Mariangela Serra

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.

Stress and neuroactive steroids.

The discovery that the endogenous steroid derivatives 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, or 3 alpha,5 alpha-TH PROG) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, or 3 alpha,5 alpha-TH DOC) elicit marked anxiolytic and anti-stress effects and selectively facilitate gamma-aminobutyric acid (GABA)-mediated neurotransmission in the central nervous system (see Chapter 3) has provided new perspectives for our understanding of the physiology and neurobiology of stress and anxiety. Evidence indicating that various stressful conditions that downregulate GABAergic transmission and induce anxiety-like states (Biggio et al., 1990) also induce marked increases in the plasma and brain concentrations of these neuroactive steroids (Biggio et al., 1996, 2000) has led to the view that stress, neurosteroids, and the function of GABAA receptors are intimately related. Changes in the brain concentrations of neurosteroids may play an important role in the modulation of emotional state as well as in the homeostatic mechanisms that counteract the neuronal overexcitation elicited by acute stress. Indeed, neurosteroids not only interact directly with GABAA receptors but also regulate the expression of genes that encode subunits of this receptor complex. This chapter summarizes observations from our laboratories and others, suggesting that neurosteroids and GABAergic transmission are important contributors to the changes in emotional state induced by environmental stress.

Neurochemical action of the general anaesthetic propofol on the chloride ion channel coupled with GABAA receptors

The effect of propofol, a novel short acting anaesthetic, on the function of the GABAA/ionophore receptor complex was studied in vitro in cortical membrane preparations from rat cerebral cortex and was compared with the action of pentobarbital and alphaxalone, two general anaesthetics known to enhance GABAergic transmission. Propofol, mimicking the action of pentobarbital and alphaxalone, increased [3H]GABA binding, reduced [35S]TBPS binding and enhanced muscimol-stimulated 36Cl- uptake in a concentration-dependent manner. While the efficacy of the drugs in affecting these biochemical parameters was similar, they differed markedly in potency being alphaxalone greater than propofol greater than pentobarbital. However, separate sites of action or different mechanisms for these drugs can be suggested by the result that the concomitant addition of propofol either with alphaxalone or pentobarbital or diazepam produced a simple additive inhibition of [35S]TBPS binding as well as an additive enhancement of [3H]GABA binding and muscimol-stimulated 36Cl- uptake. The effect of propofol at the level of the GABA/ionophore receptor complex seems to be strictly dependent on the interaction of GABA with its recognition site. In fact, the specific GABAA receptor antagonist bicuculline antagonized the decrease of [35S]TBPS binding as well as the enhancement of [3H]GABA binding and muscimol-stimulated 36Cl- uptake induced by propofol. On the other hand, propofol was able to enhance [3H]GABA binding in membranes previously incubated with the specific chloride channel blocker picrotoxin. Finally, the finding that propofol fails to affect [3H]flunitrazepam binding together with the failure of Ro 15-1788 and PK 11195 to antagonize its effect on [35S]TBPS binding excludes a direct interaction at the level of benzodiazepine recognition sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The general anesthetic propofol enhances the function of γ-aminobutyric acid-coupled chloride channel in the rat cerebral cortex

Abstract: The effect of the general anesthetic propofol on t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to unwashed membrane preparations from rat cerebral cortex was studied and compared to that of other general anesthetics (pentobarbital, alphaxalone) which are known to enhance GABAergic transmission. Propofol produced a concentration‐dependent complete inhibition of [35S]TBPS binding, an effect similar to that induced by pentobarbital and alphaxalone, although these agents differ markedly in potency (alphaxalone > propofol > pentobarbital). The concomitant addition of propofol either with alphaxalone or pentobarbital produced an additive inhibition of [35S]TBPS binding, suggesting separate sites of action or different mechanisms of these drugs. Moreover, although bicuculline (0.1 μM) completely antagonized the propofol‐induced inhibition of [35S]TBPS binding, the effect of this anesthetic was not due to a direct interaction with the γ‐aminobutyric acidA (GABAA) recognition site. In fact, propofol, like alphaxalone and pentobarbital, markedly enhanced [3H]GABA binding in the rat cerebral cortex. Finally, propofol was able to enhance [3H]GABA binding in membranes previously incubated with the specific chloride channel blocker picrotoxin. Taken together these data strongly suggest that propofol, like other anesthetics and positive modulators of GABAergic transmission, might exert its pharmacological effects by enhancing the function of the GABA‐activated chloride channel.

Physiology and gene regulation of the brain NPY Y1 receptor

Neuropeptide Y (NPY) is one of the most prominent and abundant neuropeptides in the mammalian brain where it interacts with a family of G-protein coupled receptors, including the Y(1) receptor subtype (Y(1)R). NPY-Y(1)R signalling plays a prominent role in the regulation of several behavioural and physiological functions including feeding behaviour and energy balance, sexual hormone secretion, stress response, emotional behaviour, neuronal excitability and ethanol drinking. Y(1)R expression is regulated by neuronal activity and peripheral hormones. The Y(1)R gene has been isolated from rodents and humans and it contains multiple regulatory elements that may participate in the regulation of its expression. Y(1)R expression in the hypothalamus is modulated by changes in energetic balance induced by a wide variety of conditions (fasting, pregnancy, hyperglycaemic challenge, hypophagia, diet induced obesity). Estrogens up-regulate responsiveness to NPY to stimulate preovulatory GnRH and gonadotropin surges by increasing Y(1)R gene expression both in the hypothalamus and the pituitary. Y(1)R expression is modulated by different kinds of brain insults, such as stress and seizure activity, and alteration in its expression may contribute to antidepressant action. Chronic modulation of GABA(A) receptor function by benzodiazepines or neuroactive steroids also affects Y(1)R expression in the amygdala, suggesting that a functional interaction between the GABA(A) receptor and Y(1)R mediated signalling may contribute to the regulation of emotional behaviour. In this paper, we review the state of the art concerning Y(1)R function and gene expression, including our personal contribution to many of the subjects mentioned above.

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.

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.

2-Phenyl-imidazo[1,2-a]pyridine derivatives as ligands for peripheral benzodiazepine receptors: stimulation of neurosteroid synthesis and anticonflict action in rats

Selective activation of peripheral benzodiazepine receptors (PBRs) in adrenal cells and brain oligodendrocytes promotes steroidogenesis. Three 2‐phenyl‐imidazo[1,2‐a]pyridine derivatives (CB 34, CB 50 and CB 54) have now been investigated with regard to their selectivity for PBRs and their ability to stimulate central and peripheral steroidogenesis in rats. The three CB compounds (10−10–10−4 M) potently inhibited the binding of the PBR ligand [3H]‐PK 11195 to brain and ovary membranes in vitro, without substantially affecting [3H]‐flunitrazepam binding to central benzodiazepine receptors. These compounds (10−7–10−4 M) also had little or no marked effects on GABA‐evoked Cl− currents in voltage‐clamped Xenopus oocytes expressing human α1β2γ2S GABAA receptors. In addition, they failed to affect ligands binding to GABAB, D1/D2 dopamine, muscarinic acetylcholine, N‐methyl‐D‐aspartic acid and opiate receptors. Intraperitoneal administration of CB compounds (3–50 mg kg−1) induced a dose‐dependent increase in the concentrations of neuroactive steroids in plasma and brain. The brain concentrations of pregnenolone, progesterone, allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC) showed maximal increases in 96±3, 126±14, 110±12 and 70±13% above control, respectively, 30 to 60 min after injection of CB 34 (25 mg kg−1). CB 34 also increased the brain concentrations of neuroactive steroids in adrenalectomized‐orchiectomized rats, although to a lesser extent than in sham‐operated animals, suggesting that CB compounds stimulate brain steroidogenesis independently of their effects on peripheral tissues. The increase in brain and plasma neurosteroid content induced by CB 34 was associated with a marked anticonflict effect in the Vogel test. Our results indicate that the three CB compounds tested are specific and potent agonists at peripheral benzodiazepine receptors, and that they stimulate steroidogenesis in both the brain and periphery.

Neurosteroids, GABAA receptors, and ethanol dependence

RationaleChanges in the expression of type A receptors for γ-aminobutyric acid (GABA) represent one of the mechanisms implicated in the development of tolerance to and dependence on ethanol. The impact of such changes on the function and pharmacological sensitivity of GABAA receptors (GABAARs) has remained unclear, however. Certain behavioral and electrophysiological actions of ethanol are mediated by an increase in the concentration of neuroactive steroids in the brain that results from stimulation of the hypothalamic–pituitary–adrenal (HPA) axis. Such steroids include potent modulators of GABAAR function.ObjectivesWe have investigated the effect of ethanol exposure and withdrawal on subunit expression and receptor function evaluated by subunit selective compounds, as well as the effects of short-term exposure to ethanol on both neurosteroid synthesis and GABAAR function, in isolated neurons and brain tissue.ResultsChronic treatment with and subsequent withdrawal from ethanol alter the expression of genes for specific GABAAR subunits in cultured rat neurons, and these changes are associated with alterations in receptor function and pharmacological sensitivity to neurosteroids, zaleplon, and flumazenil. Acute ethanol exposure increases the amount of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) in hippocampal slices by a local action independent of the activity of the HPA axis. This effect of ethanol was associated with an increased amplitude of GABAAR-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurons in such slices.ConclusionsChronic ethanol exposure elicits changes in the subunit composition of GABAARs, which, in turn, likely contribute to changes in receptor function associated with the altered pharmacological and behavioral sensitivity characteristic of ethanol tolerance and dependence. Ethanol may also modulate GABAAR function by increasing the de novo synthesis of neurosteroids in the brain in a manner independent of the HPA axis. This latter mechanism may play an important role in the central effects of ethanol.