Nucleus-specific modulation of phasic and tonic inhibition by endogenous neurosteroidogenesis in the murine thalamus

Neurosteroids are potent allosteric modulators of GABA_A receptors (GABA_ARs). Although the effects of exogenous neurosteroids on GABA_AR function are well documented, less is known about effects of neurosteroids produced by local endogenous biosynthesis. The neurosteroidogenic enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase are expressed in two nuclei of somatosensory thalamus, the thalamic reticular nucleus (nRT) and ventrobasal nucleus (VB). Here, the effects of acute blockade of neurosteroidogenesis by the 5α-reductase inhibitor finasteride on phasic and tonic GABA_AR-mediated currents were examined in nRT and VB of mice. In nRT, finasteride altered the decay and amplitude, but not the frequency, of phasic currents, with no effect on tonic inhibition. In VB neurons, by contrast, finasteride reduced both the size and frequency of phasic currents, and also reduced the degree of tonic inhibition. These studies thus provide novel evidence for endogenous modulation of GABA_AR function by 5α-reduced neurosteroids in the mature thalamus.     

GABAA receptor-acting neurosteroids: A role in the development and regulation of the stress response

Regulation of hypothalamic–pituitary–adrenocortical (HPA) axis activity by stress is a fundamental survival mechanism and HPA-dysfunction is implicated in psychiatric disorders. Adverse early life experiences, e.g. poor maternal care, negatively influence brain development and programs an abnormal stress response by encoding long-lasting molecular changes, which may extend to the next generation. How HPA-dysfunction leads to the development of affective disorders is complex, but may involve GABA_A receptors (GABA_ARs), as they curtail stress-induced HPA axis activation. Of particular interest are endogenous neurosteroids that potently modulate the function of GABA_ARs and exhibit stress-protective properties. Importantly, neurosteroid levels rise rapidly during acute stress, are perturbed in chronic stress and are implicated in the behavioural changes associated with early-life adversity. We will appraise how GABA_AR-active neurosteroids may impact on HPA axis development and the orchestration of the stress-evoked response. The significance of these actions will be discussed in the context of stress-associated mood disorders.     

Antidepressant-Like and Anxiolytic-Like Effects of ZBD-2, a Novel Ligand for the Translocator Protein (18 kDa)

Activation of translocator protein (18 kDa) (TSPO) plays an important role to mediate rapid anxiolytic efficacy in stress response and stress-related disorders by the production of neurosteroids. However, little is known about the ligand of TSPO on the anxiety-like and depressive behaviors and the underlying mechanisms in chronic unpredictable mild stress (UCMS) mice. In the present study, a novel ligand of TSPO, ZBD-2 [N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide] synthesized by our laboratory, was used to evaluate the anxiolytic and antidepressant efficacy and to elucidate the underlying mechanisms. ZBD-2 (3 mg/kg) significantly attenuated anxiety-like and depressive behaviors in the UCMS mice, which was blocked by TSPO antagonist PK11195 (3 mg/kg). Treatment of ZBD-2 reversed the decrease in biogenic amines (norepinephrine, dopamine, and serotonin) in the brain region of hippocampus in the UCMS mice. The decreases in TSPO, GluN2B-containing N-methyl-d-aspartate (NMDA) receptors, GluA1, p-GluA1-Ser831, p-GluA1-Ser845, PSD-95, and GABA_A-a2 were integrated with the increases of CaMKII and iNOS levels in the hippocampus of the UCMS mice. ZBD-2 significantly reversed the changes of above proteins. However, ZBD-2 or PK11195 treatment did not affect the levels of GluN2A-containing NMDA receptors and the total levels of GAD67. Our study provides strong evidences that ZBD-2 has a therapeutic effect on chronic stress-related disorders such as depression and anxiety through regulating the biogenic amine levels and the synaptic proteins in the hippocampus.     

Lack of tolerance to anxiolysis and withdrawal symptoms in mice repeatedly treated with AC-5216, a selective TSPO ligand

AC-5216, a ligand for the translocator protein (18 kDa) (TSPO), produces anxiolytic-like effects in animal models of anxiety without causing the side effects normally associated with conventional benzodiazepines. This study aimed to investigate whether repeated administration of AC-5216 induces tolerance to anxiolytic-like effects of AC-5216 and produces withdrawal on abrupt cessation, and compare the results with those of diazepam. In the tolerance experiment, AC-5216 (0.1 mg/kg, p.o.) produced significant anxiolytic-like effects in both groups of mice pretreated with the vehicle and AC-5216 twice daily for 14 days. Diazepam (0.1 mg/kg, p.o.) also retained its anxiolytic effects in mice repeatedly treated with diazepam. In the withdrawal experiment, mice were orally treated with either AC-5216 (0.1, 1 or 10 mg/kg; twice daily) or diazepam (0.1, 1 or 10 mg/kg; twice daily) for 14 days, and examined, during a treatment withdrawal period, for anxiogenic-like effects in the social interaction test, and for body weight loss as indices of emotional and somatic withdrawal symptoms, respectively. In AC-5216-treated groups, neither anxiogenic-like effects nor body weight loss was observed upon treatment withdrawal at any of the doses tested. In contrast, in diazepam 1 mg/kg- and 10 mg/kg-treated groups, treatment withdrawal not only induced anxiogenic-like effects on the second day of the withdrawal period, but also decreased body weight gain and brought about body weight loss in mice. These findings indicate that AC-5216 when repeatedly administered does not induce tolerance to its anxiolytic-like effects or withdrawal symptoms.     

Protein kinase C regulates tonic GABAA receptor‐mediated inhibition in the hippocampus and thalamus

Tonic inhibition mediated by extrasynaptic GABA_A receptors (GABA_ARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA_ARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole‐cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC‐mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA_AR‐mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA_AR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA_ARs, which represent a key extrasynaptic GABA_AR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA_ARs. The inhibitory effects of PKC activation on α4β2δ GABA_AR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC‐mediated phosphorylation can be an important physiological regulator of tonic GABA_AR‐mediated inhibition.     

Role of GABA<Subscript>A</Subscript> receptors in alcohol use disorders suggested by chronic intermittent ethanol (CIE) rodent model

GABAergic inhibitory transmission is involved in the acute and chronic effects of ethanol on the brain and behavior. One-dose ethanol exposure induces transient plastic changes in GABA_A receptor subunit levels, composition, and regional and subcellular localization. Rapid down-regulation of early responder δ subunit-containing GABA_A receptor subtypes mediating ethanol-sensitive tonic inhibitory currents in critical neuronal circuits corresponds to rapid tolerance to ethanol’s behavioral responses. Slightly slower, α1 subunit-containing GABA_A receptor subtypes mediating ethanol-insensitive synaptic inhibition are down-regulated, corresponding to tolerance to additional ethanol behaviors plus cross-tolerance to other GABAergic drugs including benzodiazepines, anesthetics, and neurosteroids, especially sedative-hypnotic effects. Compensatory up-regulation of synaptically localized α4 and α2 subunit-containing GABA_A receptor subtypes, mediating ethanol-sensitive synaptic inhibitory currents follow, but exhibit altered physio-pharmacology, seizure susceptibility, hyperexcitability, anxiety, and tolerance to GABAergic positive allosteric modulators, corresponding to heightened alcohol withdrawal syndrome. All these changes (behavioral, physiological, and biochemical) induced by ethanol administration are transient and return to normal in a few days. After chronic intermittent ethanol (CIE) treatment the same changes are observed but they become persistent after 30 or more doses, lasting for at least 120 days in the rat, and probably for life. We conclude that the ethanol-induced changes in GABA_A receptors represent aberrant plasticity contributing critically to ethanol dependence and increased voluntary consumption. We suggest that the craving, drug-seeking, and increased consumption in the rat model are tied to ethanol-induced plastic changes in GABA_A receptors, importantly the development of ethanol-sensitive synaptic GABA_A receptor-mediating inhibitory currents that participate in maintained positive reward actions of ethanol on critical neuronal circuits. These probably disinhibit nerve endings of inhibitory GABAergic neurons on dopamine reward circuit cells, and limbic system circuits mediating anxiolysis in hippocampus and amygdala. We further suggest that the GABA_A receptors contributing to alcohol dependence in the rat and presumably in human alcohol use disorders (AUD) are the ethanol-induced up-regulated subtypes containing α4 and most importantly α2 subunits. These mediate critical aspects of the positive reinforcement of ethanol in the dependent chronic user while alleviating heightened withdrawal symptoms experienced whenever ethanol is absent. The speculative conclusions based on firm observations are readily testable.     

Modulation of native and recombinant GABAA receptors by endogenous and synthetic neuroactive steroids

Upon administration, certain pregnane steroids produce clear behavioural effects including, anxiolysis, sedation, analgesia, anaesthesia and are anti-convulsant. This behavioural profile is characteristic of compounds that act to enhance the actions of GABA acting at the GABA_A receptor. In agreement, numerous studies have now demonstrated these steroids to be potent, positive allosteric modulators of the GABA_A receptor. The pregnane steroids are synthesized in the periphery by endocrine glands such as the adrenals and the ovaries, but are also made by neurons and glial cells in the central nervous system itself. Hence, these compounds could play both an endocrine and a paracrine role to influence neuronal excitability by promoting inhibition. Here we review evidence that the pregnane steroids are highly selective and extremely potent GABA_A receptor modulators and that their effects at ‘physiological’ concentrations (low nanomolar) may be influenced by the subunit composition of the GABA_A receptor. This feature may underlie recent findings demonstrating the effects of the neurosteroids on inhibitory synaptic transmission to be brain region dependent, although recent reports suggest that phosphorylation mechanisms may additionally influence neurosteroid sensitivity of the GABA_A receptor. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit the behavioural effects of the pregnane steroids and progress with this approach will be discussed. However, the demonstration that the steroids may be made within the central nervous system offers the alternative strategy of targeting the enzymes that synthesize/metabolise the neurosteroids to exploit this novel endocrine/paracrine interaction.     

Rapid decline of GABAA receptor subunit mrna expression in hippocampus following transient cerebral ischemia in the gerbil

Inhibitory neurotransmission may play an important role in neuronal degeneration following transient cerebral ischemia. We studied the effect of transient forebrain ischemia on the GABA_A receptor system in the gerbil hippocampus. Gerbils were subjected to 5 minutes of bilateral carotid occlusion and were sacrificed at various times over 4 days following reperfusion. There was a substantial loss of pyramidal cells in the CA1 area of the hippocampus, granule cell layer of the dentate gyrus, and ventroposterior medial and ventroposterior lateral nuclei of the thalamus at any time following ischemia. Examination of brain slices by in situ hybridization histochemistry revealed that a change in expression of the GABA_A receptor α₁ and β₂ subunit mRNAs occurred in two phases following onset of reperfusion. The early phase (rapid) occurred within the first 4 hours following reperfusion. The expression of mRNAs significantly decreased (up to 25%) within 1 hour after occlusion in CA1 and CA3 pyramidal cell layers of the hippocampus and in the granule cell layer of the dentate gyrus. The expression of the mRNAs in these regions continued to decrease for 4 hours (up to 43%). In the second phase, which began between 4 and 12 hours following reperfusion, mRNA expression started to return to control levels in CA3 hippocampus and in the dentate. However, expression of both mRNAs continued to decline slowly in the CA1 pyramidal cell layer (up to 85%) over the next 3 days, concomitantly with degeneration of the CA1 pyramidal cells. Expression of mRNAs in the ventroposterior medial or ventroposterior lateral nuclei of the thalamus was similar to control values. To determine if a change in GABA_A receptor distribution paralleled changes in receptor subunit mRNA expression, we also measured the binding of [³⁵S]t-butylbicylophosphorothionate to GABA_A receptor chloride channels. The t-butylibicyclophosphorothionate [³⁵S] binding decreased between 1 and 4 days after reperfusion in the dendritic fields of CA1 pyramidal cells (strata oriens, radiatum, and lacunosum-moleculare) but not in the pyramidal cell body layer. These results indicate that expression of GABA_A receptor subunit mRNAs decrease well before CA1 pyramidal cell degeneration and loss of GABA_A receptors. At present, it is not clear if an early loss of mRNA expression after an ischemic insult leads to a functional defect in GABA_A receptors. If so, a loss of GABA neurotransmission may contribute to the development of neuronal degeneration following cerebral ischemia. The maintenance of normal GABA neurotransmission in surviving cells may explain their resistance to ischemia-induced neuronal death.     

Neurosteroid replacement therapy for catamenial epilepsy

Perimenstrual catamenial epilepsy, the cyclical occurrence of seizure exacerbations near the time of menstruation, affects a high proportion of women of reproductive age with drug-refractory epilepsy. Enhanced seizure susceptibility in perimenstrual catamenial epilepsy is believed to be due to the withdrawal of the progesterone-derived GABA_A receptor modulating neurosteroid allopregnanolone as a result of the fall in progesterone at the time of menstruation. Studies in a rat pseudopregnancy model of catamenial epilepsy indicate that after neurosteroid withdrawal there is enhanced susceptibility to chemoconvulsant seizures. There is also a transitory increase in the frequency of spontaneous seizures in epileptic rats that had experienced pilocarpine-induced status epilepticus. In the catamenial epilepsy model, there is a marked reduction in the antiseizure potency of anticonvulsant drugs, including benzodiazepines and valproate, but an increase in the anticonvulsant potency and protective index of neurosteroids such as allopregnanolone and the neurosteroid analog ganaxolone. The enhanced seizure susceptibility and benzodiazepine-resistance subsequent to neurosteroid withdrawal may be related to reduced expression and altered kinetics of synaptic GABA_A receptors and increased expression of GABA_A receptor subunits (such as α4) that confer benzodiazepine insensitivity. The enhanced potency of neurosteroids may be due to a relative increase after neurosteroid withdrawal in the expression of neurosteroid-sensitive δ-subunit-containing perisynaptic or extrasynaptic GABA_A receptors. Positive allosteric modulatory neurosteroids and synthetic analogs such as ganaxolone may be administered to prevent catamenial seizure exacerbations, in what we call neurosteroid replacement therapy.     

Favouring inhibitory synaptic drive mediated by GABAA receptors in the basolateral nucleus of the amygdala efficiently reduces pain symptoms in neuropathic mice

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the ‘nociceptive amygdala’, but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically‐implanted cannulas. We found that an N‐methyl‐D‐aspartate‐type glutamate receptor antagonist (MK‐801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA_A receptor antagonist. Potentiating GABA_A receptor function using diazepam or etifoxine (a non‐benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α‐steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch‐clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA_A receptor‐mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA_A miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non‐pharmacological therapies.     

Discriminative stimulus properties of ethanol in the rat: effects of neurosteroids and picrotoxin

Tetrahydrodeoxycorticosterone (5β-THDOC; 1.3–12.0 mg/kg), a neurosteroid enhancing the GABA_A receptor-associated chloride conductance, produced predominantly ethanol-appropriate responding (>80%) in rats trained to discriminate 1.0 g/kg ethanol from saline. However, neither picrotoxin (0.25–1.5 mg/kg), nor dehydroepiandrosterone sulfate (0.01–100.0 mg/kg), a neurosteroid acting as a GABA_A receptor antagonist, attenuated the stimulus effects of ethanol. These results indicate that: (1) at least certain neurosteroids may produce subjective states similar to these induced by ethanol; (2) blockade of the GABA_A receptor-associated channel does not eliminate the ethanol interoceptive cue in rats. ©1997 Elsevier Science B.V. All rights reserved.     

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Bicuculline‐ and neurosteroid‐sensitive tonic chloride current in rat hypoglossal motoneurons and atypical dual effect of SR95531

Hypoglossal motoneurons (HMs) are known to be under ‘permanent’ bicuculline‐sensitive inhibition and to show ‘transient’ synaptic γ‐aminobutyric acid (GABA)_A and glycine inhibitory responses. The present paper describes a permanent bicuculline‐sensitive current that should contribute to their tonic inhibition. This current was recorded in brainstem slices superfused without any exogenous agonist and remained detectable with tetrodotoxin. It could also be blocked by the other GABA_A antagonists picrotoxin (PTX) and 2‐(3‐carboxypropyl)‐3‐amino‐6‐(4 methoxyphenyl)pyridazinium bromide) (SR95531; gabazine), but persisted in the presence of a specific blocker of α5‐containing GABA_A receptors. Addition of 2 μm 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol hydrochloride (THIP), known to preferentially activate GABA_A receptors devoid of a γ‐subunit, induced a sustained anionic current that could be further enhanced by neurosteroids such as allopregnanolone (100 nm ). Thus, HMs show a tonic inhibitory current carried by extrasynaptic γ‐free GABA_A receptors, highly sensitive to neurosteroids. A second result was obtained by using SR95531 at concentrations sufficiently high to rapidly block the tonic current above the chloride equilibrium potential (E_Cl). Surprisingly, below E_Cl, SR95531 (10–40 μm ) activated a sustained inward current, associated with a conductance increase, and resistant to bicuculline or PTX (100 μm ). Similarly, after blockade of the bicuculline‐sensitive current, SR95531 activated an outward current above E_Cl. The bicuculline‐resistant anionic current activated by SR95531 could be blocked by a GABA_C receptor antagonist. Thus, two types of inhibitory GABA receptors, belonging to the GABA_A and GABA_C families, are able to show a sustained activity in HMs and provide promising targets for neuroprotection under overexcitatory situations known to easily damage these particularly fragile neurons.     

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids

GABA_A receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABA_A receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABA_A receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABA_A receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2 h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABA_A receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.     

Étifoxine,neurostéroïdes et anxiété

Anxiety states are related to the hypofunctioning of GABAergic inhibitory synaptic transmission in certain regions of the encephalon. Benzodiazepines are currently used in the treatment of anxiety states, but they are associated with side effects (sedation, amnesia) and their use in long-term treatment poses problems of functional tolerance. Benzodiazepines bind to specific sites located on the GABA_A receptor channels that underlie rapid GABAergic transmission and potentiate their functioning. Neurosteroids such as allopregnanolone that are reduced in positions 3α and 5α (3α5α-NS) also potentiate GABA_A receptor function and may be considered as endogenous anxiolytics. Neurosteroids are distinguished from other steroids by the fact that they are synthesised de novo in the nervous system by neurons and glial cells independently of circulating steroids. This synthesis occurs from cholesterol imported into the mitochondrion by a multiprotein complex of which the « peripheral benzodiazepine receptor », recently renamed TSPO (translocator protein of 18 kDa), forms a part. The majority of benzodiazepines bind to this protein and stimulate the transfer of cholesterol and consequently neurosteroidogenesis. These effects can be blocked pharmacologically by PK11195, which acts as an antagonist at the mitochondrial binding site for benzodiazepines. Cholesterol is transformed in the mitochondrion to pregnenolone, the universal precursor of all steroids. Pregnenolone then leaves the mitochondrion and the nature of the steroids synthesised in the cytoplasm will depend on the type of neurosteroidogenesis enzymes expressed by the cell.Etifoxine does not possess a benzodiazepine-related structure but does exhibit anxiolytic effects. The objective of our study consequently was to understand the mechanism of action of this compound by studying its effects on the functional properties of GABA_A receptors and on GABAergic synaptic transmission. Binding tests on isolated membranes have revealed that etifoxine binds directly to the GABA_A receptor and the peripheral benzodiazepine receptor (TSPO). Electrophysiological experiments have shown that etifoxine dose-dependently potentiates membrane currents induced by the application of submaximal but not of saturating concentrations of GABA. Etifoxine also facilitates GABAergic transmission in hypothalamic neuronal cultures and increases the amplitude of a sustained GABAergic current due to the tonic activation of GABA_A receptors by a weak GABA concentration in the extracellular space. The effect of etifoxine on this current is partially antagonised by PK11195 but not by flumazenil, an antagonist of the benzodiazepine binding sites on the GABA_A receptor.All of our data indicate that etifoxine facilitates GABAergic inhibition by two types of action: [1] by directly potentiating the activity of GABA_A receptors via an allosteric modulatory different site from that of the benzodiazepines, and [2] by stimulating the production of 3α5α-NS, which in turn potentiate the activity of GABA_A receptors. These two effects are independent and additive. They therefore contribute to make GABAergic transmission more effective.     

Antinociceptive Effects of H3 (R-Methylhistamine) and GABAB (Baclofen)-Receptor Ligands in an Orofacial Model of Pain in Rats

The present study explored the antinociceptive effects of H₃ (R-alpha-methylhistamine) and GABA_B (baclofen) receptor ligands in an orofacial model of pain in rats. Orofacial pain was induced by subcutaneous injection of formalin (50 μl, 5 %) in the upper lip region, and the number of jumps and time spent face rubbing was recorded for 40 min. Formalin produced a marked biphasic pain response; first phase, 0–10 min (jumps), and second phase, 15–40 min, (rubbing). Baclofen (50 μg) injected into the rat wiskerpad 5 min before formalin administration suppressed both phases of pain whereas R-alpha-methylhistamine (12.5 μg) abolished the first phase only. Brains were taken immediately after behavioral testing was completed. HPLC/ED analysis showed that 5-hydroxytryptamine (5-HT) turnover was increased in hippocampus, thalamus, and brain stem of all formalin groups, excepting the baclofen group in which the balance of 5-HT metabolism was restored to control values. These findings demonstrate that GABA_B receptors represent peripheral targets for analgesia. Consequently, locally administered baclofen may be a useful approach in treating inflammatory trigeminal pain.     

Ethanol differentially modulates GABAA receptor-mediated chloride currents in hippocampal,cortical, and septal neurons in rat brain slices

Previous electrophysiological studies have reported conflicting results concerning the effects of ethanol on γ-aminobutyric acid-A (GABA_A) receptor-mediated responses in the brain. To examine the variables that might explain these inconsistencies, the present study was designed to determine whether ethanol modulation of synaptically evoked GABA responses is brain region dependent, to identify factors that might regulate ethanol sensitivity, and to investigate the mechanism(s) underlying ethanol modulation of GABA responses. Whole-cell voltage clamp methods were used to examine the effects of ethanol on synaptically evoked GABA_A inhibitory postsynaptic currents (IPSCs) recorded from neurons in hippocampus, cerebral cortex, and intermediate lateral and medial septum from rat brain slice preparations. Bicuculline-sensitive IPSCs elicited by local stimulation were pharmacologically isolated by pretreatment with the glutamate specific antagonists, DL-(?)-2-amino-5-phosphonovaleric acid (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). Superfused ethanol (80 mM) potentiated evoked GABA_A IPSCs in cortical neurons and in intermediate lateral and medial septal neurons but not in CA1 hippocampal neurons. However, the mechanism by which ethanol enhanced GABA_A IPSC amplitudes differed between brain regions. In cortex, ethanol induced a hyperpolarizing shift in the GABA_A IPSC reversal potential (E_IPSC) without modifying the underlying GABA_A receptor-mediated conductance (G_IPSC). In contrast, ethanol enhanced GABA_A IPSC amplitudes in lateral and medial septal neurons by increasing the G_IPSC without modifying the E_IPSC These results suggest that ethanol differentially modulates responses to endogenous GABA released during synaptic activation and that important differences between various brain regions may reflect multiple mechanisms of ethanol action. © 1994 Wiley-Liss, Inc.     

γ-Aminobutyric acid type A/benzodiazepine receptors regulate rat retina neurosteroidogenesis

It has been previously shown that retinal ganglion cells have the ability to synthesize steroids including neuroactive steroids such as pregnenolone sulfate. Since ganglion cells possess GABA_A/benzodiazepine (BZ) receptors and neurosteroids modulate retinal GABA_A receptor function, we investigated the role of these receptors in isolated rat retina neurosteroidogenesis. Ligands for central-type BZ receptors stimulated retinal pregnenolone synthesis. Clonazepam was the most potent ligand examined acting at nanomolar concentrations. Moreover, the effective steroidogenesis stimulatory dose (ED₅₀) for these ligands and theK_i to inhibit [³-H]flunitrazepam binding showed a coefficient of correlation ofr = 0.87, suggesting the involvement of the central-type BZ receptors in this event. Ro 5-4864, which preferentially binds to peripheral-type BZ receptors, was less efficacious and potent whereas PK 11195 did not affect the basal pregnenolone formation and did not antagonize the Ro 5-4864 stimulated steroid synthesis. The GABAergic agonist muscimol, stimulated neurosteroid synthesis and this effect was reversed by the GABAergic antagonists bicuculline and picrotoxinin. In addition, these antagonists decreased basal pregnenolone formation, suggesting a tonic GABAergic control of the steroidogenic pathway, and reduced clonazepam-stimulated steroidogenesis. These results, together with the reported ability of neurosteroids to modulate GABA_A receptor function, suggest a novel regulatory mechanism to control the inhibitory transmission.     

Gender and age differences in expression of GABAA receptor subunits in rat somatosensory thalamus and cortex in an absence epilepsy model

Absence epilepsy is more prevalent in females, but reasons for this gender asymmetry are unknown. We reported previously that perinatal treatment of Long–Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 causes a life-long increase in EEG spike-wave discharges (SWDs), correlated with decreased expression of GABA_A receptor subunit γ2 protein levels in thalamic reticular and ventrobasal nuclei (SS thalamus) [Li, H., Kraus, A., Wu, J., Huguenard, J.R., Fisher, R.S., 2006. Selective changes in thalamic and cortical GABA(A) receptor subunits in a model of acquired absence epilepsy in the rat. Neuropharmacology 51, 121–128]. In this study, we explored time course and gender different effects of perinatal AY9944 treatment on expression of GABA_A receptor α1 and γ2 subunits in SS thalamus and SS cortex. Perinatal AY9944 treatment-induced decreases in GABA_A γ2 receptor subunits in rat SS thalamus and increases in SS cortex are gender and age specific. The findings suggest a mechanism for the higher prevalence of absence epilepsy in female patients.