Maria Giuseppina Pisu

Social Isolation‐Induced Decreases in Both the Abundance of Neuroactive Steroids and GABAA Receptor Function in Rat Brain

The effects of social isolation on behavior, neuroactive steroid concentrations, and GABAA receptor function were investigated in rats. Animals isolated for 30 days immediately after weaning exhibited an anxiety‐like behavioral profile in the elevated plus‐maze and Vogel conflict tests. This behavior was associated with marked decreases in the cerebrocortical, hippocampal, and plasma concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone compared with those apparent for group‐housed rats ; in contrast, the plasma concentration of corticosterone was increased in the isolated animals. Acute footshock stress induced greater percentage increases in the cortical concentrations of neuroactive steroids in isolated rats than in group‐housed rats. Social isolation also reduced brain GABAA receptor function, as evaluated by measuring both GABA‐evoked Cl‐ currents in Xenopus oocytes expressing the rat receptors and tert‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes. Whereas the amplitude of GABA‐induced Cl‐ currents did not differ significantly between group‐housed and isolated animals, the potentiation of these currents by diazepam was reduced at cortical or hippocampal GABAA receptors from isolated rats compared with that apparent at receptors from group‐housed animals. Moreover, the inhibitory effect of ethyl‐β‐carboline‐3‐carboxylate, a negative allosteric modulator of GABAA receptors, on these currents was greater at cortical GABAA receptors from socially isolated animals than at those from group‐housed rats. Finally, social isolation increased the extent of [35S]TBPS binding to both cortical and hippocampal membranes. The results further suggest a psychological role for neurosteroids and GABAA receptors in the modulation of emotional behavior and mood.

Brain steroidogenesis mediates ethanol modulation of GABAA receptor activity in rat hippocampus.

An interaction with the GABA type A (GABAA) receptor has long been recognized as one of the main neurochemical mechanisms underlying many of the pharmacological actions of ethanol. However, more recent data have suggested that certain behavioral and electrophysiological actions of ethanol are mediated by an increase in brain concentration of neuroactive steroids that results from stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Neuroactive steroids such as 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) are, in fact, potent and efficacious endogenous positive modulators of GABAA receptor function. Because neurosteroids can be synthesized de novo in the brain, we have investigated whether ethanol might affect both neurosteroid synthesis and GABAA receptor function in isolated rat hippocampal tissue. Here, we show that ethanol increases the concentration of 3α,5α-THP as well as the amplitude of GABAA receptor-mediated IPSCs recorded from CA1 pyramidal neurons in isolated hippocampal slices. These effects are shared by the neurosteroid precursor progesterone, the peripheral benzodiazepine receptor-selective agonist CB34, and γ-hydroxybutyrate, all of which are known to increase the formation of neuroactive steroids in plasma and in the brain. The action of ethanol on GABAA receptor-mediated IPSC amplitude is biphasic, consisting of a rapid, direct effect on GABAA receptor activity and an indirect effect that appears to be mediated by neurosteroid synthesis. Furthermore, ethanol affects GABAA receptor activity through a presynaptic action, an effect that is not dependent on neurosteroid formation. These observations suggest that ethanol may modulate GABAA receptor function through an increase in de novo neurosteroid synthesis in the brain that is independent of the HPA axis. This novel mechanism may have a crucial role in mediating specific central effects of ethanol.

Neurosteroid secretion in panic disorder

Evidence that neurosteroids have anxiolytic effects in animal models of anxiety has suggested that alterations of neurosteroid secretion might be implicated in the pathogenetic mechanisms of anxiety disorders in humans. In 25 female patients with panic disorder (PD) and 11 healthy female controls, we measured plasma concentrations of progesterone (PROG), pregnenolone (PREG), allopregnanolone (3alpha,5alpha-tetrahydroprogesterone=3alpha,5alpha-THPROG), dehydroepiandrosterone (DHEA) and tetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC) during a drug-free month and during the following month of paroxetine therapy. The neurosteroids were measured during the early follicular phase, the mid-luteal phase and the premenstrual phase of both months (days 7, 22 and 27 from the beginning of the cycle). Significantly higher levels in patients than controls were found in PROG during the mid-luteal phase of both months, PREG in the premenstrual phase in the drug-free month, 3alpha,5alpha-THPROG during the follicular phase of the drug-free month and during the premenstrual phase of the therapy month, and 3alpha,5alpha-THDOC during the premenstrual phases of both months. DHEA levels did not differ in patients and controls. These results suggest that neurosteroids in PD are hypersecreted, possibly as an attempt to counteract the anxiogenic underlying hyperactivity of the hypothalamo-pituitary-adrenal axis and to improve a reduced GABA(A) receptor sensitivity.

2-Phenyl-imidazo(1,2-a)pyridine Compounds Containing Hydrophilic Groups as Potent and Selective Ligands for Peripheral Benzodiazepine Receptors: Synthesis, Binding Affinity and Electrophysiological Studies

A series of imidazopyridine acetamides were synthesized to evaluate the effects of structural changes at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include the introduction of polar substituents or ionizable functional groups at the 2- and 8-position of the imidazopyridine skeleton. The results suggest that substituents endowed with hydrogen bonding acceptor and/or donor properties in the para position of the phenyl ring lead to high affinity for PBR. In electrophysiological studies, it was found that compounds 9, 12, 13, and 28 markedly enhanced GABA-evoked Cl (-) currents in Xenopus oocytes expressing alpha 1beta 2gamma 2 GABA A receptors. The capability of flumazenil to reduce the stimulatory effect exerted by compound 9 supports the conclusion that the modulatory effects of the examined compounds occur involving the CBR. The ability of compound 16 to increase GABA A receptor-mediated miniature inhibitory postsynaptic currents in CA1 pyramidal neurons is indicative of its ability to stimulate the local synthesis and secretion of neurosteroids.

Increased neuroactive steroid concentrations in women with bipolar disorder or major depressive disorder.

Abstract: Changes in the plasma concentrations of neuroactive steroids have been associated with various neuropsychiatric disorders. However, the possible role of neuroactive steroids in bipolar disorder (BD) has remained unknown. We therefore determined the plasma levels of neuroactive steroids during the luteal phase of the menstrual cycle in women with BD or major depressive disorder (MDD). The plasma concentrations of 3&agr;-hydroxy-5&agr;-pregnan-20-one (3&agr;,5&agr;-THPROG), 3&agr;,21-dihydroxy-5&agr;-pregnan-20-one, progesterone, and cortisol were determined in 17 outpatients with BD, 14 outpatients with MDD, and 16 healthy control subjects. All patients were in a state of well-being and without relapse or recurrence for at least 3 months. Plasma concentrations of progesterone and 3&agr;,5&agr;-THPROG were significantly greater in patients than in controls, also being higher in BD patients than in MDD patients. Drug-free patients with BD or MDD showed similar differences in steroid concentrations relative to controls, as did drug-treated patients. Comorbidity with panic disorder, obsessive-compulsive disorder, or eating disorder had no effect on the association of mood disorders with steroid concentrations. Women with BD or MDD in a state of well-being showed higher plasma concentrations of progesterone and 3&agr;,5&agr;-THPROG in the luteal phase than did healthy controls. These differences did not seem to be attributable simply to drug treatment or to comorbidity with other psychiatric conditions in the patients.

Social isolation‐induced increase in α4 and δ subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABAA receptor function

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3α‐hydroxy‐5α‐pregnan‐20‐one (3α,5α‐TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol‐induced increase in 3α,5α‐TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid‐induced convulsions is greater in isolated rats than in group‐housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABAA receptor‐mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group‐housed rats, an effect inhibited by finasteride. The amounts of the α4 and δ subunits of the GABAA receptor in the hippocampus were increased in isolated rats as were GABAA receptor‐mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABAA receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABAA receptor function and associated behaviour.

Plasma concentrations of anxiolytic neuroactive steroids in men with panic disorder

Plasma concentrations of neuroactive steroids in men with panic disorder (PD) were measured to evaluate their relations to psychopathology both before and during treatment. Participants comprised 13 men with PD and 10 normal controls. Patients were evaluated while drug-free as well as after 1 and 2 months of paroxetine therapy. Psychopathology was assessed by the State-Trait Anxiety Inventory (STAI), the Panic-Associated Symptom Scale, and the Fear Questionnaire total score. Plasma concentrations of steroids were measured by radioimmunoassay. The plasma concentrations of progesterone and dehydroepiandrosterone were greater in drug-free patients than in controls, whereas those of allopregnanolone and tetrahydrodeoxycorticosterone did not differ between the two groups. Paroxetine treatment for 2 months significantly increased the plasma concentration of allopregnanolone but did not affect those of the other steroids. At 2 months of therapy, allopregnanolone concentrations in patients were significantly greater than those in controls. The plasma concentrations of progesterone and tetrahydrodeoxycorticosterone correlated with the STAI state score in patients before treatment. Our data suggest that neuroactive steroids may play a role in PD in men.

Increased expression of the neuropeptide Y receptor Y(1) gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone.

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABAA receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y1R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y1 receptor for NPY linked to the lacZ gene, we previously showed that long‐term treatment with benzodiazepine receptor ligands modulates Y1 receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y1R/LacZ transgene expression, as determined by quantitative histochemical analysis of β‐galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and β‐galactosidase expression in the medial amygdala. Finasteride, a 5α‐reductase inhibitor, prevented both of these effects. Long‐term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected β‐galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y1 receptor gene expression through modulation of GABAA receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.

Down-regulation of hippocampal BDNF and Arc associated with improvement in aversive spatial memory performance in socially isolated rats

Rats deprived of social contact with other rats at a young age experience a form of prolonged stress that leads to long-lasting changes in behavioral profile. Such isolation is thought to be anxiogenic for these normally gregarious animals, and the abnormal reactivity of isolated rats to environmental stimuli is thought to be a product of prolonged stress. We now show that isolation of rats at weaning reduced immobility time in the forced swim test, decreased sucrose intake and preference, and down-regulated both brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeletal associated protein (Arc) in the hippocampus. In the Morris water maze, isolated rats showed a reduced latency to reach the hidden platform during training, indicative of an improved learning performance, compared with group-housed rats. The cumulative search error during place training trials indicated a reliable difference between isolated and group-housed rats on days 4 and 5. The probe trial revealed a significant decrease of the average proximity to the target location in the isolated rats suggesting an improvement in spatial memory. Isolated rats also showed an increase in the plasma level of corticosterone on the 5th day of training and increased expression of BDNF and Arc in the hippocampus on both days 1 and 5. These results show that social isolation from weaning in rats results in development of depressive-like behavior but has a positive effect on spatial learning, supporting the existence of a facilitating effect of stress on cognitive function.

Effects of voluntary ethanol consumption on emotional state and stress responsiveness in socially isolated rats

Social isolation of rats immediately after weaning is thought to represent an animal model of anxiety-like disorders. This mildly stressful condition reduces the cerebrocortical and plasma concentrations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) as well as increases the sensitivity of rats to the effects of acute ethanol administration on the concentrations of this neuroactive steroid. We further investigated the effects of voluntary consumption of ethanol at concentrations increasing from 2.5 to 10% over 4 weeks of isolation. Isolated rats showed a reduced ethanol preference compared with group-housed animals. Ethanol consumption did not affect the isolation-induced down-regulation of BDNF or Arc, but it attenuated the increase in the cerebrocortical concentration of 3α,5α-TH PROG induced by foot-shock stress in both isolated and group-housed animals as well as increased the percentage of number of entries made by socially isolated rats into the open arms in the elevated plus-maze test. Ethanol consumption did not affect expression of the α₄ subunit of the GABA(A) receptor in the hippocampus of group-housed or isolated rats, whereas it up-regulated the δ subunit throughout the hippocampus under both conditions. The results suggest that low consumption of ethanol may ameliorate some negative effects of social isolation on stress sensitivity and behavior.