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J. Neurochem. (2010) 113, 1285–1295. Gonadal steroids, in particular estradiol, exert important actions during pre- and perinatal periods in the regulation of sexual dimorphism and development of the nervous system. We have now examined the effects of neonatal estradiol administration in female rats on brain concentrations of the neuroactive steroids allopregnanolone and tetrahydrodeoxycorticosterone, expression of GABAA receptor subunits, and behavioral sensitivity to benzodiazepines and allopregnanolone. Administration of β-estradiol 3-benzoate on the day of birth resulted in marked decreases in the concentrations of progesterone and allopregnanolone in the cerebral cortex at 21, 60, and 180 days after birth. The concentrations of tetrahydrodeoxycorticosterone, 17β-estradiol, and dehydroepiandrosterone in the brain at 60 days were not affected by such treatment. Neonatal administration of β-estradiol 3-benzoate also increased the cerebrocortical abundance of α1, α2, and γ2 subunits of the GABAA receptor without affecting that of α3, α4, α5, or δ subunits. Diazepam induced a greater reduction in locomotor activity as well as a more pronounced anxiolytic-like effect in the elevated plus-maze test in rats subjected to neonatal treatment with β-estradiol 3-benzoate than in vehicle-treated controls, while allopregnanolone induced a similar effect in both groups. These effects of estradiol suggest that it plays a major role in regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system. [ABSTRACT FROM AUTHOR]

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABAA-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABAA-Rs, and a tonic component mediated by extrasynaptic GABAA-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3α,5α-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the δ subunit of the GABAA-R and a concomitant decrease in that of the γ2 subunit in the hippocampus at P19. Expression of the α4 subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5α-reductase inhibitor finasteride prevented the changes in tonic current and in δ and γ2 subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABAA-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids. [ABSTRACT FROM AUTHOR]

Neuroactive steroids such as 3α,5α‐THP are reduced metabolites of progesterone and are considered to play an important physiological role to locally modulate neuronal excitability by “fine‐tuning” the action of GABA acting at GABAA receptors (GABAA‐Rs). In different brain regions, such as the hippocampus, different subpopulations of nerve cells exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABAA‐Rs, and a tonic component mediated by “ambient” GABA acting at extrasynaptic GABAA‐Rs mainly containing the δ subunit and endowed with a higher sensitivity to neuroactive steroids compared to synaptic receptors. It is also well accepted that fluctuations in brain neuroactive steroid levels may result in plastic changes of GABAA‐Rs. In this article we review some of our results obtained with the model of pregnancy in rats. Pregnancy, in fact, is characterized by a marked and progressive increase in plasma and brain levels of neuroactive steroids which could contribute to changes in mood, anxiety as well as other psychiatric conditions. Such elevation in brain neuroactive steroid concentrations during pregnancy, in turn, is accompanied by alterations in both gene expression and function of synaptic and extrasynaptic GABAA‐Rs in the hippocampus as well as other areas. [Copyright &y& Elsevier]

Previous studies have shown that GABAergic neuroactive steroids increase Y1 receptor (Y1R) gene expression in the amygdala of Y 1 R/ LacZ transgenic mice, harbouring the murine Y1R gene promoter linked to a LacZ reporter gene. As ethanol is known to increase GABAergic neuroactive steroids, we investigated the relationship between fluctuations in the brain content of neuroactive steroids induced by chronic voluntary ethanol consumption or ethanol discontinuation and both the level of neuropeptide Y (NPY) immunoreactivity and Y1R gene expression in the amygdala of Y 1 R/ LacZ transgenic mice. Ethanol discontinuation (48 h) after voluntary consumption of consecutive solutions of 3%, 6%, 10% and 20% (v/v) ethanol over 4 weeks produced an anxiety-like behaviour as measured by elevated plus maze. Voluntary ethanol intake increased the cerebrocortical concentration of the progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) that returned to control level 48 h after discontinuation of ethanol intake. Ethanol discontinuation significantly decreased NPY immunoreactivity and concomitantly increased Y 1 R/ LacZ transgene expression in the amygdala, whereas chronic ethanol intake failed to affect these parameters. The 5α-reductase inhibitor finasteride prevented both the increase in the cerebrocortical concentration of 3α,5α-TH PROG apparent after 4 weeks of ethanol intake and the changes in NPY immunoreactivity and transgene expression induced by ethanol discontinuation. Data suggest that 3α,5α-TH PROG plays an important role in the changes in NPY–Y1R signalling in the amygdala during ethanol discontinuation. [ABSTRACT FROM AUTHOR]

Patents for biologic agents first marketed in the 1980s are now beginning to expire, opening the door for 'non-proprietary' versions of these agents to enter the market. However, there are fundamental differences between biologics and traditional pharmaceuticals that preclude the extrapolation of existing regulatory processes for traditional generic agents to these new biologic products. These include differences in dimensions (molecular weight), synthesis, purification, stability, and immunogenicity. There is also controversy over the terminology of these biologic agents, with a number of terms put forward. European regulatory authorities have adopted the term 'biosimilars,' while the US FDA prefers the term 'follow-on biologics.' It is important that these terms are not used as synonyms for 'generics,' and already there are moves to prevent classification of these products as 'generics.' In this review, we focus on the differences that exist between generics and biosimilars, and assess the current scenario of problems and opportunities. Furthermore, we also attempt to highlight the problems with establishing regulatory guidelines and those associated with the introduction of these drugs into clinical practice. [ABSTRACT FROM AUTHOR]

Abstract: Neurosteroids play a crucial role in stress, alcohol dependence and withdrawal, and other physiological and pharmacological actions by potentiating or inhibiting neurotransmitter action. This review article focuses on data showing that the interaction among stress, ethanol, and neuroactive steroids may result in plastic molecular and functional changes of GABAergic inhibitory neurotransmission. The molecular mechanisms by which stress–ethanol–neuroactive steroids interactions can produce plastic changes in GABAA receptors have been studied using different experimental models in vivo and in vitro in order to provide useful evidence and new insights into the mechanisms through which acute and chronic ethanol and stress exposure modulate the activity of GABAergic synapses. We show detailed data on a) the effect of acute and chronic stress on peripheral and brain neurosteroid levels and GABAA receptor gene expression and function; b) ethanol-stimulated brain steroidogenesis; c) plasticity of GABAA receptor after acute and chronic ethanol exposure. The implications of these new mechanistic insights to our understanding of the effects of ethanol during stress are also discussed. The understanding of these neurochemical and molecular mechanisms may shed new light on the physiopathology of diseases, such as anxiety, in which GABAergic transmission plays a pivotal role. These data may also lead to the need for new anxiolytic, hypnotic and anticonvulsant selective drugs devoid of side effects. [Copyright &y& Elsevier]

The influence of the estrous cycle on dopamine levels in the rat medial prefrontal cortex under basal and ethanol-stimulated conditions was evaluated by microdialysis. The basal dopamine concentration in the dialysate varied markedly during the estrous cycle, being highest in estrus and lowest in proestrus. Furthermore, a challenge intraperitoneal administration of ethanol (0.5 g/kg) induced a significant increase in dopaminergic output (+50%) during estrus but had no effect in diestrus or proestrus. Ovariectomy or pretreatment with either finasteride (a 5α-reductase inhibitor) or clomiphene (an estrogen receptor antagonist) prevented this ethanol-induced increase in dopamine concentration. The effect of ethanol was restored in ovariectomized rats by pretreatment with estrogen but not by that with progesterone. Our results thus show that the basal levels of dopamine in the prefrontal cortex are dependent on the phase of the estrous cycle. Furthermore, this dependence appears to be attributable to the effects of ovarian steroid hormones and results in a differential sensitivity of the dopaminergic neurons to ethanol. The hormone-induced changes in the activity of these neurons might contribute to the differences in drug sensitivity and mood state apparent among phases of the estrous cycle and between the sexes.Neuropsychopharmacology (2007) 32, 892–901. doi:10.1038/sj.npp.1301150; published online 12 July 2006 [ABSTRACT FROM AUTHOR]

Type A receptors for GABA (GABAA receptors) that contain the δ subunit are located predominantly at extrasynaptic sites and are implicated in modulation of neuronal excitability through tonic inhibition. We have examined the effects of chronic exposure to and subsequent withdrawal of progesterone or the progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-THPROG) on expression of the d subunit of GABAA receptors and on receptor function in cultured rat hippocampal neurons. Progesterone treatment for 1 day increased the amounts of both d subunit mRNA and protein, whereas such treatment for 6 days induced marked decreases in the abundance of both them RNA and protein. Subsequent progesterone withdrawal up-regulated expression of the d subunit, which was significantly increased at 9-12 h after withdrawal. These effects of progesterone were mimicked by 3α,5α-THPROG and blocked by the 5α-reductase inhibitor finasteride. They were also accompanied by parallel changes in the function of GABAA receptors in hippocampal neurons. These results show that chronic exposure to and withdrawal of progesterone induce differential effects on both expression of the d subunit of GABAA receptors and receptor function that are mediated by 3α,5α-THPROG. They are consistent with the notion that this progesterone metabolite plays a key physiological role in modulation of GABAergic synapses. [ABSTRACT FROM AUTHOR]

Abstract: A deregulation in the peripheral and brain concentrations of neuroactive steroids has been found in certain pathological conditions characterized by emotional or affective disturbances, including major depression and anxiety disorders. In this article we summarize data pertaining to the modulatory effects of oral contraceptive treatment on neuroactive steroids in women and rats. Given that the neuroactive steroids concentrations are reduced by oral contraceptives, together with the evidence that a subset of women taking oral contraceptives experience negative mood symptoms, we propose the use of this pharmacological treatment as a putative model to study the role of neuroactive steroids in the etiopathology of mood disorders. Moreover, since neuroactive steroids are potent modulators of GABAA receptor function and plasticity, the treatment with oral contraceptives might also represent a useful experimental model to further investigate the physiological role of these steroids in the modulation of GABAergic transmission. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Examines the effects of acute treatment with chronic exposure to nicotine or morphine on the concentrations of allopregnanolone and its precursors, pregnenolone and progesterone, in the cerebral cortex and plasma of rats. Multiple pharmacological characteristics shared by nicotine and morphine; Stages of drug dependent implicating neurosteroids; Effects of nicotine and morphine on the body.

Examines the effect of ethanol exposure and withdrawal on subunit expression and receptor function and the effects of short-term exposure to ethanol on both neurosteroid synthesis and GABAAR function. Functions of GABA receptors; Effects of chronic ethanol exposure on GABAARs; Role of neurosteroids in the effects of ethanol on GABAARs; Vital determinant of alcohol withdrawal syndrome.

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABAAR) for γ-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABAAR plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the δ subunit of GABAARs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of δ subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated δ subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABAARs. GABAARs containing the δ subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABAARs that contain the δ subunit in different neuronal types. [ABSTRACT FROM AUTHOR]

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 m. [ABSTRACT FROM AUTHOR]

A sustained increase in the brain concentrations of neuroactive steroids was previously shown to induce Y1 receptor gene expression in the amygdala of Y1R/LacZ transgenic mice which harbour a construct comprising the murine Y1 receptor gene promoter and the lacZ reporter gene. We now investigated the effects of restraint stress on both the cere- brocortical concentrations of neuroactive steroids and Y1 receptor gene expression in the amygdala and hypothalamic paraventricular nucleus (PVN) of Y1R/LacZ transgenic mice. The cerebrocortical concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone were significantly increased immediately after a 1-h exposure to restraint stress and had returned to control values within 30 min. Expression of Y1R/LacZ was increased in the amygdala and PVN 6 h after restraint. The 5α-reductase inhibitor finasteride, that prevented the increase in neuroactive steroid concentrations, did not block that in transgene expression induced by 1-h restraint. Daily exposure to restraint for 10 days also increased the cerebrocortical concentrations of neuroactive steroids but failed to affect transgene expression. Acute but not repeated restraint thus increases Y1 receptor gene expression in the amygdala and PVN, suggesting that tolerance develops towards this stressor. The effect of acute restraint is not mediated by the increase in the brain concentrations of neuroactive steroids but may rather reflect a ligand-induced increase in Y1 receptor gene transcription. Data support a role of Y1 receptors in the behavioural and neuroendocrine responses to stress. [ABSTRACT FROM AUTHOR]

Changes in the expression of subunits of the GABA type A (GABA[subA]) receptor are implicated in the development of ethanol tolerance and dependence as well as in the central hyperexcitability associated with ethanol withdrawal. The impact of such changes on GABA[subA] receptor function and pharmacological sensitivity was investigated with cultured rat hippocampal neurons exposed to ethanol for 5 d and then subjected to ethanol withdrawal. Both ethanol treatment and withdrawal were associated with a marked decrease in the maximal density of GABA-evoked Cl[sup-] currents, whereas the potency of GABA was unaffected. Ethanol exposure also reduced the modulatory efficacy of the benzodiazepine receptor agonists lorazepam, zolpidem, and zaleplon as well as that of the inverse agonists Ro 15-4513 and FG 7142, effects that were associated with a reduced abundance of mRNAs encoding the receptor subunits α1, α3, γ2L, and γ2S. Ethanol withdrawal restored the efficacy of lorazepam, but not that of low concentrations of zolpidem or zaleplon, to control values. Flumazenil, which was ineffective in control neurons, and Ro 15-4513 each potentiated the GABA response after ethanol withdrawal. These effects of withdrawal were accompanied by upregulation of the α2, α3, and α4 subunit mRNAs as well as of the α4 protein. Diazepam or γ-hydroxybutyrate, but not baclofen, prevented the changes in both GABA[subA] receptor pharmacology and subunit mRNA levels induced by ethanol withdrawal. Changes in GABA[subA] receptor gene expression induced by prolonged exposure to and withdrawal of ethanol are thus associated with altered GABA[subA] receptor function and pharmacological sensitivity. [ABSTRACT FROM AUTHOR]

Here, we summarize recent data pertaining to the effects of GABAA receptor modulators on the receptor gene expression in order to elucidate the molecular mechanisms behind tolerance and dependence induced by these drugs. Drug selectivity and intrinsic activity seems to be important to evidence at the molecular level the GABAA receptor tolerance. On the contrary, we suggested that all drug tested are equally potentially prone to induce dependence. Our results demonstrate that long-lasting exposure of GABAA receptors to endogenous steroids, benzodiazepines and ethanol, as well as their withdrawal, induce marked effects on receptor structure and function. These results suggest the possible synergic action between endogenous steroids and these drugs in modulating the functional activity of specific neuronal populations. We report here that endogenous steroids may play a crucial role in the action of ethanol on dopaminergic neurons. [Copyright &y& Elsevier]

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 (3α,5α-tetrahydroprogesterone=3α,5α-THPROG), dehydroepiandrosterone (DHEA) and tetrahydrodeoxycorticosterone (3α,5α-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, 3α,5α-THPROG during the follicular phase of the drug-free month and during the premenstrual phase of the therapy month, and 3α,5α-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 GABAA receptor sensitivity. [Copyright &y& Elsevier]

Abstract Social isolation of rats for 30 days immediately after weaning results in marked decreases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG), and 3α,5α-tetrahydrodeoxycorticosterone (3α,5α-TH DOC), as well as a moderate increase in the plasma concentration of corticosterone. This mildly stressful condition has now been shown to increase the sensitivity of rats to the effect of acute ethanol administration on the cerebrocortical and plasma concentrations of neuroactive steroids. The percentage increases in the brain and plasma concentrations of pregnenolone, progesterone, 3α,5α-TH PROG, and 3α,5α-TH DOC, apparent 20 min after a single intraperitoneal injection of ethanol (1 g/kg), were thus markedly greater in isolated rats than in group-housed animals. A subcutaneous injection of isoniazid (300 mg/kg) also induced greater percentage increases in the concentrations of these steroids in isolated rats than in group-housed animals. These results suggest that mild chronic stress, such as that induced by social isolation, enhances the steroidogenic effect of ethanol, a drug abused by humans under stress or affected by neuropsychiatric disorders. Social isolation also induced hyper-responsiveness of the hypothalamic–pituitary–adrenal (HPA) axis, as was apparent after reduction of GABA-mediated inhibitory tone by isoniazid administration. [ABSTRACT FROM AUTHOR]