The results presented here show that a mild stress such as the handling ma­nipulation during the experimental procedure decreases the density of low affinity GABA receptors in the rat cerebral cortex. These data indicate that, in order to study the involvement of the GABAergic system in the physiological response to stress, we have to take into account the emotional state of the animals during the experiment. Accordingly, electrical foot-shock decreases the total number of low affinity GABA receptors in the cerebral cortex of handling-habituated rats, but is inef­fective in the cerebral cortex of naive animals. Most likely, the reason that explains the failure of foot-shock to affect GABA receptors in naive rats is that, for rats not habituated to handling, the manipulation before sacrifice constitutes a stressful stimulus responsible for the supramaximal decrease in low affinity GABA re­ceptors. It has been proposed that benzodiazepines exert their antianxiety effect by enhancing the GABAergic transmission in the brain (4,7,35). The finding that the in vitro addition of diazepam, ZK 93423, and ZK 91296 reverses the changes produced by foot-shock on GABA receptors inc’icates that benzodiazepine re­ceptors are involved in the action of stress on GABA receptors. This conclusion is also supported by the finding that those /3-carboline esters that bind with high affinity to the benzodiazepine receptors (5,6) and produce experimental anxiety in animals (14,15,23,25,27,30) and panic attacks in humans (16), share with stress (handling or foot-shock) the capability to down-regulate GABA receptors. Altogether these results indicate that cortical low affinity GABA receptors, coupled to benzodiazepine receptors, play a critical role in the regulation of emotional states. Compelling evidence indicates that in neuronal membranes the GABA receptor is part of a supramolecular complex that includes a chloride ionophore (24). Our experiments have shown that the function of the GABA receptor-coupled chloride channel is also modified by stress. Thus foot-shock reduces СГ efflux from cortical synaptoneurosome preparations of handling-habituated rats, but is ineffective in the same preparation from naive rats. These results, taken together with our previous findings, indicate that the GABAergic system plays a major role in anxiety and suggest that some emotional states related to stress and anxiety may result from a diminished GABAergic transmission at the level of the GABA/ benzodiazepine receptor/chloride ionophore complex.
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It has been suggested that the facilitation of the GABAergic transmission in­duced by benzodiazepines plays an important role in the antistress and antianxiety effects of these drugs (4,7,35). On the basis of this hypothesis, we evaluated whether anxiolytic benzodiazepine receptor ligands were able to antagonize the decrease in the density of low affinity GABA receptors induced by electrical foot-shock stress in rats. As shown in Table 3, the in vitro addition of diazepam (5 mM to cortical membrane preparations from foot-shocked rats increased the density of low affinity GABA receptors to the level found in nonstressed animals. This finding indicates that in the rat cerebral cortex, benzodiazepine receptors are functionally linked to the low affinity GABA receptors and may play a critical role in the regulation of emotional states. Recently, a novel series of b-carboline derivatives with pharmacological prop­erties opposite to those of anxiogenic b-carbolines have been synthetized. Among
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Department of Experimental Biology, Chair of Pharmacology, University of Cagliari, 09100 Cagliari, Italy
Benzodiazepines, the drugs most widely used in the treatment of anxiety, produce their pharmacological effects by regulating the interaction of GABA with its recognition site at the level of the GABA/benzodiazepine receptor com­plex (17,18). In fact, it has been shown that benzodiazepines favor, through an allosteric mechanism, the interaction between endogenously released GABA and its recognition site. Thus the anxiolytic effect of benzodiazepines may be con­sidered the consequence of the activation of the GABA receptor induced by these drugs. Accordingly, in different animal models of anxiety, the anticonflict effect of benzodiazepines is antagonized by picrotoxin and bicuculline (4,35), drugs that reduce the responsiveness of GABA receptors and have an anxiogenic effect on their own (12,27), whereas it is mimicked and potentiated by muscimol, a specific GABA receptor agonist (7). Moreover, it has been found that /S-carboline derivatives, benzodiazepine receptor ligands (5) that modulate the GABAergic function in the opposite direction to the benzodiazepines (2,6,8,11,21,26,29) have an anxiogenic effect in several animal species, man included (14-16,23, 25,27,30). Although the above studies have clearly demonstrated that the GABA/ben­zodiazepine receptor complex participates in the anxiolytic effect of benzodi­azepines, the molecular events involved in the physiopathology of stress and anxiety have still to be clarified. With the aim of clarifying this problem we have recently found that stress selectively reduces the density of low affinity GABA receptors in the cerebral cortex of the rat (1,3,9,13). This finding is consistent with the hypothesis that some emotional states related to stress and anxiety may result from a diminished GABAergic transmission at the level of the GABA/benzodiazepine receptor complex. The finding that brain GABA receptors may be modified by the emotional state of the animal before sacrifice was obtained a few years ago in our laboratory when we found that the cerebral cortex of rats habituated to the manipulations that precede sacrifice by guillotine has a higher density of low affinity GABA receptors than that of naive, nonhabituated animals (Table 1). On the basis of this result we speculated that the handling manipulation con­stitutes, for a naive animal, a stressful stimulus sufficient to induce a decrease in the density of GABA receptors. To confirm this hypothesis we studied the effect of electrical foot-shock on the binding of 3 H-GABA in the cerebral cortex of naive and handling-habituated rats. Foot-shock, delivered just before sacrifice to these two groups of rats, decreased the density of 3 H-GABA binding sites in cortical membrane preparations from handling-habituated rats, but failed to further decrease 3 H-GABA binding in those of naive ones (Table 1). From these results we concluded that handling-habituated rats represent a relatively nonstressed condition, whereas the handling of naive animals just before sacrifice or the electrical foot-shock are stressful stimuli able to cause a decrease in GABA receptors. To further clarify the molecular mechanism that mediates the decrease of GABA receptors elicited by stress we studied whether the effect of stress was mimicked by anxiogenic /J-carboline derivatives, benzodiazepine receptor ligands that down-regulate the GABAergic function (2,6,8,11,21,26,29). As shown in Table 2, the in vitro addition of different /3-carboline derivatives to cortical membrane preparations from unstressed (handling-habituated) rats decreased the number of low affinity GABA receptors to approximately the same (more…)

Several lines of evidence indicate that the GABA/benzodiazepine receptor complex is coupled to a chloride ionophore (24). Thus electrophysiological studies have demonstrated that GABA produces its inhibitory effect on neurotransmis­sion by enhancing the flux of chloride ions across its specific channel on the nerve membrane (22). Recently it has been shown that using the synaptoneurosomes, a novel sub­cellular brain membrane preparation, it is possible to study the membrane per
meability to chloride ions by measuring the changes in chloride efflux induced by different drugs acting at the level of the GABA/benzodiazepine receptor / СП ionophore complex (31). Hence, by using this biochemical technique, we eval­uated the effect of foot-shock stress on 36 C1 efflux from cerebral cortex synap-toneurosomes of handling-habituated and naive rats. As shown in Table 4 foot-shock stress decreased 36 C1 efflux from synapto-neurosomes of handling-habituated rats, but failed to modify the 36 СГ exchange from the membranes of naive rats. The presence in the dilution buffer of pentobarbital, a drug that facilitates the chloride transport by increasing the opening time of the GAB A receptor-coupled chloride channel (21,34) reversed the stress-induced decrease of 36 С l efflux from synaptoneurosomes of handling-habituated rats. Moreover, pentobarbital also stimulated 36 Cl efflux from cerebral cortex synaptoneurosomes of naive rats. Thus the function of the СГ channel coupled to the GABA/benzodiazepine receptors is decreased by foot-shock stress. This finding is in line with our previous results showing that foot-shock stress markedly decreases the density of low affinity GABA receptors in the cerebral cortex of handling-habituated rats. (more…)