Costantino Motzo

Inhibition of basal and stress-induced dopamine release in the cerebral cortex and nucleus accumbens of freely moving rats by the neurosteroid allopregnanolone

The neurosteroid allopregnanolone is a potent and efficacious modulator of γ-aminobutyric acid (GABA) type A receptors. The effects of intracerebroventricular injection of allopregnanolone (5 to 15 μg in 5 μl) on basal and stress-induced changes in the extracellular concentrations of dopamine were investigated by microdialysis in various brain areas of freely moving rats and compared with those of the benzodiazepine midazolam (1 to 10 μg in 5 μl). Allopregnanolone reduced (by a maximum of 65 to 75%) basal dopamine content in the prefrontal cortex and nucleus accumbens in a dose-dependent manner, but had no effect on dopamine output in the striatum. Allopregnanolone (10 to 15 μg) also completely prevented the increase in extracellular dopamine concentrations in the nucleus accumbens and cerebral cortex induced by foot-shock stress. Midazolam reduced basal dopamine content in all three brain regions studied as well as the stress- induced increase in dopamine content in the nucleus accumbens and cerebral cortex with a greater potency than allopregnanolone. These results suggest that endogenous neurosteroids may participate in the GABAergic modulation of dopaminergic transmission in the rat cerebral cortex and nucleus accumbens, two brain areas which are important in the regulation of emotional processes. These agents do not appear to affect striatal dopaminergic transmission which modulates motor function.

NMDA Receptor Function Is Enhanced in the Hippocampus of Aged Rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [3H]CGP 39653 and [3H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [3H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 μM glycine and 10 μM glutamate increased [3H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [3H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [3H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [3H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.

Characterization of the electrophysiological and pharmacological effects of 4‐iodo‐2,6‐diisopropylphenol, a propofol analogue devoid of sedative‐anaesthetic properties

Several derivatives and analogues of the general anaesthetic 2,6‐diisopropylphenol (propofol) have been recently synthesised with the aim of exploring the structure‐activity relationships. In the present study, the effects of one such compound, 4‐iodo‐2,6‐diisopropylphenol (4‐I‐Pro), on γ‐aminobutyric acid type A (GABAA) receptors in vitro were compared with its in vivo effects in rodents. Human GABAA receptors were expressed in Xenopus oocytes, and the actions of 4‐I‐Pro on receptor function were compared with those of propofol by two‐electrode voltage‐clamp recording. Similar to propofol, 4‐I‐Pro directly activated Cl− currents in the absence of GABA at all combinations of receptor subunits tested. However, the efficacy of 4‐I‐Pro in inducing direct activation of α1β2γ2S receptors was markedly less than that of propofol. Similarly to propofol, 4‐I‐Pro potentiated in a concentration‐dependent manner GABA‐evoked Cl− currents measured at different GABAA receptor constructs. As expected, intraperitoneal injection of propofol induced sedation, ataxia, and loss of the righting reflex in rats. In contrast, administration of 4‐I‐Pro failed to produce any of these behavioural effects. Administration of 4‐I‐Pro to rats reduced in a dose‐dependent manner the incidence of tonic‐clonic seizures induced by pentylenetetrazol and induced an anticonflict effect as measured in the Vogel test. Microdialysis revealed that, like propofol, administration of 4‐I‐Pro reduced acetylcholine release in the hippocampus of freely moving rats. These results demonstrate that para‐substitution of the phenol ring of propofol with iodine yields a compound that exhibits anticonvulsant and anticonflict effects, but is devoid of sedative‐hypnotic and anaesthetic properties. Thus, 4‐I‐Pro possesses pharmacological characteristics more similar to anxiolytic and anticonvulsant drugs than to general anaesthetics.

Long-term treatment with abecarnil fails to induce tolerance in mice.

The effects of long-term treatment (3 times a day for 4 weeks) with a pharmacologically active dose (0.1 mg/kg i.p.) of the novel anxiolytic, abecarnil, on exploratory behaviour and [35S]TBPS (t-butylbicyclophosphorothionate) binding were compared to those of diazepam (1 mg/kg i.p.) in mice. A challenge dose (0.1 mg/kg) of abecarnil given 12 h after the last administration of the treatment protocol markedly inhibited exploratory behaviour in animals treated chronically with abecarnil (-62%) or vehicle (-87%). Consistent with this behavioural effect, the same challenge dose of abecarnil significantly reduced [35S]TBPS binding to unwashed cerebral cortical membranes from mice treated chronically with abecarnil (-28%) or vehicle (-30%). In contrast, a challenge dose (1 mg/kg) of diazepam failed to affect motor behaviour and [35S]TBPS binding in mice chronically exposed to diazepam; in animals chronically treated with vehicle, diazepam markedly inhibited both exploratory behaviour (-55%) and [35S]TBPS binding (-21%). These results indicate that long-term treatment with abecarnil failed to induce tolerance to the effect of this drug on gamma-aminobutyric acid type A (GABAA) receptor function. Accordingly, [35S]TBPS binding was increased (+15-26%) 12 and 48 h after discontinuation of long-term diazepam administration while no such increase in [35S]TBPS binding was observed for mice chronically treated with abecarnil. Moreover, whereas a significant decrease (-15%) in [35S]TBPS binding was observed 96 h after discontinuation of long-term diazepam treatment, chronic treatment with abecarnil did not modify this parameter. Together, these data indicate that long-term treatment with a pharmacologically effective dose of abecarnil did not induce tolerance or the discontinuation syndrome in mice.

Chronic administration of an anticonvulsant dose of imidazenil fails to induce tolerance of GABAA receptor function in mice

The ability of an anticonvulsant dose (0.1 mg/kg i.p.) of imidazenil, a new partial agonist of benzodiazepine receptors, to antagonize the convulsions and the increase in t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the gamma-aminobutyric acid type A (GABAA) receptor elicited by isoniazid, an inhibitor of central GABAergic function, was evaluated in mice chronically treated (3 times daily for 30 days) with the same dose of imidazenil. The challenge dose of imidazenil, administered 36 h after the last injection of the chronic treatment protocol, reduced both isoniazid-induced convulsions and the isoniazid-induced increase in [35S]TBPS binding to the same marked extent as in control mice. These results indicate that long-term treatment with a pharmacologically effective dose of imidazenil failed to induce tolerance to both the anticonvulsant effect and the positive modulatory action on GABAA receptor function of this drug in mouse brain.

Expression of Native GABAA Receptors in Xenopus Oocytes Injected With Rat Brain Synaptosomes

Abstract: Oocytes from the frog Xenopus laevis were shown recently to express native nicotinic acetylcholine receptors after injection with purified Torpedo electroplaque membrane vesicles. Injection of Xenopus oocytes with rat cortical or nigral synaptosomes has now been shown to result in the expression of γ‐aminobutyric acid type A (GABAA) receptor‐mediated Cl− currents. Electrophysiological characterization of the responses of these receptors to GABA and other agents revealed that they were incorporated into the oocyte membrane and that they retained their original pharmacological properties, such as sensitivity to Cl− channel blockers, benzodiazepines, and general anesthetics. These results suggest that this approach to the expression of heterologous proteins in Xenopus oocytes may facilitate the study of native synaptic proteins derived from brain tissue.

Failure of flumazenil to precipitate a withdrawal syndrome in cats chronically treated with the new anxioselective beta-carboline derivative abecarnil.

The effect of chronic administration of the novel anxiolytic β-carboline derivative, abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-β-carboline-3-carboxylate), was examined and compared with the capability of diazepam to induce physical dependence in cats. The acute administration of the benzodiazepine receptor antagonist, flumazenil (20 mg/kg i.p.), to cats treated for 2 weeks with diazepam (7 mg/kg i.p., three times daily), induced a severe withdrawal syndrome characterized by the appearance of severe physical signs. Within minutes all cats displayed tremors, increased muscle tone, fear response, repeated vocalization and salivation. On the contrary, in all cats treated chronically (2 weeks) with abecarnil (7 mg/kg i.p. three times daily) the challenge dose of flumazenil failed to precipitate a clear abstinence syndrome. In fact, a pupillary dilatation and a mild fear response were the only signs present 15–30 min after flumazenil administration. This finding indicates that abecarnil, a new potential therapeutic agent for anxiety disorders and seizures, might have advantages over classical benzodiazepines with regard to development of physical dependence.

Functional changes in rat nigral GABAA receptors induced by degeneration of the striatonigral GABAergic pathway : An electrophysiological study of receptors incorporated into Xenopus oocytes

Abstract: Expression of rat brain γ‐aminobutyric acid type A (GABAA) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABAA receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABAA receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage‐clamp conditions. The maximal amplitude of GABA‐induced Cl− currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half‐maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA‐induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ∼65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half‐maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up‐regulation of nigral GABAA receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β‐carbolines.

Enhancement by flumazenil of dopamine release in the nucleus accumbens of rats repeatedly exposed to diazepam or imidazenil

Abstract The effect of long-term treatment (three times daily for 3 weeks) with a behaviorally relevant dose of the benzodiazepine receptor partial agonist imidazenil (0.5 mg/kg, IP) on basal dopamine release in the nucleus accumbens of freely moving rats was compared with that of diazepam (3 mg/kg, IP), a benzodiazepine receptor full agonist. Challenge doses of imidazenil and diazepam decreased the extracellular dopamine concentration in the nucleus accumbens by approximately the same extent in animals repeatedly exposed to vehicle or to the respective drug. Moreover, the abrupt discontinuation of long-term treatment with diazepam or imidazenil failed to affect basal dopamine release in this brain area during the first 5 days of withdrawal. In contrast, administration of the benzodiazepine receptor antagonist flumazenil (4 mg/kg, IP) elicited a marked increase (95 or 60%) in dopamine release in the nucleus accumbens 6h after withdrawal of diazepam or imidazenil, respectively. Flumazenil induced a similar but smaller effect (50% increase) 5 days after diazepam withdrawal but had no effect 5 days after discontinuation of imidazenil treatment. The resultssupport an involvement of the mesoaccumbens dopaminergic neurons in the withdrawal syndrome precipitated by flumazenil and allow further differentiation of benzodiazepine receptor partial and full agonists with respect to dependence liability of dopaminergic neurons in the nucleus accumbens.

Autoimmunity in gestational diabetes mellitus in Sardinia: a preliminary case-control report

BackgroundWe previously reported a high prevalence (22.3%) of gestational diabetes mellitus (GDM) in a large group of Sardinian women, in contrast with the prevalence of Type 2 diabetes. Sardinia has an unusual distribution of haplotypes and genotypes, with the highest population frequency of HLA DR3 in the world, and after Finland, the highest prevalence of Type 1 diabetes and Autoimmune-related Diseases. In this study we preliminarily tested the prevalence of serological markers of Type 1 diabetes in a group of Sardinian GDM patients.MethodsWe determined glutamic decarboxylase antibodies (anti-GAD65), protein tyrosine phosphatase ICA 512 (IA2) antibodies (anti-IA2), and IAA in 62 GDM patients, and in 56 controls with matching age, gestational age and parity.ResultsWe found a high prevalence and very unusual distribution of antibodies in GDM patients (38.8%), the anti-IA2 being the most frequent antibody. Out of all our GDM patients, 38.8% (24 of 62) were positive for at least one antibody. Anti-IA2 was present in 29.0 % (18 out of 62) vs. 7.1% (4 out of 56) in the controls (P < 0.001). IAA was present in 14.5% (9 out of 62) of our GDM patients, and absent in the control subjects (P < 0.001). Anti-GAD65 was also present in GDM patients, with a prevalence of 3.2% (2 out of 62) while it was absent in the control group (P = NS). Pre-gestational weight was significantly lower (57.78 ± 9.8 vs 65.9 ± 17.3 P = 0.04) in auto-antibodies- positive GDM patients.ConclusionThese results are in contrast with the very low prevalence of all antibodies reported in Italy. If confirmed, they could indicate that a large proportion of GDM patients in Sardinia have an autoimmune origin, in accordance with the high prevalence of Type 1 diabetes.