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Described in this unit are ligand-binding assays for GABAA , GABAB , and the homomeric ρ GABAA (formerly GABAC ) receptor recognition sites in brain tissue. Although GABA binding sites are present in peripheral organs, most research is directed toward examining these receptors in the CNS. These assays may also be used to determine the affinity of an unlabeled compound for the GABA binding sites. Excluded from the unit are ligand-binding assays for other components of the GABAA receptor complex, such as the benzodiazepine or ion-channel binding sites., (Copyright © 2013 John Wiley & Sons, Inc.)

Modification in gamma-aminobutyric acid-B (GABA(B)) receptors may contribute to the symptoms of some neurological and psychiatric disorders and to the clinical response to psychotherapeutics. The present study was undertaken to determine whether chronic administration of tranylcypromine (TCP), an antidepressant, and chronic stress influence GABA(B) receptor function in rat brain. The results indicate that TCP treatment, but not stress, increases GABA(B) receptor activity in the cerebral cortex, as measured by baclofen-stimulated GTPgammaS binding. In addition, chronic administration of TCP enhances significantly the locomotor response to a single dose of amphetamine, an effect that is abolished by restraint stress. These results indicate that although TCP administration modifies brain GABA(B) receptor activity, which may contribute to the antidepressant response to this agent, this effect is unrelated to the interaction of stress and TCP treatment on the locomotor response to amphetamine.

Pharmacologic and biochemical data indicate a variety of gamma-aminobutyric acidA receptor subtypes. The identification of gamma-aminobutyric acidA receptor subunits and their isoforms has yielded a more refined characterization of the pharmacologic selectivity of these sites. Such structural diversity may explain the different responses noted with drugs acting through gamma-aminobutyric acidA receptors and makes possible the design of new agents capable of selectively manipulating this neurotransmitter system.

The discovery of sigma binding sites has prompted investigation into the functional role of these sites. Binding studies have revealed that sigma sites exhibit a unique pharmacological profile, and have provided evidence favoring the existence of a multiplicity of sigma binding sites in central nervous system. However, the findings that chemicals having diverse structures and therapeutic applications are all potent sigma agents, and that sigma binding sites are present in peripheral tissues, have raised concerns about the physiological and pharmacological relevances of this site. Furthermore, an endogenous ligand for the sigma binding site has not yet been identified. Finally, there is a lack of data regarding the functional coupling of sigma binding sites, although recent studies have provided some clues as to a possible signalling mechanism (Karbon et al., 1990). Besides raising questions about the relevance of sigma sites, the paucity of information on their functional properties has made it difficult to distinguish sigma agonists from and antagonists. While haloperidol is assumed to be a sigma antagonist, the finding that prolonged administration of this neuroleptic decreases the number of sigma sites would seem to argue in favor of it being an agonist for this site. Regardless of the precise nature of the sigma binding site, studies have suggested that it may represent the site of action for a number of important drugs. For example, haloperidol, a butyrophenone antipsychotic, exhibits high affinity for sigma binding sites, and several psychotomimetics, including PCP and (+)-benzomorphans, also bind this site.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro incubation of rat cerebral cortex slices with antidepressant drugs reduced beta-adrenergic receptor binding of 3H-dihydroalprenolol by 30%. The decrease was maximum after 60 min, and was reversible after 120 min. (-)-Isoproterenol incubation caused a rapid and reversible loss (60%) of beta-receptor binding. Both effects were due to a decrease in beta-receptor sites. In vitro beta-receptor subsensitivity due to desipramine and isoproterenol was non-additive. The observed reversible loss of beta-receptor sites may be an initial phase of the beta-receptor down-regulation by antidepressants seen in vivo.

Receptor binding studies were undertaken in an attempt to identify and characterize pharmacologically and functionally distinct receptor sites for gamma-aminobutyric acid (GABA) in rat brain. The results indicated that the potency of bicuculline, a GABA receptor antagonist, to displace membrane-bound [3H]GABA varies significantly among different brain regions, with the greatest potency found in the cerebral cortex and midbrain. In addition, in the presence of 50 mM ammonium thiocyanate, the potency of bicuculline to displace specifically bound [3H]GABA was increased significantly, with the magnitude of this increase being greater in some brain areas than others. The biological relevance of this thiocyanate-induced shift in the potency of bicuculline to inhibit [3H]GABA binding was indicated by the finding that ammonium thiocyanate also increased the potency of bicuculline to inhibit GABA-activated benzodiazepine receptor binding, a biochemical measure of GABA receptor function. Receptor site saturation analysis revealed that ammonium thiocyanate selectively abolished the high affinity GABA binding site without affecting either the low affinity component or GABA-activated benzodiazepine receptor binding. These findings provide further evidence for the existence of pharmacologically distinct GABA receptor sites, with some being more sensitive to the blocking action of bicuculline than others. Furthermore, the data provide direct evidence to support the hypothesis that only low affinity GABA receptor sites are linked to the benzodiazepine receptor, indicating that the kinetically different GABA binding sites are also functionally distinct. The discovery that ammonium thiocyanate selectively destroys high affinity GABA receptor binding may be useful for further defining the pharmacological, biochemical, and functional differences between GABA receptors in brain.

Neurophysiological, biochemical and histochemical analyses have indicated a heterogeneous distribution of the GABA-ergic system in mammalian brain. The poor correlation between the regional distributions of presynaptic markers and synaptic receptors for GABA suggests that it is impossible to predict the functional importance of this neurotransmitter system in any particular brain region using either pre- or post-synaptic determinations alone. The possibility that the receptor affinity for GABA may vary significantly among different brain regions suggests that this may be another mechanism whereby the brain regulates the activity level of this transmitter system. Thus, in any given brain region, the storage and uptake capacities for GABA, the rates of its synthesis and release, and the number and affinity of GABA receptor recognition sites can all play a role in regulating GABA-ergic tone. Subtle alterations in any one or more of these parameters can lead to dysfunction, whose manifestation will depend upon the brain region involved. Regional distribution studies can also yield insights into the possible mechanisms of action of centrally-active drugs. Thus, a significant correlation between the regional distribution of a neurotransmitter receptor and a particular class of drug provides circumstantial evidence that the drug interacts with this receptor site. Such a situation appears to exist for the benzodiazepines and the GABA-receptor.

Bilateral adrenalectomy (ADX) induces a significant, regionally selective, increase in GABA, but not cholinergic muscarinic or alpha1-adrenergic, receptor binding in rat brain. The increase in GABA receptor binding in the midbrain occurs within 72 h of surgery, whereas that found in the corpus striatum becomes evident between 1 and 2 weeks later. These ADX-induced receptor changes are counteracted by the administration of corticosterone, a reversal which can occur within 24 h following a single administration of steroid. Unlike ADX, hypophysectomy causes a significant reduction in [3H]GABA receptor binding in these two brain areas, an action that is not reversed by corticosterone treatment. Furthermore, systemic administration of either ACTH1-39 or ACTH4-10 in unoperated animals causes an increase in midbrain and striatal GABA receptor binding similar to that observed in ADX animals. The increase in [3H]GABA binding observed after ACTH administration appears to be due to the appearance of low affinity, high capacity binding sites not observed in untreated animals. ADX had no effect on high affinity of GABA uptake, glutamic acid decarboxylase or GABA content in the brain regions where receptor modifications were noted. These findings indicate that GABA receptor binding in rat brain can be modified by changes in the circulating levels of ACTH.

Baclofen and other gamma-aminobutyric acid B (GABAB) agonists potentiate the cyclic AMP response in rat brain slices that occurs during exposure to norepinephrine, isoproterenol, adenosine, vasoactive intestinal peptide, and histamine. By themselves the GABAB agonists have only a slight effect on basal cyclic AMP levels. Dose-response and time-course studies revealed that baclofen has little influence on neurotransmitter recognition site affinity, but rather enhances the synthesis or accumulation of second messenger that occurs in response to these agents. Baclofen appears to be neither an inhibitor of phosphodiesterases nor does it require adenosine to promote the response to other transmitters. The synergistic interaction between baclofen and catecholamines is a calcium-dependent process and is evident only in the rat brain cerebral cortex, hippocampus and corpus striatum, being undetectable in the pons-midbrain, cerebellum, and spinal cord. In contrast to the findings with neurotransmitter receptor stimulants, GABAB agonists inhibited the cyclic AMP response to forskolin. It remains unclear whether this action is related to the neurotransmitter potentiating effect of baclofen. These data suggest that GABAB agonists may modulate neurotransmitter receptor function by influencing a component of the cyclic nucleotide-generating system beyond the level of the hormone recognition site.

[3H]Reserpine was administered (i.v.) to rats and they were killed 7 days later. At this time, the regional localization of radioactivity paralleled the distribution of specific binding sites observed in other laboratories. Autoradiographic studies of certain areas displayed a striking localization of radioactivity. There was a marked association of autoradiographic grains with areas containing catecholamine systems. In particular, the locus ceruleus, the caudate-putamen, the nucleus accumbens, the dorsolateral septum and the infundibulum had high grain densities. In the caudateputamen, there was a clear localization of grains to the neuropil. There was also a striking association of autoradiographic grains with certain hypothalamic nuclear, i.e., the dorsal premammillary nucleus, the prelateral mammillary nucleus and the lateral mammillary nucleus. In these areas, the grains were clearly localized in the cytoplasm of the cell bodies. All of the above localizations of autoradiographic grains were blocked by administration of unlabeled reserpine before injection of [3H]reserpine. The significance of these findings and their relationship to the clinical actions of reserpine are discussed.

The augmentation of isoproterenol or vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in rat brain slices by the GABAB agonist baclofen was compared to that mediated by tumor-promoting phorbol esters. The protein kinase C inhibitor H7 and desensitization of protein kinase C reduced the cyclic AMP augmenting effect of the phorbol ester, but not baclofen. Incubation of brain slices in the presence of both baclofen and a phorbol ester amplified the cyclic AMP response to isoproterenol or VIP to a greater degree than that found with either baclofen or the phorbol ester alone, with the increased augmentation appearing to be additive. These findings indicate that although stimulation of GABAB receptors or protein kinase C activation by phorbol esters have similar effects on transmitter-stimulated cyclic AMP production in brain, these augmenting actions appear to be independently mediated.

The development of anxioselective agents has made it possible to examine the biochemical basis of anxiety. Electrophysiological analysis revealed that benzodiazepines selectively enhance gamma-aminobutyric acid (GABA) neurotransmission. Subsequent work demonstrated the presence of a specific population of benzodiazepine binding sites on neuronal membranes. These sites appear to be linked to certain GABA receptors such that occupation of the benzodiazepine component reveals a group of GABA recognition sites that may be more sensitive to the neurotransmitter. These data, coupled with the findings that barbiturates may act, at least in part, by interacting with the GABA receptor-coupled chloride channel, suggest that pharmacological manipulations of the GABA system can alleviate the symptoms of anxiety. The anxioselectivity of the benzodiazepines may be related to the fact that they activate only a certain population of GABA receptors, whereas barbiturates can potentiate the majority of these sites. These discoveries point to the possibility that alterations in the GABA system may partially explain the neurochemical basis of anxiety.

While there are homeostatic mechanisms to protect the brain against wide fluctuations in the availability of essential nutrients, food deprivation is known to influence brain neurochemistry. Given the growing problem of infant undernutrition and the fact that the developing nervous system appears to be especially vulnerable to this type of insult, numerous studies have been conducted to define the relationship between nutritional factors and cellular growth and maturation in the brain. The data suggest that the development of both neural and nonneural elements are significantly affected by undernutrition. This includes processes and substances important for neurotransmission such as transmitter synthesis, degradation and receptor sites. Because many neuropsychiatric conditions can be traced to dysfunctions in synaptic neurochemistry, it is possible that some of the central nervous system abnormalities which result from childhood undernutrition may be a consequence of a modification in synaptic biochemistry. The present report reviews data relating to this issue with the aim of assessing its relevance to developmental neurobiology.

Examination of various biochemical characteristics of the GABAnergic nervous system in the rat brain was made between 15 days of gestation and adulthood. At birth, the concentration of GABA in whole brain and most regions is approximately 50% of adult levels, whereas the medulla-pons has achieved adult levels by this time. Compared to GABA levels, there is a marked lag in the development of the activity of glutamic acid decarboxylase in all areas studied; however, the activity of the enzyme increases in a linear fashion from birth to adulthood. The development of the uptake of GABA into particulate fractions prepared from whole brain and regions differs markedly from that of GABA and glutamic acid decarboxylase, with uptake near adult levels by birth, peaking considerably above that of the adult between one to two weeks after birth and then declining toward adult activity by 4 weeks after birth. Examination of the kinetics of GABA uptake into resuspended P2 fractions demonstrates that the developmental changes in the uptake reflects differences in the Vmax whereas the Kt remains constant; Studies on the development of the apparent postsynaptic receptor for GABA reveals that in all regions binding is approximately 25% of adult up to 8 days after birth, at which time it increases dramatically, approximating adult levels by 4 weeks after birth. The rise in the density of the apparent postsynaptic GABA receptor after 8 days postpartum correlates best with the increase in the activity of glutamic acid decarboxylase, a presynaptic marker.

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.

High (17 nM) and low (603 nM) affinity binding sites for [3H]tetrahydrotrazodone ([3H] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [3H]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [3H] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [3H]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors.

The alpha 2-adrenergic receptor regulation of cyclic adenosine monophosphate (cAMP) accumulation in rat brain slices was examined. using a prelabeling technique for measuring second messenger production. The mixed alpha-adrenergic agonist 6-fluoronorepinephrine, as well as the more selective alpha 2-agonists clonidine and UK-14,304, caused a concentration-dependent inhibition of forskolin-stimulated cAMP accumulation in cerebral cortical slices, whereas phenylephrine, a selective alpha 1-adrenergic agonist, had no inhibitory effect in this system. Moreover, alpha 2-adrenergic receptor antagonists were more potent than alpha 1-adrenergic antagonists in blocking the inhibitory response to UK-14,304. Neither alpha 1- nor alpha 2-adrenergic agonists displayed any inhibitory effect when cAMP accumulation was stimulated by isoproterenol, vasoactive intestinal peptide or 2-chloroadenosine. The results provide further evidence that some alpha 2-adrenergic receptors are negatively coupled to adenylate cyclase in brain, and yield a procedure for studying this phenomenon in intact central nervous system tissue.

Antinociception produced by the GABA uptake inhibitors d,l- SKF-89976A and SKF-100330A was characterized and compared to that produced by other types of GABAergic drugs. Using the mouse tail-immersion assay it was found that the antinociception produced by the uptake inhibitors was antagonized by scopolamine, a cholinergic muscarinic receptor antagonist. However, neither SKF compound demonstrated any significant affinity for muscarinic receptor binding sites suggesting that they are not direct-acting cholinomimetics. In vitro uptake experiments revealed that the SKF compounds selectively inhibit GABA transport, having no effect on the accumulation of aspartic acid, glutamic acid, beta-alanine or glycine. Moreover, antinociception and GABA uptake inhibition were stereoselective for SKF-89976A, with the d-isomer being more active in both tests. When comparing antinociceptive responses at maximally effective doses it was also found that the SKF compounds were substantially more efficacious than direct-acting GABA receptor agonists or a GABA transaminase inhibitor. These data suggest that uptake inhibitors may be facilitating GABA transmission in a system that is less affected by other types of GABAergic compounds.

Experiments were undertaken to determine whether alterations in rat brain monoaminergic tone influence the manner in which mianserin administration modifies beta-adrenergic receptor function. Whereas the chronic administration of mianserin had little effect on isoproterenol-stimulated cAMP accumulation in control cerebral cortical slices, treatment with this antidepressant prevented or reversed beta-adrenergic receptor super-sensitivity resulting from reserpine or 6-hydroxydopamine administration. In contrast, 5,7 dihydroxytryptamine treatment had no effect on beta-adrenergic receptor activity, or on the ability of mianserin to modify isoproterenol-stimulated cAMP accumulation. The results indicate that antidepressant-induced alterations in beta-adrenergic receptor activity may, in some cases, be a function of receptor sensitivity at the time of drug administration.

Chronic (21 days) antidepressant administration to rats results in a decrease in both serotonin and beta-adrenergic, but not cholinergic muscarinic, receptor binding in selected brain regions, with the frontal cortex appearing to be somewhat more sensitive to this effect. Neither nisoxetine nor fluoxetine, potent and specific inhibitors of norepinephrine and serotonin uptake respectively, caused receptor binding changes after chronic administration, suggesting that inhibition of transmitter uptake, in itself, is insufficient to cause receptor subsensitivity. In vitro experiments indicated that antidepressants are relatively weak alpha 2-receptor blocking agents, but some are potent on the alpha 1-receptor system indicating that the norepinephrine releasing potency of some antidepressants may not be mediated by blockade of presynaptic autoreceptors.

The effect of early undernutrition and dietary rehabilitation on [3H]gamma-aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein-deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high- and low-affinity components in the undernourished group, whereas control animals possessed only a low-affinity site. The concentration of low-affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X-100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA-activated benzodiazepine binding suggests the possibility that neither the high- nor the low-affinity GABA binding sites are coupled to this receptor component.

Long-term administration of imipramine caused a decrease in serotonin2 receptor binding in rat brain cerebral cortex, an effect that was abolished by ovariectomy. In contrast, ovariectomy had no effect on imipramine-induced decreases in hippocampal serotonin or in cerebral cortical and hippocampal beta-adrenergic receptor binding. Administration of estradiol or progesterone separately or in combination reestablished the effect of imipramine treatment on cortical serotonin2 receptors. These results suggest that ovarian steroids may play an important, but subtle, role in the neurochemical and perhaps clinical response to this drug.

Pre- and postsynaptic cholinergic markers were studied in various brain regions of mice and rats aged 6 to 30 months in an attempt to determine whether alterations in this transmitter system occur during the normal aging process. Reliable decreases in cholinergic receptor binding and choline acetyltransferase (CAT) activity were found in the cerebral cortex and corpus striatum. These alterations in the cholinergic system were typically more consistent and robust than changes involving glutamic acid decarboxylase, and enzyme marker for GABA neurons. No statistically significant changes in any markers were found in the hippocampus of either species. Significant age-related changes in retention of passive avoidance learning and locomotor activity were also observed in these same animals. These findings demonstrate that changes in the cholinergic system occur naturally in aged mice and rats and that both the loss of cholinergic receptors and decrease in cat activity may contribute to the motor and mental impairments that often accompany old age.

Using a new rat conflict test it was found that 30% of the subjects failed to respond to benzodiazepines and other anxiolytic agents. This value is similar to that reported using more classical procedures such as the Geller-Seifter and Vogel conflict tests. Biochemical analysis of various brain regions from responder (R) and non-responder (NR) subjects revealed no significant differences in 5-HT1, 5-HT2, GABA receptor binding or GABA-activated benzodiazepine binding. However, a small, but significant, increase in basal benzodiazepine binding was noted in the hippocampus of NR rats. These findings suggest that the insensitivity of these animals to anxiolytics is probably unrelated to an alteration in serotonin, GABA or benzodiazepine binding sites in brain.

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbachol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M1 receptor antagonist, in competing for [3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M2 receptors in brain.

Alpha-Adrenergic and GABAB receptor agonists regulate adenylate cyclase either negatively, by direct inhibition of the enzyme, or positively, by augmenting agonist-stimulated production of cyclic AMP. While the inhibition of adenylate cyclase is most likely to be mediated by a direct stimulation of Gi protein, the enhancement of production of cyclic nucleotide appears to involve protein kinase C and perhaps PLA2. alpha-Adrenergic and GABAB receptor augmentation of accumulation of cyclic AMP is also influenced by pituitary-adrenal hormones, suggesting a link between brain function and the endocrine system. The number of components associated with the modulation of neurotransmitter receptor-coupled second messenger production provides multiple targets for the pharmacological manipulation of this system. By influencing the modulatory response rather than receptor activity directly, it may be possible to produce subtle alterations in the function of the central nervous system yielding safer and perhaps more effective therapies for the treatment of mental illness.

Incubation of rat cerebral cortical slices with the beta-adrenergic agonist isoproterenol causes an increase in alpha 2-adrenergic receptor binding in addition to a decrease in beta-adrenergic receptor binding. The effects are rapid and reversible, show a parallel time course, and are blocked by sotalol, a beta-adrenergic receptor antagonist. The beta-mediated regulation of alpha 2-receptor sensitivity at brain norepinephrine synapses may be a mechanism for the homeostatic control of central noradrenergic activity.

Bicuculline-sensitive [3H]GABA receptor binding was studied in membrane fractions prepared from vertebrate whole brain or invertebrate cephalic ganglia. In tissue not treated with Triton X-100, a significant amount of bicuculline-displaceable [3H]GABA binding was detected in the brains of all vertebrates studied, with the hagfish brain binding over twice as much [3H]GABA as the spiny dogfish, the next oldest species. All other vertebrates bound similar amounts of [3H]GABA, being one-third to one-fourth that observed in the hagfish. In contrast, after Triton treatment, the hagfish displayed the least amount of bicuculline-sensitive [3H]GABA binding and, under those conditions, the amount of binding observed increased in an evolutionary fashion. No measurable bicuculline-sensitive GABA receptor binding was noted in any invertebrate studied. These results suggest that bicuculline-sensitive GABA receptors are present in the brains of all vertebrates and that during the course of evolution there developed a Triton-sensitive substance(s) whose presence modifies the kinetic properties of this receptor site.

The binding of GABA to crude synaptic membrane fractions of the rat central nervous system in the absence of sodium appears to involve synaptic receptor sites for GABA. The sodium-independent GABA receptor binding differs in a number of features from sodium-dependent GABA binding. In fresh tissue sodium-dependent GABA binding is about 10 times greater than sodium-independent binding, while freezing and thawing the brain membranes virtually abolishes sodium-dependent binding but increases sodium-independent GABA receptor binding about 2-fold. Both sodium-dependent and -independent GABA binding are saturable processes with dissociation constants of 1.2 muM and 0.37 muM respectively. Sodium-dependent GABA binding is much more sensitive to extremes of temperature and pH than is sodium-dependent GABA binding. In addition, regional variations in synaptosomal uptake of GABA, sodium-dependent and sodium-independent GABA binding are markedly different. Also, amino acids and drugs differ in their potencies as inhibitors of sodium-dependent and sodium-independent GABA binding, with the relative affinity of several agents for sodium-independent GABA receptor sites closely paralleling their abilities to mimic neurophysiologic effects of GABA at postsynaptic receptors. Though there are some similarities between the influence of drugs and amino acid analogues on sodium-dependent binding and synaptosomal accumulation of GABA there are also marked differences such as a much greater affinity of beta-alanine for the sodium-dependent GABA binding sites than for synaptosomal GABA uptake. This suggests that the sodium-dependent GABA binding may involve uptake sites for the GABA accumulating system of glia rather than for neuronal GABA transport.

GABAA (bicuculline-sensitive) and GABAB (bicuculline-insensitive) receptor binding were examined in an attempt to determine whether either site was present on noradrenergic terminals in rat brain cerebral cortex. Equilibrium binding saturation studies revealed the presence of two GABAB binding sites in brain tissue, with affinity constants of 60 and 229 nM. Regional distribution studies in the cow brain revealed that the ratio of the high and low affinity GABAB binding sites differed among the areas examined. Both GABAA and GABAB binding were examined in the rat brain cortex 12 days following a unilateral lesion of the dorsal noradrenergic bundle. While GABAA and high affinity GABAB binding were unaffected by the lesion, the number of low affinity GABAB sites was significantly reduced. These results indicate that high and low affinity GABAB binding sites may be anatomically distinct, with only the latter being associated with cerebral cortical noradrenergic nerve terminals.

[3H]Muscimol binding to crude synaptic membrane fractions of the rat central nervous is saturable with a high affinity dissociation constant of 2.2 nM. The regional distribution of this binding in rat brain and the effects of freezing, sodium and Triton X-100 are similar to those previously reported for [3H]GABA binding to the the synaptic GABA receptor site. Also, the substrate specificity of [3H]muscimol binding is identifical to that observed for the GABA receptor. Thus, [3H]muscimol is displaced, stereospecifically, only by those drugs and amino acids which are known to neurophysiologically interact with the synaptic GABA receptor but is unaffected by agents which activate or inhibit other neurotransmitter receptors. This suggests that the behavioral effects observed after the systemic administration of muscimol are probably the result of GABA receptor activation.

Continuous treatment (1-3 weeks) with imipramine or adrenocorticotropin (ACTH) decreases the responsiveness of the norepinephrine-coupled cyclic nucleotide generating system in rat brain cerebral cortex. Experiments were undertaken to determine which component of the second messenger system is influenced by the hormone and antidepressant. Neither treatment modified the amount or function of extractable stimulatory guanine nucleotide binding protein or the activities of adenylate cyclase or phosphodiesterase. While both imipramine and ACTH treatment decreased the cyclic AMP response to norepinephrine, only imipramine administration influenced the response to isoproterenol. ACTH treatment was found to reduce the alpha adrenergic potentiation of isoproterenol- and 2-chloroadenosine-stimulated cyclic AMP production, as well as reduce the sensitivity of the norepinephrine response to prazosin. These findings indicate that imipramine and ACTH treatments decrease the responsiveness of the rat brain norepinephrine-stimulated cyclic AMP generating system through actions on the alpha and beta adrenergic receptor components. The results suggest that noradrenergic receptor activity may be under the control of adrenal and/or pituitary hormones.