existed, as GABA was bound to a greater extent to particles of cerebral cortex than to those of spinal cord; “regional specificity” existed, as more GABA than glycine was bound to particles of cerebral cortex. Further studies on P2 fractions in the presence of 32 mM NaCl revealed the following order of binding potency: GABA, cortex > GABA, cord 2 glycine, cord > glycine, cortex. For cerebral cortex, GABA binding occurred with a K, 2 1.8 x lo-’ M and a B,,,, g 65 nanomoles/g cortex, whereas glycine was bound to spinal particles with a K, g 3.3 x lo-’ M and a I?,,,, 2 43 nanomoles/g cord (DeFeudis, 1974, 1975b). At saturation, the capacity of the GABA binding mechanism of cerebral cortex (about 1.6 x lo- l9 mole GABA/nerve ending) was sufficient to account for’ both the depressant action of iontophoretically-applied GABA and for inactivation of the amount of GABA required to produce this effect. The binding of both GABA and glycine was then shown to occur by triple-@nity processes to particles of cerebral cortex and by double-ufinity processes to those of spinal cord (DeFeudis and Schiff, ,1974, 1975; see also Section 3.2 and Fig. 4). B,,, values for GABA and glycine reflected their different endogenous concentrations in cortex and spinal cord. “High-affinity” processes (DeFeudis, 1974) and “intermediate-affinity” processes (DeFeudis and Schiff, 1975) for GABA binding to rat cerebral cortex possessed K, values which were rather identical to K, values reported for “high-affinity” energymediated uptakes of GABA and glycine by small slices or homogenates of rat cerebral cortex (e.g., Iversen and Neal, 1968; Balcar and Johnston, 1973; Peterson and Raghupathy, 1973). Thus, “high-affinity” uptake and Na+-dependent binding of GABA could be identical processes (DeFeudis, 1973~). GABA was bound to a greater extent to synaptosome-enriched fractions of cerebral and cerebellar cortices than to those prepared from several other regions of the feline CNS (Balfagon et al., 1975) further supporting iontophoretic findings (see e.g., reviews by Curtis and Watkins, 1965; Curtis and Crawford, 1969; Krnjevic, 1974; DeFeudis, 1975a). Zukin et al. (1974) observed that “specific”, bicuculline-displaceable, 3H-GABA (3.2 x 10e8 M) binding to membrane fractions in the absence of added Na+ was greatest in cerebellum, least in spinal cord and medulla oblongata-pons, and of intermediate potency in several other regions of rat CNS. It seems noteworthy that in the presence of a physiological concentration of Na+ the binding of 3H-GABA to synaptosomal-mitochondrial fractions of cerebellar cortex was also found to be as great (Balfagon et al., 1975) or greater (Sagarra et al., 1975) than its binding to other CNS regions. Na+-independent GABA binding did not correlate closely with regional variations in endogenous GABA content (Zukin et al., 1974). A further study revealed that marked differences existed between Na+-dependent and Na+-independent GABA binding processes among eight regions of rat CNS and that regional variations in synaptosomal GABA uptake correlated neither with Na+-dependent nor with Na+-independent GABA (2.5 x 1Om8M) binding (Enna and Snyder, 1975). However, a rather close correlation among Na+-independent GABA binding, GAD activity and synaptosomal GABA uptake was. revealed among thirty-one regions of monkey (Macaca mulatta) CNS (Enna et al., 1975). The number of sites and affinities for GABA binding mechanisms in cerebellar cortex, frontal cerebral cortex and thalamus or putamen were also determined by Enna et al. (1975). No regional differences among the KB values for Na+-dependent (see also Bond, 1973) or Na+-independent GABA binding were found, and although the total number of binding sites (B,,,) for both Naf-dependent and Na+-independent GABA binding did exhibit regional variation, this was similar to variations in binding obtained with single concentrations of GABA. The conclusion of Enna et al. (1975) based on studies with frozen/thawed membrane fractions in the absence of Na+, that variations in GABA binding appeared to reflect differences in B,,,, rather than in affinity, did not differ

136

F. V. DEFEUDIS

from that drawn from studies on Na+-dependent GABA binding to freshly-prepared synaptosome-enriched fractions (DeFeudis and Schiff, 1975). Recent studies (Lloyd et ul., 1977) have revealed a regional dependency of Nat-independent 3H-GABA (2.5 x lo-* M) binding to membranes prepared from human brain. In normal brain, cerebellar cortex had the greatest number of binding sites, and this was followed by hippocampus > cerebral cortex areas; GABA binding in several basal ganglia regions was lower than that of cerebral cortex. Recent reviews on the regional distribution of GABA, GAD activity (Fahn, 1976; Okada, 1976) and GABA uptake (Martin, 1976) permit further assessment of these studies on the regional dependency of GABA binding. Fahn’s (1976) analysis revealed that GABA content and GAD activity correlate remarkably well among various CNS regions of several species. GABA contents of CNS regions of monkey and rat are also strikingly similar, leading one to suspect that the regional variation in GABA binding in these two species should also be similar. However, though close correlations were shown among Na+-independent GABA binding, GAD activity, and synaptosomal GABA uptake among regions of monkey CNS, this was not the case between this binding and GABA uptake regions of rat CNS (see above). Also, the finding that K,, values for both Na+-dependent and Nat-independent GABA binding in monkey brain (Enna et al., 1975) were only about 1/8th of those in rat brain (Enna and Snyder. 1975) is not in accord with the very similar regional distribution of GABA among CNS regions of these two species. 3.5. EFFECTSOF MATURATION AND ENVIRONMENT ON GABA BINDING 3.5.1. Maturatim Naf-dependent “binding” of GABA to subcellular particles of rat brain changes during maturation (DeFeudis, 1972b, 1973a, d). 3H-GABA “binding” occurred to a greater extent to P2 fractions prepared from the brains of ll- or 15-day-old rats than to those prepared from rats of other ages, whereas in “crude nuclear” fractions it increased until 21-22 days of age, thereafter remaining rather constant. Maximal Na+-dependent GABA binding to P, fractions coincided with the period during which cerebral growth rate (Davison and Dobbing, 1968), synaptogenesis (Aghajanian and Bloom, 1967), and excitability (Timiras et al., 1968) are also maximal. However, since similar age-related changes existed for the binding of glutamate, glycine and arginine (DeFeudis, 1973a), such changes do not appear to be related specifically to GABA or to other putative amino acid transmitters. In chick embryo brain both GAD activity and Na’-independent GABA binding were barely detectable up to 9 days of conceptual age, and thereafter both systems increased rapidly, but no appreciable time difference existed between the development of GAD activity and Na+-independent GABA binding (Enna et al., 1976b; Fig. 5). The greatest increases in GAD activity and Naf-independent GABA binding correlated temporally with the appearance of morphologically-distinct synapses and with mature cerebral electrical activity and motor coordination (e.g., Rogers et al., 1960; Foelix and Oppenheim, 1973). Coyle and Enna (1976) showed that the development of “high-affinity”, Na+dependent GABA uptake by particulate fractions of rat brain differed markedly from that of GAD activity, uptake being near adult levels at birth, peaking considerably above adult levels at l-2 weeks after birth, and then declining toward the adult level by 4 weeks after birth. Developmentally related changes in Na+-dependent GABA uptake (Coyle and Enna, 1976) and in Nat-dependent binding of GABA (DeFeudis, 1972b, 1973a, d) were remarkably similar. In the studies of Coyle and Enna (1976), Nat-independent GABA binding increased dramatically after 8 days following birth, approximated adult levels by 4 weeks of age, and correlated best with the increase in GAD activity in all brain regions studied. Hence, development of the “GABA-recep-

137

GABA-RECEPTORS

6

14

IO

Conceptual

6

IO

I4

Conceptual

16 age,

22

26

days

18

22

oge,

doys

26

FIG. 5. Development of glutamate-cc-decarboxylase (GAD) activity, the postsynaptic receptor for GABA and orotein content in chick brain expressed per whole brain (a) and as a function of protein (b). For receptor experiments, tissue homogenates were incubated at 4°C for 5min with 25 x 10m9 M 3H-GABA (5OO,OOOcounts/min), with lo-“ M bicuculline added to half the tubes serving to displace specific 3H-GABA binding. GAD was assayed according to Albers and Brady (1959). (Reproduced with permission from Enna et al., 1976b).

to?, as measured, appeared to correlate best with that of presynaptic “GABA-ergic” elements. These studies have revealed that developmentally-linked changes in GABA binding have a morphological basis. 3.5.2. Environment In the presence of Na+, the binding of both GABA and glycine to synaptosomeenriched (PJ fractions of the brains of male mice was lower in animals that had been kept “isolated” for 8-10 weeks than in their “aggregated” counterparts (DeFeudis et al., 1976a; see also DeFeudis, 1972a). Experiments with discontinuous sucrose gradient centrifugation revealed that both GABA and glycine were bound to a lesser extent to a “heavy synaptosomal fraction” of the brains of “isolated” mice when the data were expressed as mole amino acid/fraction, but since the protein contents of fractions from “isolated” mice were also lower than those of their “aggregated” counterparts, no difference in amino acid binding existed when these data were expressed as mole amino acid/mg protein (DeFeudis et al., 1976b). Analyses of polypeptide profiles (Conde and DeFeudis, 1977) revealed that this environmentally-induced difference was of a quantitative nature. These findings gain additional significance when it is considered that only GABA, among the seven amino acids measured by Osborne et al. (1976), was found to be relatively concentrated in a “heavy” synaptosomal fraction of rat olfactory lobe. Thus, isolation-rearing had induced a morphological change in the brain. The binding of GABA and glycine provided a measure of the number (or development) of cerebral nerve endings which can be affected by long-term changes in external environ-

13X

F. V. DEFEUDIS

ment. The brains of individually-housed mice, by having decreased capacities for the binding of GABA and glycine, could have less inhibitory nerve endings and/or receptors, a change which could be related to the increased aggressiveness of male mice produced by environmental impoverishment. These results support the notion that the brain exhibits structural plasticity when an animal is adapting to conditions which elicit dramatic changes in its behavior. Further studies with amino acid-antagonists should reveal the receptor components of such environmentally-induced changes.

4. Concluding

Remarks

Much progress has been made toward elucidating vertebrate GABA-receptors. In the CNS, it now seems likely that such receptors exist and that though the binding of GABA, itself, to its receptors may not be dependent upon Na+, the actions of its antagonists (e.g., bicuculline) may possess reduced affinities in the absence of Na+. Recent binding studies have revealed that the K,, for specific GABA binding and the ECUS for BMI inhibition of this binding are within the range predicted from in ciao iontophoretic studies. The best available GABA-antagonists, bicuculline and picrotoxin, may act indirectly by affecting GABA-ionophores. Since Hill coefficients calculated from irk citro data on GABA binding or from in cliuo data on receptor-activation varied from about one to three, it is not yet clear whether GABA-receptors are activated by one or more molecules of GABA. Such differences could be related to the experimental techniques employed, to differences in the properties of GABA-receptors when studied in citro vs irz nivo, and/or to differences based on the species or nervous tissues that were studied. It seems possible now to measure GABA-receptors in relation to the major determinants of normal and abnormal behaviors; i.e., to study the properties of GABA-receptors in relation to maturation, genetics and environmental modification. Such studies, together with those concerning the changes in GABA-receptors that might be associated with various neurological disorders in man, should provide further information about synaptic plasticity and might reveal the physiological significance of GABA in the CNS.

Acknowledgements Thanks for typing

are due to Mrs Maria Pascual for the art work several drafts of this manuscript.

References

and to Miss Gaynor

Fox

,

ADAMS, P. R. and BROWN, D. A. (1973) Action of ;j-aminobutyric acid (GABA) on rat sympathetic ganglion cells. Br. J. Pharmac. 47, 639%64OP. . AGHAJANIAN. G. K. and BLOOM, F. E. (1967) The formation of synaptic junctions in developing rat brain: A quantitative electron microscopic study. Brain Res. 6. 71&727. ALBERS, R. W. and BRADY, R. 0. (1959) The distribution of glutamic decarboxylase in the nervous system of the rhesus monkey. J. Biol. Chem. 234, 926928. ALTMANP‘~.H., BRUGGENCATE, G. TEN, PICKELMANN. P. and STEINBERG, R. (1976) Effects of GABA, glycine, picrotoxin and bicuculline methochloride on rubrospinal neurones in cats. Brain Res. 111, 337-345. BALCAR, V. J. and JOHNSTON, G. A. R. (1973) High affinity uptake of transmitters: Studies on the uptake of L-aspartate, GABA, L-glutamate and glycine in cat spinal cord. J. Neurochem. 20, 529-539. BALFA&N. G., GERVAS-CAMACHO, J., GADEA-CIRIA, M., SOMOZA, G. and DEFEUDIS, F. V. (1975) Comparison of GABA binding to synaptosomal fractions of seventeen regions of the feline central nervous system. E.xp. Neural. 48, 383-386. BANNA, N. R. and JABBUR, S. J. (1971) The effects of depleting GABA on cuneate presynaptic inhibition. Brain Res. 33, 53&532. BARKER. J. L. and NICOLL, R. A. (1972) Gamma-aminobutyric acid: Role in primary afferent depolarization. Science 176, 1043S1045. BARKER, J. L. and NICOLL, R. A. (1973) The pharmacology and ionic dependency of amino acid responses in the frog spinal cord. J. Physiol. (Land.) 228, 259-277. BARKER, J. L., NICOLL, R. A. and PADJEN, A. (1975a) Studies on convulsants in the isolated frog spinal cord. I. Antagonism of amino acid responses. J. Physiol. (Lond.) 245, 521-536.

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GABA-receptors in the vertebrate nervous system.

Progrrn in Ncurohioloy~. 1977. Vol. 9, pp. 123-145. GABA-RECEPTORS Pergamon Press. Prmted in Great IN THE VERTEBRATE SYSTEM F. V. “Ram& Britai...
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