Replacement with GABAergic steroid precursors restores the acute ethanol withdrawal profile in adrenalectomy/gonadectomy mice

The neurosteroid allopregnanolone (ALLO) is a progesterone metabolite that is one of a family of neuroactive steroids (NAS) that are potent positive allosteric modulators of γ-aminobutyric acid_A (GABA_A) receptors. These GABAergic NAS are produced peripherally (in the adrenals and gonads) and centrally in the brain. Peripherally produced NAS modulate some effects of ethanol intoxication (e.g., anxiolytic, antidepressant, and anticonvulsant effects) in rodents. We have found that NAS also may be involved in the rebound neural hyperexcitability following a high ethanol dose. Removal of the adrenals and gonads (ADX/GDX) increased withdrawal severity following 4 g/kg ethanol, as measured by handling-induced convulsions (HICs) in male and female DBA/2J mice. NAS are produced through the metabolism of progesterone (PROG), deoxycorticosterone (DOC), or testosterone, which can be blocked with the administration of finasteride (FIN), a 5α-reductase enzyme inhibitor. The current investigation was undertaken to clarify the step(s) in the biosynthetic NAS pathway that were sufficient to restore the acute ethanol withdrawal profile in ADX/GDX mice to that seen in intact animals. Male and female DBA/2J mice underwent ADX/GDX or SHAM surgery. After recovery, separate groups of animals were administered PROG, DOC, PROG+FIN, DOC+FIN, FIN, ALLO, ganaxalone (a synthetic ALLO derivative), corticosterone, or vehicle. Animals were then administered a 4 g/kg ethanol dose and allowed to undergo withdrawal. HICs were measured for 12 h and again at 24 h. The results indicate that replacement with PROG and DOC restored the withdrawal profile in ADX/GDX animals to SHAM levels, and that this effect was blocked with co-administration of FIN. Administration of FIN alone increased the withdrawal profile in both SHAM and ADX/GDX males. These findings indicate that the increase in acute withdrawal severity after ADX/GDX may be due to the loss of GABAergic NAS, providing insight into the contribution of endogenous GABAergic NAS to ethanol withdrawal severity.     

Neurosteroid modulation of allopregnanolone and GABA effect on the GABA-A receptor

The neurosteroid allopregnanolone (ALLO) or 3α-OH-5α-pregnane-20-one interacts with the GABA type A receptor chloride ion channel complex and enhances the effect of GABA. Animal and human studies suggest that ALLO plays an important role in several disorders including premenstrual syndrome, anxiety, and memory impairment. In contrast to ALLO, steroids with a hydroxy group in the 3β position usually exert a reducing effect and have recently attracted interest due to their suggested role in counteracting the negative action of ALLO. In this study, five different 3β-steroids were tested for their ability to modulate GABA-mediated chloride ion uptake in the absence and presence of ALLO in rat brain microsacs preparations. In addition, the effects of the 3β-steroids and their interaction with ALLO were investigated by patch-clamp recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) in rat hypothalamic neurons from the medial preoptic nucleus (MPN). All tested 3β-steroids reduced the ALLO-enhanced GABA response in cerebral cortex, in hippocampus and in MPN. In cerebellum, only one had this effect. However, in the absence of ALLO, two of the 3β-steroids potentiated GABA-evoked chloride ion uptake and prolonged the sIPSCs decay time, whereas the others had little or no effect. Therefore, it is possible that at least some 3β-steroids can act as positive GABA_A receptor modulators as well as negative modulators depending on whether or not ALLO is present. Finally, these results suggest that the 3β-steroids could be of interest as pharmacological agents that could counteract the negative effects of ALLO.     

Inflammatory mediators potentiate high affinity GABAA currents in rat dorsal root ganglion neurons

Following acute tissue injury action potentials may be initiated in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery, a process referred to as a dorsal root reflex (DRR). The DRR is dependent on the activation of GABA_A receptors. The prevailing hypothesis is that DRR is due to a depolarizing shift in the chloride equilibrium potential (E_Cl) following an injury-induced activation of the Na⁺–K⁺–Cl⁻-cotransporter. Because inflammatory mediators (IM), such as prostaglandin E₂ are also released in the spinal cord following tissue injury, as well as evidence that E_Cl is already depolarized in primary afferents, an alternative hypothesis is that an IM-induced increase in GABA_A receptor mediated current (I_GABA) could underlie the injury-induced increase in DRR. To test this hypothesis, we explored the impact of IM (prostaglandin E₂ (1 μM), bradykinin (10 μM), and histamine (1 μM)) on I_GABA in dissociated rat dorsal root ganglion (DRG) neurons with standard whole cell patch clamp techniques. IM potentiated I_GABA in a subpopulation of medium to large diameter capsaicin insensitive DRG neurons. This effect was dependent on the concentration of GABA, manifest only at low concentrations (<10 μM). THIP evoked current were also potentiated by IM and GABA (1 μM) induced tonic currents enhanced by IM were resistant to gabazine (20 μM). The present data are consistent with the hypothesis that an acute increase in I_GABA contributes to the emergence of injury-induced DRR.     

GABAA receptor inhibition does not affect mGluR-dependent LTD at hippocampal Schaffer collateral-CA1 synapses

Hippocampal synaptic plasticity between Schaffer collaterals and CA1 pyramidal neurons can be induced by activation of N-methyl-d-aspartate receptors (NMDARs) or of metabotropic glutamate receptors (mGluRs). Inhibitory GABAergic interneurons in this region abundantly terminate on pyramidal neurons and may thus influence synaptic plasticity. Although NMDAR-dependent synaptic plasticity is known to be influenced by inhibitory interneurons, little is known about the role of GABA on mGluR-dependent plasticity. Here, we used field potential recordings of the Schaffer collateral-CA1 synapses in rat hippocampal slices in order to study the effect of GABA_A receptor (GABA_AR) inhibition on mGluR-dependent long-term depression (LTD). Without GABA_AR blockade, mGluR-dependent LTD was induced pharmacologically by the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG, 100 μM, 10 min) as well as electrically by paired-pulse low-frequency stimulation (PP-LFS, 900 paired pulses at 1 Hz) resulting in a stable depression of the field response lasting at least 80 min after LTD induction. The GABA_AR antagonist gabazine (5 μM) itself caused an increase of field responses suggesting an endogenous GABA release inhibiting CA1 field potentials. However, when either DHPG or PP-LFS was applied during GABA_AR inhibition, the field responses were significantly reduced. Moreover, normalizing these responses to experiments without GABA_AR blockade, there was no significant effect of gabazine on both DHPG- and PP-LFS-induced LTD. Thus, our results show that mGluR-dependent LTD at Schaffer collateral-CA1 synapses is unaffected by GABA_AR mediated synaptic transmission.     

Vesicular GABA release delays the onset of the Purkinje cell terminal depolarization without affecting tissue swelling in cerebellar slices during simulated ischemia

Neurosteroids that can enhance GABA_A receptor sensitivity protect cerebellar Purkinje cells against transient episodes of global brain ischemia, but little is known about how ischemia affects GABAergic transmission onto Purkinje cells. Here we use patch-clamp recording from Purkinje cells in acutely prepared slices of rat cerebellum to determine how ischemia affects GABAergic signaling to Purkinje cells. In voltage-clamped Purkinje cells, exposing slices to solutions designed to simulate brain ischemia caused an early, partial suppression of the frequency of spontaneous inhibitory post synaptic currents (sIPSCs), but after 5–8 min GABA accumulated in the extracellular space around Purkinje cells, generating a large (∼17 nS), sustained GABA_A receptor-mediated conductance. The sustained GABA_A conductance occurred in parallel with an even larger (∼117 nS) glutamate receptor-mediated conductance, but blocking GABA_A receptors did not affect the timing or magnitude of the glutamate conductance, and blocking glutamate receptors did not affect the timing or magnitude of the GABA_A conductance. Despite the lack of interaction between GABA and glutamate, blocking GABA_A receptors significantly accelerated the onset of the Purkinje cell “ischemic” depolarization (ID), as assessed with current-clamp recordings from Purkinje cells or field potential recordings in the dendritic field of the Purkinje cells. The Purkinje cell ID occurred ∼2 min prior to the sustained glutamate release under control conditions and a further 1–2 min earlier when GABA_A receptors were blocked. Tissue swelling, as assessed by monitoring light transmittance through the slice, peaked just after the ID, prior to the sustained glutamate release, but was not affected by blocking GABA_A receptors. These data indicate that ischemia induces the Purkinje cell ID and tissue swelling prior to the sustained glutamate release, and that blocking GABA_A receptors accelerates the onset of the ID without affecting tissue swelling. Taken together these data may explain why Purkinje cells are one of the most ischemia sensitive neurons in the brain despite lacking NMDA receptors, and why neurosteroids that enhance GABA_A receptor function protect Purkinje cells against transient episodes of global brain ischemia.     

Enhanced activity of GABA receptors inhibits glutamate release induced by focal cerebral ischemia in rat striatum

Cerebral ischemia causes an excess release of glutamate, which can injure neurons. The striatum is one of the important regions vulnerable to hypoxia and ischemia. Using push–pull perfusion technique, we investigated the regulatory role of γ-aminobutyric acid (GABA) and its receptors in modifying the amount of glutamate in rat striatum with ischemia. Perfusion with exogenous GABA (1 mM) inhibited cerebral ischemia-induced glutamate release by as much as 47%. We further characterized relative roles of subtype receptors of GABA on glutamate release by using pharmacological tools. While baclofen (500 μM), a GABA_B receptor agonist, suppressed ischemia-induced glutamate release by 52%, GABA_B receptor antagonist saclofen (500 μM) failed to produce a significant increase of glutamate release. The GABA_A receptor agonist muscimol (500 μM) also reduced by 38% the release of glutamate induced by cerebral ischemia but the GABA_A receptor antagonist bicuculline (500 μM) had very little effect. The present study demonstrates that the excessive release of glutamate or the overly activated glutamate receptor, triggered by cerebral ischemia, can be down-regulated by exogenous GABA or by increased activity of GABA receptors, especially the presynaptic GABA_B receptors, which might be one of the important mechanisms to protect against striatum neuronal damage from over stimulation by excessive glutamate during ischemia.     

Tonic GABA inhibition in hippocampal dentate granule cells: its regulation and function in temporal lobe epilepsies

Both human and experimental evidence strongly supports the view of brain region‐ and cell‐specific changes in tonic GABA inhibition in temporal lobe epilepsies (TLE). This ‘tonic’ form of signalling is not time‐locked to presynaptic action potentials, which depends upon detection of ambient GABA by extrasynaptic GABA_A receptors (GABA_ARs). Extrasynaptic GABA_ARs have distinct physiological and pharmacological features, including high GABA‐binding affinity and low desensitization and a variety of the specific subunit combinations (α₄δ‐,α₆δ‐,α₅γ‐,ε‐containing receptors). These features closely contribute to the function of tonic GABA current, which is preserved properly or increased in dentate gyrus in models of TLE, even in the face of a loss of synaptic inhibition and inhibitory interneurones. Markedly reduced tonic GABA inhibition may facilitate an episode of epilepsy, while persistent elevated tonic inhibition may contribute to the onset of spontaneous recurrent seizures. In dentate granule cells, tonic GABA inhibition is positively modulated by endogenous neurosteroids and other factors, which undergo changes related to hormonal status after TLE. Tonic inhibition regulates neuronal excitability through its effects on membrane potential by both offsetting the threshold and reducing the frequency of action potentials and input resistance. Therefore, extrasynaptic GABA_ARs are expected to be the most important pharmacological targets in TLE. It is likely that both elevate the ambient GABA concentration and potentiate the tonic currents, contributing to the antiepileptic effects.     

Melatonin potentiates the GABAA receptor-mediated current in cultured chick spinal cord neurons

The effect of melatonin on the γ-aminobutyric acid_A (GABA_A) receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Melatonin rapidly and reversibly potentiated the GABA-induced current in a dose-dependent fashion, with an EC₅₀ of 766 μM and a maximal potentiation of 148%. Potentiation of the GABA response by melatonin was mediated by increasing the potency of GABA rather than the efficacy. Prolonged exposure to a saturating concentration of the disulfide-reducing agent dithiothreitol did not attentuate the effect of melatonin on the GABA response, indicating that melatonin does not act through the redox site. Furthermore, our results demonstrate that melatonin and 5α-pregnan-3α-ol-20-one (a positive steroid modulator of the GABA_A receptor) act through different sites.     

Effect of saikosaponin A on maintenance of intravenous morphine self-administration

In this study, we investigated the effects of saikosaponin A (SSA), a major compound of Bupleurum falcatum L., on morphine self-administration behavior. Male Sprague–Dawley rats were trained to self-administer intravenous morphine (0.1 mg/kg per injection over 5 s) during daily 1-h sessions under a fixed-ratio 1 schedule. Rats were pretreated with SSA (0.25, 0.5, 1.0 mg/kg) by intraperitoneal injection 30 min prior to the start of the test session. Results demonstrated that pretreatment with SSA reduced morphine-maintained responding dose-dependently. Additionally, SSA inhibition of morphine-reinforced behavior was blocked by the selective GABA_B receptor antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911), but not the selective GABA_A receptor antagonist bicuculline. Together, these results suggest that SSA may effectively suppress morphine-reinforced behavior by activating GABA_B receptors.     

Neuroprotection of ethanol against ischemia/reperfusion-induced brain injury through decreasing c-Jun N-terminal kinase 3 (JNK3) activation by enhancing GABA release

Our latest study indicated that ethanol could attenuate cerebral ischemia/reperfusion-induced brain injury through activating Ionotropic glutamate receptors Kainate Family (Gluk1)–kainate (KA) receptors and gamma-aminobutyric acid (GABA) receptors. However, the possible mechanism of the neuroprotective effects of ethanol remains unclear. In this study we report that ethanol shows neuroprotective effects against ischemic brain injury through enhancing GABA release and then decreasing c-Jun N-terminal kinase 3 (JNK3) activation. Electrophysiologic recording indicated that ethanol enhances GABA release from presynaptic neurons and the released GABA subsequently inhibits the KA receptor–mediated whole-cell currents. Moreover, our data show that ethanol can inhibit the increased assembly of the Gluk2–PSD-95–MLK3 (postsynaptic density protein-95, PSD-95 and mixed-lineage kinase 3, MLK3) module induced by cerebral ischemia and the activation of the MLK3-MKK4/7-JNK (mitogen-activated protein kinase kinase 4/7, MKK4/7) cascade. Pretreatment of the GABA_A receptor antagonist bicuculline and antagonist of VGCC (a broad-spectrum blocker of the voltage-gated calcium channel [VGCC]) Chromic (CdCl₂) can demolish the neuroprotective effects of ethanol. The results suggest that during ischemia-reperfusion, ethanol may activate presynaptic Gluk1-KA and facilitate Ca²⁺-dependent GABA release. The released GABA activates postsynaptic GABA_A receptors, which suppress the ischemic depolarization and decrease the association of signaling module Gluk2–PSD-95–MLK3 induced by the activation of postsynaptic Gluk2-KA receptors. There is a raised possibility that ethanol inhibiting the JNK3 apoptotic pathway (MLK3/MKK4/7/JNK3/c-Jun/Fas-L) performs a neuroprotective function against ischemic brain injury.     

Neurosteroid modulation of GABAergic neurotransmission in the central amygdala: a role for NMDA receptors

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone or ALLO) positively modulates GABA(A) receptors, an action that may contribute to the anxiolytic effects of ALLO. Recent evidence suggests that ALLO's anxiolytic effects appear to be mediated by the amygdala, a key neural structure for emotional and cognitive behaviors. However, little is known regarding ALLO effects on amygdala physiology. We therefore explored ALLO effects on GABA neurotransmission in the central nucleus (Ce) of the amygdala, a major output nucleus involved in fear and anxiety. We recorded evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in Ce neurons using whole-cell patch clamp techniques. We observed that ALLO significantly reduced the amplitude of evoked GABA(A) receptor-mediated IPSCs. However, the effect of ALLO was occluded by the NMDA receptor antagonist D-APV. D-APV alone also reduced evoked IPSCs in Ce neurons. These results suggest that ALLO-induced reduction of GABAergic transmission in Ce appears to depend on neural network activity, possibly involving an NMDA receptor-mediated mechanism. These ALLO effects on GABAergic transmission in the central amygdala may play a role in mediating its anxiolytic actions.     

The role of GABAergic neuron on NMDA- and SP-induced phase delays in the suprachiasmatic nucleus neuronal activity rhythm in vitro

Gamma-aminobutyric acid (GABA), and its biosynthetic enzyme, glutamic decarboxylase, are widely distributed in the suprachiasmatic nucleus (SCN). In the present study, we examined the role of the GABA_A receptor on in vitro SCN responses to photic-like signals. We found that 100 μM GABA_A receptor antagonist bicuculline partially blocked field potentials evoked by optic nerve stimulation. NMDA- and SP-induced phase shifts of SCN neuronal activity rhythms, were blocked with 10 μM bicuculline. Application of 100 μM bicuculline alone induced phase advance of SCN neuronal activity rhythm. These results show that NMDA- and SP-induced phase shifts are blocked by bicuculline and suggest GABA has an important role as neurotransmitter in the neuronal network regulating phase shifts of the circadian clock.     

Multiple roles for the first transmembrane domain of GABAA receptor subunits in neurosteroid modulation and spontaneous channel activity

Neurosteroids exert potent physiological effects by allosterically modulating synaptic and extrasynaptic GABA_A receptors. Some endogenous neurosteroids, such as 3α, 21-dihydroxy-5β-pregnan-20-one (5α, 3α-THDOC), potentiate GABA_A receptor function by interacting with a binding pocket defined by conserved residues in the first and fourth transmembrane (TM) domains of α subunits. Others, such as pregnenolone sulfate (PS), inhibit GABA_A receptor function through as-yet unidentified binding sites. Here we investigate the mechanisms of PS inhibition of mammalian GABA_A receptors, based on studies of PS inhibition of the UNC-49 GABA receptor, a GABA_A-like receptor from Caenorhabditis elegans. In UNC-49, a 19 residue segment of TM1 can be mutated to increase or decrease PS sensitivity over a 20-fold range. Surprisingly, substituting these UNC-49 sequences into mammalian α₁, β₂, and γ₂ subunits did not produce the corresponding effects on PS sensitivity of the resulting chimeric receptors. Therefore, it is unlikely that a conserved PS binding pocket is formed at this site. However we observed several interesting unexpected effects. First, chimeric γ2 subunits caused increased efficacy of 5α, 3α-THDOC potentiation; second, spontaneous gating of α₆β₂δ receptors was blocked by PS, and reduced by chimeric β₂ subunits; and third, direct activation of α₆β₂δ receptors by 5α, 3α-THDOC was reduced by chimeric β₂ subunits. These results reveal novel roles for non-α subunits in neurosteroid modulation and direct activation, and show that the β subunit TM1 domain is important for spontaneous activity of extrasynaptic GABA_A receptors.     

Longer term progesterone treatment induces changes of GABAA receptor levels in forebrain sites in the female hamster: quantitative autoradiography study

Summary Quantitative receptor autoradiography was applied to evaluate the effects of one and three injections of 1 mg progesterone (P) on ³H muscimol binding levels in the different forebrain areas of the female hamster. The overall effect of P resulted in substantial increases in ³H muscimol binding in brain areas containing gonadal steroid receptors: medial preoptic area and ventromedial hypothalamic nucleus as well as in bed nucleus stria terminalis and subiculum. Similarly, the caudate putamen, a region where gonadal steroid receptors are not abundant, also showed substantial increases of ³H muscimol binding receptor levels. Moreover, female hamsters treated with P for 3 days presented altered ³H muscimol binding levels in the amygdala and thalamic nucleus that were, in some cases, not produced by one dose of P. P treatment also decreased GABA_A binding in two areas of the thalamus. These results are consistent with the proposal that P may alter GABAergic inhibitory activity via changes in the levels of GABA_A receptors in certain forebrain areas in the female hamster, changes which may be linked to the mediation of anxiolytic effects and to the inhibition of aggressive behavior. These data also suggest that P treatment increases the binding of high affinity GABA receptors in some forebrain sites and may be responsible for maintenance of the anxiolytic effects.     

Role for GABA agonists in the nucleus accumbens in regulating morphine self-administration

In the present study, functional roles of GABA receptors in the nucleus accumbens on morphine self-administration behavior were investigated. Male Sprague–Dawley rats were trained to press lever for morphine (0.1 mg/kg per infusion) during daily 1-h self-administration session. After establishing stable baseline responses, rats were given microinjections of the GABA_A receptor agonist muscimol (0, 250 and 500 ng/μl, bilateral) or the GABA_B receptor agonist baclofen (0, 100 and 250 ng/μl, bilateral) into the nucleus accumbens immediately before the morphine self-administration. Microinjection of muscimol (250 and 500 ng/μl) into the nucleus accumbens, but not baclofen, decreased morphine self-administration responses. These results suggest that activation of GABA_A receptors, but not GABA_B receptors, in the nucleus accumbens plays a critical role in modulating the reinforcing effects of morphine.     

The behavioural and neuronal effects of the chronic administration of benzodiazepine anxiolytic and hypnotic drugs

Benzodiazepine anxiolytic and hypnotic drugs are some of the most widely prescribed drugs in the Western world. Despite this fact, the mechanisms that underlie the development of tolerance to, and dependence upon, benzodiazepines are poorly understood. The aim of this review is to summarize and critically evaluate the experimental evidence relating to the chronic behavioural and neuronal effects of benzodiazepines. Behavioural studies in animals generally indicate that tolerance gradually develops to the muscle relaxant, ataxic, locomotor and anticonvulsant effects of benzodiazepines. The evidence relating to the development of tolerance to the anxiolytic effects of benzodiazepines is less clear. The literature on the possible mechanisms of benzodiazepine tolerance and dependence is large, highly complex and difficult to interpret. The effect of chronic benzodiazepine treatment varies enormously as a function of the benzodiazepine used and the treatment schedule employed. Many studies have demonstrated a down-regulation of benzodiazepine binding sites, although affinity is usually unchanged. The evidence relating to the number and affinity of GABA_A binding sites is unclear. Some studies suggest that chronic benzodiazepine administration results in a reduction in the number of C⁻ channels associated with the GABA_A receptor complex, although it is not clear that the efficacy of the GABA binding site in operating the Cl⁻ channel necessarily changes. There is, however, substantial evidence to support the hypothesis that chronic benzodiazepine treatment results in a reduction in the coupling between the GABA_A and benzodiazepine binding sites (the “functional uncoupling hypothesis”). Although some electrophysiological studies suggest that chronic benzodiazepine treatment results in a subsensitivity to GABA, this effect seems to be highly area-specific.     

Antinociceptive effects of the neuroactive steroid, 3α-hydroxy-5α-pregnan-20-one and progesterone in the land snail,Cepaea nemoralis

Results of investigations with vertebrates have implicated neuroactive steroids and in particular 5α-reduced metabolites of progesterone such as 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP/3A5P and originally allopregnanolone) in the rapid modulation of diverse functions including that of nociceptive sensitivity. These effects have been indicated to involve modulation of GABA receptors. Results of recent phylogenetic studies have revealed the presence of GABA receptors in invertebrates that may also be subject to modulation by steroids and neuroactive steroids. The present study examined the effects of the neuroactive steroid, 3α-hydroxy-5α-pregnan-20-one, as well as progesterone on aversive thermal (nociceptive) responses in a mollusc, the land snail, Cepaea nemoralis. 3α-Hydroxy-5α-pregnan-20-one had significant dose-related (0.01–1.0 μg) antinociceptive effects in Cepaea increasing the latency of response to a 40°C surface, with maximum effects being evident 15–30 min after administration. These effects of 3α-hydroxy-5α-pregnan-20-one were stereospecific, with the stereoisomer 3β-hydroxy-5α-pregnan-20-one (3B5P) failing to affect nociceptive responses. Progesterone also had significant dose-related (0.10–10 μg) antinociceptive effects that, however, were delayed in onset and relatively prolonged (60–120 min), suggestive of the formation of active metabolites. The presence of endogenous progesterone (12.36±0.17 ng/g tissue) was ascertained by a radioimmunoassay further supporting a functional role for steroids in Cepaea. The antinociceptive effects of 3α-hydroxy-5α-pregnan-20-one and progesterone were blocked by the GABA antagonists, bicuculline and picrotoxin, while being relatively insensitive to opioid and N-methyl-d-aspartate antagonists.These results suggest an early evolutionary development and phylogenetic continuity of neuroactive steroid and GABA involvement in the mediation of nociception.     

Activation of the dorsal vagal nucleus increases pancreatic exocrine secretion in the rat

Pancreatic secretion is regulated by the dorsal vagal nucleus (DVN) which is modulated by several neurotransmitters and diverse synaptic inputs. The inhibitory neurotransmitter GABA is a major modulator of the vagal output to the gastrointestinal tract. The present study investigated the effects of GABA_A receptor blockade in the DVN, using bicuculline methiodide (BIM, GABA_A receptor antagonist, 100 pmol/25 nl), on pancreatic exocrine secretion (PES). Male Sprague–Dawley rats anaesthetised with isoflurane were used in all experiments. PES was collected from the common bile-pancreatic duct and was used to determine the pancreatic protein output (PPO). PES and PPO were measured prior to, and after, microinjection of BIM into the DVN. Bilateral microinjection of BIM into the DVN significantly increased PES and PPO from 23.4 ± 3.2 μl/h to 66.1 ± 17.5 μl/h and 19.3 ± 1.7 μg/h to 35.7 ± 3.0 μg/h (P < 0.05), respectively. Atropine methonitrate (100 μg/(kg min), i.v.) blocked the excitatory effect of BIM microinjection on PES and PPO. These results suggest that activation of DVN neurons stimulates pancreatic secretion via a cholinergic muscarinic mechanism.     

GABAA receptors involved in sleep and anaesthesia: β1- versus β3-containing assemblies

The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus—TMN) control wakefulness, and their silencing through activation of GABA_A receptors (GABA_AR) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the β1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA_AR in TMN neurons. In recombinant receptors containing the β3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and β3N265M mice, FDD and propofol, we explored the relative contributions of β1- and β3-containing GABA_AR to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In β3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA_AR by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed β1-type pharmacology, with the same effects achieved by 3 μM propofol and 10 μM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 μM, and in all cells at 10 and 250 μM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1–6 μM) or significantly reduced (12 μM) in β3N265M mice, whereas gating and modulation of GABA_AR by PI24513 as well as modulation by propofol were unchanged. In conclusion, β1-containing (FDD-sensitive) GABA_AR represent the major receptor pool in TMN neurons responding to GABA, while β3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA_AR types.     

Only high concentrations of ethanol affect GABAA receptors of rat cerebellum granule cells in culture

In the experiments described in the present report, we evaluated the effects of ethanol on the activity of GABA_A receptors of cerebellar granule cells in culture. Only very high ethanol concentrations (100–300 mM) showed a clear and significant stimulatory effect on the activity of such receptors. This result was unexpected. In fact, previous reports from other groups would have suggested high ethanol sensitivity of at least one population of GABA_A receptors expressed by granule cells.