Ontogeny of diazepam binding inhibitor/acyl-CoA binding protein mRNA and peripheral benzodiazepine receptor mRNA expression in the rat

The Diazepam Binding Inhibitor/Acyl-CoA Binding Protein (DBI/ACBP) has been implicated in different functions, as acyl-CoA transporter and as an endogenous ligand at the GABA_A receptor and the peripheral benzodiazepine receptor (PBR). The latter is thought to be involved in control of steroidogenesis. We studied the ontogeny of DBI/ACBP and PBR mRNA expression in embryos and offspring of time-pregnant Long Evans rats by in-situ hybridization with ³³P-endlabelled oligonucleotides. Both mRNAs were present in embryo and placenta at gestational day (G)11, the earliest stage studied. DBI/ACBP mRNA was strongly expressed from embryonic through mid-foetal stages in central nervous system (maximum in neuroepithelium), cranial and sympathetic ganglia, anterior pituitary, adrenal cortex, thyroid, thymus, liver and (late foetal) brown adipose tissue, moderately in testis, heart, lung and kidney. In brain, a late foetal decrease of DBI/ACBP mRNA was followed by an increase at postnatal day 6. Peripheral benzodiazepine receptor mRNA expression started very low and increased to moderate levels in adrenal cortex and medulla, testis, thyroid, brown adipose tissue, liver, heart, lung, salivary gland at mid- to late-foetal stages. Data suggest a significant role of DBI/ACBP at early developmental stages. Both proteins may be involved in the control of foetal steroidogenesis. However, differences in developmental patterns indicate that additional functions may be equally important during ontogeny, such as the involvement in lipid metabolism in the case of DBI/ACBP.     

Involvement of Neurosteroids in the Effect of the Endogenous Benzodiazepine Receptor Ligand Octadecaneuropeptide (ODN) on Gonadotropin-Releasing Hormone Gene Expression in Rat Brain

We have recently demonstrated that different activators of the GABA_A receptor complex including reduced progesterone metabolites and the endozepine octodecaneuropeptide (ODN) exert an inhibitory influence on GnRH gene expression. In order to investigate the possible involvement of neurosteroids, especially progesterone metabolites in the effect of ODN, we have evaluated in adrenalectomized and castrated male rats the influence of pretreatment with an inhibitor of 3 β -hydroxysteroid dehydrogenase (3 β -HSD) trilostane (TRIL) and an inhibitor of 5 α -reductase MK-906 on GnRH mRNA levels in ODN-treated rats. TRIL completely prevented the inhibitory influence of ODN on GnRH mRNA. It was also found that the inhibitor of 3 β -HSD as well as pregnenolone sulfate (PREG-S), which has been shown to be increased following TRIL treatment, could induce an increase in GnRH mRNA. MK-906 could also completely reverse the negative influence of ODN. When administered alone, this antagonist of 5 α -reductase induced an increase in GnRH mRNA. These results clearly indicate that the inhibition of two key enzymes for the synthesis of reduced progesterone metabolites can completely prevent the inhibitory influence of the endozepine ODN, suggesting that the effect of this endogenous ligand might be completely or partially mediated by an activation of the synthesis of active progesterone metabolites. On the other hand, it remains possible that, as a consequence of enzymatic inhibition, an increase in some precursors known as antagonists of GABA_A may play a role in the prevention of the ODN effect by the two enzyme antagonists.     

Regulation of cardiac sympathetic afferent reflex by GABA(A) and GABA(B) receptors in paraventricular nucleus in rats

The aim of the present study was to determine the role of GABA_A and GABA_B receptors in paraventricular nucleus (PVN) in regulating cardiac sympathetic afferent reflex (CSAR). Under urethane (800 mg/kg) and α-chloralose (40 mg/kg) anesthesia, renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded in sinoaortic-denervated and cervical-vagotomized rats. CSAR was evaluated based in the response of RSNA to epicardial application of capsaicin (0.3 nmol) or bradykinin (1 nmol). Bilateral PVN microinjection of the GABA_A receptor agonist isoguvacine (10 nmol) attenuated CSAR, while the GABA_B receptor agonist baclofen (1 nmol) abolished CSAR. Both isoguvacine and baclofen greatly decreased baseline RSNA and MAP. The GABA_A receptor antagonist gabazine (0.1 nmol) had no significant effect on CSAR, but the GABA_B receptor antagonist CGP-35348 (10 nmol) enhanced CSAR. Gabazine caused greater increases in baseline RSNA and MAP than CGP-35348. Vigabatrin (10 nmol), a selective GABA-transaminase inhibitor which increases endogenous GABA level, abolished CSAR, and decreased baseline RSNA, MAP and HR. The effects of vigabatrin were antagonized by combined gabazine (0.1 nmol) and CGP-35348 (10 nmol). The results indicate that activation of either GABA_A or GABA_B receptors in the PVN inhibits CSAR, while blockage of GABA_B receptors in the PVN enhances CSAR. Endogenous GABA in the PVN could have an important role in regulating CSAR.     

Regulation of proopiomelanocortin gene expression by endogenous ligands of the GABAA receptor complex as evaluated by in situ hybridization in the rat pars intermedia

The neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on proopiomelanocortin (POMC) neurons in the hypothalamus as well as on the melanotrope cells of the intermediate lobe (IL) of the pituitary gland. Moreover, the activation of the GABA_A receptor complex by different ligands has been shown to exert a negative influence on the POMC gene expression at the hypothalamic level. In order to elucidate the in vivo regulation of the POMC mRNA levels in the intermediate lobe of the pituitary by endogenous ligands of the GABA_A receptor complex, we have studied the effect of intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI). The possible involvement of neurosteroids in the action of ODN on melanotropic cells was evaluated following inhibition of two enzymes involved in the biosynthesis of neurosteroids known as activators of G3BA_A receptor complex: trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), and MK-906, an inhibitor of 5-reductase. The i.v. injection of ODN produced a dose-dependent inhibition of POMC gene expression in the IL. The i.c.v. injection of ODN also depressed POMC mRNA. These effects were completely reversed by the concomitant administration of the GABA_A antagonist picrotoxin. Similar results were obtained in POMC neurons in the arcuate nucleus (AN) of the hypothalamus. Trilostane administration induced an increase in POMC mRNA and also prevented the inhibitory influence of ODN. The neurosteroid pregnenolone-sulfate, a negative modulator of the GABA_A receptor, also stimulated POMC gene expression. On the other hand, MK-906 produced a decrease in mRNA levels and could not reverse the effect of ODN. The results indicate that activation of the GABA_A receptor complex by the endogenous benzodiazepine receptor ligand ODN can induce a negative regulation of POMC gene expression in the IL of the pituitary and neurons in the AN. The present results do not provide clear evidence that neurosteroids are involved in the action of ODN on POMC gene expression in the IL. ©1997 Elsevier Science B.V. All rights reserved     

GABA receptor proteins within lipid rafts in the AY-9944 model of atypical absence seizures

Purpose:   The inhibition of cholesterol synthesis with AY-9944 (AY) results in chronic recurrent atypical absence seizures in rodents. We hypothesized that cholesterol inhibition during the course of creating the AY model of atypical absence seizures results in an alteration of the entry of γ-aminobutyric acid (GABA)_A and GABA_B receptors into lipid rafts that contributes to epileptogenesis in this model.
Methods:   The cholesterol synthesis inhibitor AY (7.5 mg/kg) was administered on postnatal day (P) 2, P8, P14, and P20 in Long-Evans hooded rats. The incorporation of GABA_A and GABA_B receptor proteins into lipid rafts of the brain was then determined.
Results:   AY produced a shift of both GABA_A and GABA_B receptors in the examined detergent-resistant membranes (DRMs) and the soluble fractions. The percentage of the GABA_A and GABA_B receptors that shifted out of the DRMs varied between 17% and 50%, but the proportion of receptors in DRMs were decreased to levels around that of P5 animals or even lower. The shift observed in the AY-treated versus control animals was statistically significant (p < 0.01) for both GABA_A and GABA_B receptors.
Conclusion:   Cholesterol synthesis inhibition during rat brain development that is induced by AY leads to chronic atypical absence seizures and is associated with an alteration of GABA_A and GABA_B receptor proteins within lipid rafts. These data suggest a novel avenue of investigation into the epileptogenesis of experimental chronic atypical absence seizures.     

Hypoglycaemia-Induced Inhibition of Pulsatile Luteinizing Hormone Secretion in Female Rats: Role of Oestradiol, EndogenousOpioids and the Adrenal Medulla

Oestradiol (E₂) has been shown to exacerbate the inhibitory effect of hypoglycaemic stress on gonadotrophin-releasing hormone pulse generator (GnRH) activity in primates. The mechanism by which this is mediated is not yet known. We therefore aimed to establish whether there is a sensitizing influence of E₂ on the suppression of LH pulsatility in response to hypoglycaemia in the female rat, thus providing a more amenable model in which to study this phenomenon. In ovariectomized Wistar rats with E₂ replacement, insulin-induced hypoglycaemia (0.5  U/kg iv) resulted in an interruption of pulsatile LH secretion. Induction of the same degree of hypoglycaemia in ovariectomized rats without E₂ replacement was without effect on LH pulsatility. Naloxone administration prevented the hypoglycaemia-induced inhibition of LH pulses. Because hypoglycaemia is a potent activator of the sympathetic nervous system, we also tested the hypothesis that the adrenal medulla is involved in this suppression of LH pulses in the rat. Adrenomedullectomy completely prevented this inhibitory response to hypoglycaemic stress. These data are consistent with the hypothesis that E₂ sensitizes the GnRH pulse generator to the inhibitory influences of hypoglycaemic stress in the rat. Furthermore, a clear role for both endogenous opioid peptides and the adrenal medulla in the stress-induced suppression of LH pulsatility is identified.     

Differential Effects of Neuroactive Steroids on Somatostatin and Dopamine Secretion from Primary Hypothalamic Cell Cultures

This study investigated the effects of neuroactive steroids, which have been reported to modulate GABA-ergic transmission, on the secretion of somatostatin (SRIH) and also dopamine (DA) from primary rat hypothalamic cell cultures, where the release of both substances is regulated by a GABA_A receptor-mediated inhibitory tone. Pregnenolone sulphate (PS), a negative allosteric modulator at the GABA_A receptor, enhanced SRIH secretion in a time and dose-dependent manner (10⁻¹²–10⁻⁸  M). This effect was reversed by muscimol (10⁻⁸  M) and enhanced by bicuculline (10⁻⁶  M), thus supporting an action of PS at the GABA_A receptor. The release of endogenously synthesized dopamine (DA) was, however, unaffected by PS. A number of other steroids were also tested for their potential actions on SRIH and DA secretion. Allopregnanolone had slight but significant stimulatory actions on SRIH secretion, whereas tetrahydro-deoxycorticosterone (TH-DOC) markedly stimulated SRIH secretion with a bell-shaped dose response curve resembling that found for PS. The release of DA was unaffected by these neuroactive steroids but, unlike SRIH, DA release was stimulated by dehydroepiandrosterone sulphate (DHEAS). The results support the view that neuroactive steroids may play an important role in regulating some aspects of neuroendocrine function and they also provide the first demonstration of differential activities of neuroactive steroids within the hypothalamus at low, physiologically relevant concentrations. The results also raise the possibility that certain hypothalamic neuronal populations may posses uniquely different GABA_A receptors and that such mechanisms may contribute to the functional development of the neuroendocrine system.     

Developmental Patterns in the Regulation of Chloride Homeostasis and GABAA Receptor Signaling by Seizures

Summary:  GABA_A receptors have dual functions during development. They depolarize immature neurons but hyperpolarize more mature neurons. This functional switch has been attributed to age-related differences in the relative abundance of cation chloride cotransporters, such as KCC2 and NKCC1, which regulate chloride homeostasis. Certain insults, such as trauma, ischemia, and seizures, if they occur when GABA_Aergic signaling is hyperpolarizing, such as in the adult brain, can lead to reappearance of the immature, depolarizing synaptic responses to GABA_A receptor activation. In certain cases, this has been associated with either reduced expression of KCC2 or increase in NKCC1. In epilepsy, the depolarizing effects of GABA_A receptors have been proposed to be important for the acquisition and/or maintenance of the epileptic state. Using the kainic acid model of status epilepticus, we have studied the effects of repetitive neonatal episodes of status epilepticus on the expression of cation chloride cotransporter KCC2 in the neonatal hippocampus. In contrast to adults, seizures increased KCC2 mRNA expression in the CA3 region of the neonatal hippocampus. The contrasting patterns of regulation of KCC2 by seizures in mature and immature neurons may be one of the age-related factors that protect the neonatal brain against the development of epilepsy.     

Oxcarbazepine, not its active metabolite, potentiates GABAA activation and aggravates absence seizures

Purpose:   Studies in genetic absence epileptic rats from Strasbourg (GAERS) indicate that enhancement of γ aminobutyric acid (GABA_A) receptor activity is a critical mechanism in the aggravation of seizures by carbamazepine (CBZ). We examined whether structural analogs of CBZ, oxcarbazepine (OXC), and its active metabolite, monohydroxy derivative (MHD), also potentiate GABA_A receptor current and aggravate seizures.
Methods:   In vitro studies in Xenopus oocytes compared the three drugs' effect on GABA_A receptor currents. In vivo studies compared seizure activity in GAERS after intraperitoneal drug administration.
Results:   OXC potentiated GABA_A receptor current and aggravated seizures in GAERS, similarly to the effect of CBZ. Conversely, MHD showed only a minor potentiation of GABA_A receptor current and did not aggravate seizures.
Discussion:   A hydroxyl group at the C-10 position on the CBZ tricyclic structure in MHD reduces GABA_A receptor potentiation and seizure aggravation. Reports of the aggravation of absence seizures in patients taking OXC may result from circulating unmetabolized OXC rather than MHD.     

Dexamethasone Increases Growth Hormone (GH)-Releasing Hormone (GRH) Receptor mRNA Levels in Cultured Rat Anterior Pituitary Cells

To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24  h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5  nM after 6- and 24  h-incubations, and the maximal effect was found at 25  nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of β -actin and prolactin at 5  nM for 24  h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5  nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.     

Timing of the Developmental Switch in GABAA Mediated Signaling from Excitation to Inhibition in CA3 Rat Hippocampus Using Gramicidin Perforated Patch and Extracellular Recordings

Summary:  The timing of the developmental switch in the GABA_A mediated responses from excitatory to inhibitory was studied in Wistar rat CA3 hippocampal pyramidal cells using gramicidin perforated patch-clamp and extracellular recordings. Gramicidin perforated patch recordings revealed a gradual developmental shift in the reversal potential of synaptic and isoguvacine-induced GABA_A mediated responses from –55 ± 4 mV at postnatal days P0–2 to −74 ± 3 mV at P13–15 with a midpoint of disappearance of the excitatory effects of GABA at around P8. Extracellular recordings in CA3 pyramidal cell layer revealed that the effect of isoguvacine on multiple unit activity (MUA) switched from an increase to a decrease at around P10. The effect of synaptic GABA_A mediated responses on MUA switched from an increase to a decrease at around P8. It is concluded that the developmental switch in the action of GABA via GABA_A receptors from excitatory to inhibitory occurs in Wistar rat CA3 pyramidal cells at around P8–10, an age that coincides with the transition from immature to mature hippocampal rhythms. We propose that excitatory GABA contributes to enhanced excitability and ictogenesis in the neonatal rat hippocampus.     

Role of GABAA receptors in the physiology and pharmacology of sleep

Most sedative-hypnotics used in insomnia treatment target the γ-aminobutyric acid (GABA)_A receptors. A vast repertoire of GABA_A receptor subtypes has been identified and displays specific electrophysiological and functional properties. GABA_A-mediated inhibition traditionally refers to 'phasic' inhibition, arising from synaptic GABA_A receptors which transiently inhibit neurons. However, there is growing evidence that peri- or extra-synaptic GABA_A receptors are continuously activated by low GABA concentrations and mediate a 'tonic' conductance. This slower type of signaling appears to play a key role in controlling cell excitability. This review aims at summarizing recent knowledge on GABA transmission, including the emergence of tonic conductance, and highlighting the importance of GABA_A receptor heterogeneity. The mechanism of action of sedative-hypnotic drugs and their effects on sleep and the electroencephalogram will be reported. Furthermore, studies using genetically engineered mice will be emphasized, providing insights into the role of GABA_A receptors in mechanisms underlying physiological and pharmacological sleep. Finally, we will address the potential of GABA_A receptor pharmacology for the treatment of insomnia.     

Activation of the GABAA Receptor Inhibits the Proliferative Effects of bFGF in Cortical Progenitor Cells

Recent studies have localized γ-aminobutyric acid (GABA)-containing neurons and identified cells that express subunits of the GABA_A receptor in the proliferative zone of the developing cerebral cortex and have demonstrated a role for GABA in cortical neurogenesis. We examined here the interactions between a number of neurotrophic factors, known to be involved in cortical cell proliferation and differentiation, and the GABAergic system (GABA and GABA_A receptors) in the regulation of cell production in dissociated cortical cell cultures. We found that basic fibroblast growth factor (bFGF) increased the number of cells labelled for the α₁ subunit of the GABA_A receptor but not for the α₂, α₃ or α₅ subunits. The α₁ subunit was expressed by the majority of proliferating neuroepithelial cells as well as by differentiated neurons. We also found that activation of the GABA_A receptor by GABA or muscimol inhibited the proliferative effects of bFGF on cortical progenitors, leading to an increased number of differentiated neurons. These results suggest that bFGF stimulates cell proliferation and GABA_A receptor expression in cultured progenitor cells of the developing neocortex, and that GABA regulates cell production by providing a feedback signal that terminates cell division.     

Regulation of Activin Type-II Receptor mRNA Levels in Rat Hypothalamus by Estradiol in vivo

A solution-hybridization S1-nuclease protection assay was used to evaluate the expression of messenger RNAs for the activin β _A subunit and type II activin receptor in adult rat brain. Results indicate the presence of β _A subunit mRNA in both hypothalamus and brainstem, with approximately two-fold higher levels in brainstem. Levels of activin type II receptor mRNA were similar in the hypothalamus of young virgin and 15-day lactating females, and in females in which pups were removed after a 5-day lactation period. Male rats castrated prepubertally (30  days p.n.) had approximately 220% higher ( P <0.05) hypothalamic activin type II receptor mRNA levels than postpubertal, 3-month old age-matched sham controls. Two month treatment of castrate rats with estradiol (200  ng/g, i.p. every 2 days) reduced hypothalamic activin type II receptor mRNA expression to control levels; the same dose of testosterone had no effect. The expression of the hypothalamic activin type II receptor gene may be estrogen-regulated in vivo .     

Synchronous GABAA-receptor-dependent potentials in limbic areas of the in-vitro isolated adult guinea pig brain

Epileptiform discharges are known to reflect the hypersynchronous glutamatergic activation of cortical neurons. However, experimental evidence has revealed that epileptiform synchronization is also contributed to by population events mediated by GABA_A receptors. Here, we analysed the spatial distribution of GABA_A-receptor-dependent interictal events in the hippocampal/parahippocampal region of the adult guinea pig brain isolated in vitro . We found that arterial perfusion of this preparation with 4-aminopyridine caused the appearance of glutamatergic-independent interictal potentials that were reversibly abolished by GABA_A receptor antagonism. Laminar profiles and current source density analysis performed in different limbic areas demonstrated that these GABA_A-receptor-mediated events were independently generated in different areas of the hippocampal/parahippocampal formation (most often in the medial entorhinal cortex) and propagated between interconnected limbic structures of both hemispheres. Finally, intracellular recordings from principal neurons of the medial entorhinal cortex demonstrated that the GABAergic field potential correlated to inhibitory postsynaptic potentials (membrane potential reversal, −68.12 ± 8.01 mV, n  = 5) that were interrupted by ectopic spiking. Our findings demonstrate that, in an acute seizure model developed in the adult guinea pig brain, hypersynchronous GABA_A-receptor-mediated interictal events are generated from independent sources and propagate within limbic cortices in the absence of excitatory synaptic transmission. As spared or enhanced inhibition was reported in models of epilepsy, our data may support a role of GABA-mediated signaling in ictogenesis and epileptogenesis.     

Role of GABAA receptors in rat hindbrain nuclei controlling gastric motor function

It has been shown in cats that gastric motor control by the dorsal vagal complex and nucleus ambiguus is under a tonic GABAergic influence. Since much more work has been performed in rats to define vago-vagal reflexes controlling gastrointestinal function, an understanding of the potential inhibition by candidate neurotransmitters such as GABA (gamma aminobutyric acid) in the rat dorsal vagal complex (DVC) is essential to assess. Multiple-barrelled micropipettes were used to apply to the dorsal vagal complex the GABA_A antagonist, bicuculline methiodide (0.1–1 nmol), and a GABA_A agonist, muscimol (10 nmol) prior to micro-injection of the GABA_A antagonist. Micro-injections of bicuculline (353 pmol and 1 nmol), which were localized primarily in the dorsal motor nucleus of the vagus, produced significant increases in intragastric pressure and pyloric motility. These responses were abolished by vagotomy and by a prior micro-injection of muscimol. To determine whether GABAergic blockade in the dorsal vagal complex results in gastric motor excitation through excitatory amino acid receptors, kynurenic acid (5 nmol), a kainate/NMDA (N-methyl D -aspartic acid) receptor antagonist, was micro-injected prior to bicuculline. This abolished the increase in gastric motor function normally evoked by bicuculline. In the other two important hindbrain nuclei controlling gastric function, the nucleus raphe obscurus and nucleus ambiguus, bicuculline (353 pmol) significantly increased intragastric pressure via vagally mediated pathways. These data demonstrate that all three rat hindbrain nuclei known to influence gastric function via the vagus nerve are under tonic GABAergic control. In addition, in the dorsal vagal complex, relief from GABAergic inhibition results in increases in gastric motor function through kainate/NMDA receptor-mediated excitation.     

Prostaglandin E2 Acts via the Hypothalamus to Stimulate ACTH Secretion in the Fetal Sheep

Prostaglandin (PG) E₂ is secreted into the fetal circulation during late gestation in the sheep. Exogenous infusion of PGE₂ is associated with robust increases in the circulating concentrations of ACTH and cortisol and it has therefore been proposed that endogenous PGE₂ modulates the activity of the fetal HPA axis, which is the primary determinant of parturition and essential for the maturation of vital organ systems. The sites of action of PGE₂ within the HPA axis have not been clearly established. We have compared the effects of PGE₂ infusion on ACTH and cortisol concentrations in intact fetuses and in those whose pituitaries were surgically disconnected from hypothalamic control (hypothalamo-pituitary disconnected; HPD fetuses operated at 111–113 days gestational age). The effect of advancing gestational age on these responses was investigated by infusing PGE₂ at 121, 131, 141 and 148 days of gestation. The functional integrity of the pituitary corticotrophs was tested by injecting CRH (1  μg) into intact and HPD fetuses at day 125. This test was repeated for the HPD fetuses at 160 days.The responsiveness of the adrenal glands was also tested by injecting 2.5  μg/kg synthetic ACTH_1–24 (Synacthen) into both groups of fetuses at day 135, with the HPD group retested at day 155.