M.G. Corda

Rapid changes in GABA binding induced by stress in different areas of the rat brain.

Rats habituated to handling preceding sacrifice have higher [3H]GABA receptor binding in different brain areas (cerebellum, frontal cortex, caudate nucleus) than naive animals. The increase in GABA binding in handling-habituated rats is due to an increase in the number of receptors (Bmax) with no changes in the affinity of GABA binding for its ligand (Kd). Foot shock causes a sudden fall in GABA binding in handling-habituated rats but does not, or only slightly, in naive ones. The results indicate that stress causes a rapid decrease in GABA receptor binding in the central system and that the GABA binding values which are usually considered as normal are, in fact, values decreased by the handling manoeuvers preceding sacrifice.

Decrease in the function of the γ-aminobutyric acid-coupled chloride channel produced by the repeated administration of pentylenetetrazol to rats

Abstract: The acute administration of pentylenetetrazol (PTZ; 25–75 mg/kg i.p.) failed to modify the specific binding of t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [33S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of 7‐amino‐butyric acid (GABA)‐stimulated 36CI− uptake was measured in the cerebral cortex of PTZ‐treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA‐coupled chloride channel activity that may be related to the chemical kindling produced by this compound.

Stress and β-carbolines decrease the density of low affinity gaba binding sites: An effect reversed by diazepam

Cerebral cortex membranes from rats habituated to manipulations preceding decapitation (habituated rats) had 40% higher GABA binding than membranes from naive animals. Diazepam (5 X 10(-6) M), added to membranes from naive rats, increased GABA binding to the level of habituated rats, but failed to induce any further increase in membranes from the latter animals. Vice versa, beta-carbolines (FG 7142, beta-CCE, DMCM) added to membranes from habituated rats lowered GABA binding to the level of naive animals, but caused no further decrease in the membranes from this last group. Diazepam removed the effect of beta-carbolines in membranes from habituated rats. It is suggested that handling represents a stressful stimulus for naive animals and that stress lowers GABA binding by releasing an endogenous ligand for benzodiazepine receptors possessing similar properties to beta-carbolines. Finally, the results indicate that the emotional status of animals from which brain tissue is obtained should be considered when connections between GABA and benzodiazepine receptors are studied.

Pentylenetetrazol-induced kindling in rats: effect of GABA function inhibitors

The repeated administration of subconvulsant doses of pentylenetetrazol (PTZ) produced a progressive sensitization to the effects of this compound (i.e., chemical kindling) in the rat. A very similar time-course for PTZ-induced kindling was observed using two different treatment schedules: 1) one injection every day (30 mg/kg, IP), and 2) one injection (30 mg/kg, IP) every second day. When these treatment schedules were used for eight consecutive weeks, more than 80% of the rats displayed convulsions by the end of treatment. In contrast, only 20% of the rats were sensitized if PTZ was administered twice daily at the dose of 15 mg/kg, IP. The increased sensitivity to the convulsant effect of PTZ was still present one year after completion of the chronic treatment. Moreover, rats kindled with PTZ showed an enhanced susceptibility to convulsions induced by different inhibitors of central GABAergic function, such as the chloride channel blocker picrotoxin, the benzodiazepine receptor ligands FG 7142 and Ro 15-4513, and the inhibitor of GABA synthesis isoniazid. In contrast, the sensitivity to the convulsant action of the glycine receptor antagonist strychnine was unchanged by repeated PTZ administration. It is suggested that kindling produced by PTZ may be associated with a persistent reduction in the inhibitory function of the GABAergic system in the brain.

Inhibition of copulatory behavior in male rats by D-ALA2-Met-Enkephalinamide

Abstract D-Ala 2 -Met-Enkephalinamide (DALA), a synthetic analog of met-enkephaline resistant to enzymatic degradation, was injected intraventricularly to sexually experienced male rats paired with receptive females. A dose of DALA of 6 μg which did not influence spontaneous motor activity, completely suppressed the copulatory behavior of all animals tested. A dose of 3 μg significantly increase mounting and intromission latencies, but did not influence other measures of the copulatory behavior. The effect of DALA was prevented by naloxone (1 mg/Kg), a specific inhibitor of opioid receptors. The results suggest that enkephalins may play a role in the regulation of copulatory behavior.

Ro 15-4513, a partial inverse agonist for benzodiazepine recognition sites, has proconflict and proconvulsant effects in the rat

The present report describes the effects of Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-(1,5-a) (1,4)-benzodiazepine-3-carboxylate) in the conflict test, on convulsions induced by isoniazid and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) and on the binding of [3H]gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. Ro 15-4513 produced a dose-dependent proconflict effect that was prevented by the administration of the benzodiazepine antagonist, Ro 15-1788. In addition, Ro 15-4513 was not convulsant per se but enhanced the convulsions produced by isoniazid and completely blocked the convulsions induced by the full inverse agonist, DMCM. In vitro, Ro 15-4513, like ethyl-beta-carboline-3-carboxylate (beta CCE), antagonized the increase in [3H]GABA binding induced by diazepam. The results indicate that Ro 15-4513 is anxiogenic and interacts with benzodiazepine recognition sites as a partial inverse agonist.

Enhanced sensitivity to β-carboline inverse agonists in rats chronically treated with FG 7142

The biochemical and behavioural effects of the chronic administration of the beta-carboline inverse agonist FG 7142 were studied in the rat. Repeated administration of FG 7142 (15 mg/kg IP, twice daily for 10 consecutive days) induced sensitization to the effects of this drug, which from proconvulsant became a full convulsant. Thus, myoclonic seizures were observed in 30% and 80% of the animals by the third and the eighth day of treatment, respectively. The sensitization to the convulsant effect of FG 7142 persisted for up to 50 days after withdrawal and was completely prevented by the concurrent administration of the benzodiazepine receptor antagonist Ro15-1788 (15 mg/kg IP, twice a day for 10 days). Moreover, four to twelve days after withdrawal from chronic treatment with FG 7142, an increased sensitivity to the proconvulsant beta CCE and to the convulsant DMCM was observed. In addition, convulsions induced by isoniazid (350 mg/kg, SC) were potentiated in rats chronically treated with FG 7142 at 5 and 20 days after withdrawal. These pharmacological effects were paralleled by a decrease in the density of low affinity GABA receptors in the cerebral cortex and cerebellum. These results are consistent with the view that repeated administration of FG 7142 induces a long-lasting down-regulation of the GABAergic function which results in an increased sensitivity to beta-carboline inverse agonists and isoniazid. The possibility that a concomitant decrease in the responsiveness to benzodiazepines and Ro15-1788 takes place after chronic treatment with FG 7142 is also discussed.

Preferential affinity of 3H-2-oxo-quazepam for type I benzodiazepine recognition sites in the human brain

The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of 3H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of 3H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of 3H-flunitrazepam (3H-FNT) binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for 3H-FNT and 3H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum (95% of total sites), cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of 3H-2-oxo-quaz (2 nM) and 3H-FNT (0.5 nM) using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing 3H-2-oxo-quaz than 3H-FNT from cerebral cortex membrane preparations. The results suggest that 3H-2-oxo-quaz may be used for selectively studying Type I BZ recognition sites in the human brain.

Behavioral and biochemical evidence for a long-lasting decrease in GABAergic function elicited by chronic administration of FG 7142

Chronic treatment with the beta-carboline derivative FG 7142 (15 mg/kg i.p. twice a day for 10 consecutive days) produced a long-lasting enhancement of shock-induced suppression of drinking in rats, without affecting unpunished behaviour. This proconflict effect was observed up to 15 days after withdrawal from FG 7142. A significant sensitization to seizures induced by isoniazid, a drug known to inhibit GABAergic transmission, was also found to occur after long-term (25 days) withdrawal. Moreover, the density of low-affinity GABA receptors was decreased by 30% in the cerebral cortex of rats repeatedly injected with FG 7142 at 5 and 15 days after withdrawal. The capacity of high-affinity GABA receptors, as well as the apparent dissociation constants for both high- and low-affinity GABA receptors were unchanged. Similar modifications in [3H]GABA binding were also observed in the cerebellum. The enhancement of punishment suppressed behaviour, the sensitization to isoniazid-induced convulsions and the decrease in the density of low-affinity GABA receptors suggest that chronic administration of FG 7142 induces a persistent down-regulation of GABAergic transmission in the central nervous system.

Denervation supersensitivity for benzodiazepine receptors in the rat substantia nigra

The injection of kainic acid into the substantia nigra causes, 3 weeks after treatment, a 40% decrease in the total number of binding sites for [3H]diazepam with an increase in the dissociation constant. This decrease was restored to approximately normal by the subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The injection of kainic acid into the striatum of intact animals failed to modify the number of [3H]diazepam binding sites but increased the KD. The results indicate that benzodiazepine binding sites in the substantia nigra are partly located on kainic acid sensitive elements (probably interneurons) and, partly, on kainic acid resistant ones. These binding sites become supersensitive after degeneration of striato-nigral pathways. The possible role of GABAergic denervation of the substantia nigra in the development of benzodiazepine binding sites supersensitivity is discussed.