Temperature-dependent effect of zolpidem on the GABAA receptor-mediated response at recombinant human GABAA receptor subtypes

The effects of zolpidem on the two forms of recombinant human GABA_A receptors (α1β2γ2s and α3β2γ2s) at different temperatures were functionally investigated, using the whole-cell patch recording configuration. In both forms, zolpidem potentiated the response to GABA in a concentration-dependent manner. At 16°C, the apparent dissociation constant (K_D) values for the α1β2γ2s and α3β2γ2s forms were 3.7×10⁻⁸ and 5.6×10⁻⁷ M, respectively. When the temperature was increased to 36°C, the K_D values for the α1β2γ2s and α3β2γ2s forms were 2.1×10⁻⁷ and 1.5×10⁻⁶ M, respectively. Although the affinity ratio was reduced from 15.1 to 7.1-fold the selectivity of zolpidem for the α1β2γ2s still remained at 36°C.     

Localization of GABAA and GABAB receptor subtypes on serotonergic neurons

The effect of selective destruction of serotonin (5-HT)-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT) on [3H] muscimol and (-)-[3H]baclofen binding was investigated in various rat brain regions. Ten days after intracerebroventricular 5,7-DHT, serotonin levels and [3H]imipramine binding were markedly decreased. 5,7-DHT reduced [3H]muscimol binding only in the mesencephalon, and (-)-[3H]baclofen binding was unmodified in all the areas considered. These results suggest that except in the mesencephalon GABA receptors may not be localized on serotonergic nerve terminals.     

Excitotoxic lesions of the prefrontal cortex attenuate the potentiation of amphetamine-induced locomotion by repeated neurotensin receptor activation

This study was aimed at determining the role of prefrontal cortex neurons in the development of the potentiation of amphetamine-induced locomotor activity by repeated central injections of D-Tyr[11]neurotensin. Excitotoxic lesions of the prefrontal cortex were made by injecting bilaterally at three anterior-posterior placements 2 microg/microl of ibotenic acid. Ten days after surgery, locomotor responses to an intracerebroventricular injection of 0.18 or 18 nmol/10 microl of D-Tyr[11]neurotensin, or vehicle-saline, were measured in different groups of lesioned and sham rats. Ambulatory, non-ambulatory and vertical movements were measured for 2 h in activity cages starting immediately after the injection. This training phase was repeated on four occasions, every second day. One week after the last day of the training phase (day 14), locomotor responses to a single injection of amphetamine (0.75 mg/kg, IP) were measured in all rats. Results show that D-Tyr[11]neurotensin produced in sham animals a dose-dependent initial suppression of locomotor activity followed by an augmentation. The latter behavioral effect tended to be smaller in the lesioned rats, but not statistically different than in sham, suggesting that prefrontal cortex neurons do not play a major role in the stimulant effect of neurotensin on locomotor activity. However, sham rats pre-exposed to the high dose of D-Tyr[11]neurotensin showed stronger non-ambulatory and vertical movements than saline pre-exposed rats when tested with amphetamine; this sensitization effect was not observed in lesioned rats. The present results show that prefrontal cortex neurons are part of the neural circuitry involved in the development of amphetamine sensitization by repeated activation of central neurotensin receptors.     

Benzodiazepine- and barbiturate-interactions with GABAA receptor responses on lactotrophes

Modulation of the biphasic effect of muscimol on prolactin secretion by benzodiazepines and secobarbital was investigated, using an in vitro superfusion system. The stimulatory effect of low concentrations of muscimol was potentiated by both classes of drugs, and the effect of benzodiazepines appeared to be mediated by central-type benzodiazepine receptors. Neither benzodiazepines nor secobarbital affected the inhibitory response to muscimol. Clonazepam reduced the potency of bicuculline methiodide as an antagonist of the stimulatory effect, but did not alter the potency of picrotoxinin. These results demonstrate a selective potentiation of one component of the GABAA receptor effect on lactotrophs by benzodiazepines and barbiturates and provide evidence for a functional effect of these drugs at a site without the CNS.     

Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex

In the present communication we have investigated the pharmacological properties of the GABA_A receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABA_A receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABA_A receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [³H]flunitrazepam revealed that neither the K_d values or the B_max were modified during aging. Moreover, Cl 218 872 displacement of [³H]flunitrazepam showed no age-related modifications on either the K_is or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [³H]muscimol demonstrated a decrease in the B_max of the low affinity [³H]muscimol binding sites in aged rats (4.3 ± 0.8 pmol/mg protein vs. 2.3 ± 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABA_A receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.     

Calcineurin-mediated GABAA receptor dephosphorylation in rats after kainic acid-induced status epilepticus

Calcineurin (CaN) is a neuronally enriched, calcium-dependent phosphatase, which plays an important role in a number of neuronal processes including development of learning and memory, and modulation of receptor's function and neuronal excitability as well as induction of apoptosis. It has been established in kindling model that the status epilepticus (SE)-induced increase in CaN activity is involved in the development of seizures through down-regulation of γ-aminobutyric acid A receptor (GABA_AR) activation. However, the mechanism by which CaN mediates GABA_A receptor dephosphorylation in SE is not fully understood. Here, using a model of kainic acid (KA)-induced SE and CaN inhibitor FK506, we observed the behaviors induced by KA and levels of CaN activity and CaN expression in hippocampus by immunobloting. The results showed that the SE-induced CaN activity was time-dependent, with a peak at 2 h and a return to basal level at 24 h, whereas a significant increase in CaN expression was seen at 24 h after SE. It is proposed that the rapid elevation in CaN activity after KA-induced SE is not likely due to an increase in CaN expression but rather an increase in CaN activation state or kinetics. In addition, we also demonstrated that pre-treatment with FK506 remarkably suppressed the SE-induced CaN activity and its expression, and reversed the SE-induced dephosphorylation of GABA_AR 2/3 subunits. Taken together, our data suggest that down-regulation in inhibition of GABA_AR 2/3 by CaN activity contributes to an elevation in neuronal excitability of hippocampus, which may be involved in development of chronic processes of seizures.     

GABAA and GABAB receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO_x⁻) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA_A receptor antagonist, or saclofen, a GABA_B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO_x⁻ levels. Higher-dose perfusion of either muscimol (50 μM), a GABA_A receptor agonist, or baclofen (250 μM), a GABA_B receptor agonist resulted in a significant decrease in NO_x⁻ levels. The elevated levels of NO_x⁻ after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO_x⁻ levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA_A and GABA_B receptors in the mPFC.     

Changes in neuroactive steroid content during social isolation stress modulate GABAA receptor plasticity and function

Rats deprived of social contact with other rats at a young age experience a form of prolonged stress that leads to long-lasting alteration in their behavior profile. This chronic stress paradigm is thus thought to be anxiogenic for these normally gregarious animals and their abnormal reactivity to environmental stimuli, when reared under this condition, is thought to be a product of prolonged stress. Neurochemical, molecular, and electrophysiological evidences demonstrate that social isolation is associated with alteration in the structure and function of GABA_A receptors and suggest that endogenous content of the progesterone metabolite 3α,5α-TH PROG may be an important determinant in regulating brain excitability and sensitivity to stimuli and point out its possible role in psychiatric and neurological disorder.     

Propofol increases agonist efficacy at the GABAA receptor

Using the whole-cell patch-clamp technique, we have determined that propofol, but not midazolam, increases the efficacy of piperidine-4-sulphonic acid (P4S), a partial agonist at α₁β₁γ_2s GABA_A receptors expressed in HEK 293 cells. These findings are consistent with the idea that propofol facilitates receptor gating, while midazolam increases receptor occupancy by the agonist.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Subunit dependent modulation of GABAA receptor function by neuroactive steroids

Neurosteroids are potent, endogenous modulators of GABA_A receptor function in the central nervous system. The endogenous progesterone metabolite allopregnanolone (ALP) and the synthetic steroid compound alphaxalone (AFX) have been shown to both directly activate and potentiate GABA_A receptor-activated membrane current (I_GABA). The role of different α and γ subunit subtypes in modulation of I_GABA by ALP and AFX was investigated using recombinant GABA_A receptor isoforms expressed in Xenopus oocytes. Changing or removal of the α subunit subtype altered the efficacy of both ALP and AFX (α2β1γ2L>α1β1γ2Lβ1γ2L) to potentiate I_GABA, but did not alter the potency of the neuroactive steroids at these receptor isoforms. The efficacy of ALP to enhance I_GABA was also dependent on the γ subunit subtype (α1β1γ3>α1β1γ2L=α1β1γ1). AFX also had higher efficacy in the α1β1γ3 receptor isoform compared to α1β1γ1. In contrast to ALP, the potency of AFX was greater in the α1β1γ3 and α1β1γ1 receptor isoforms compared to α1β1γ2L. This study provides evidence that the α subunit subtype determines the efficacy, but not the potency, of these neuroactive steroids to potentiate I_GABA. The γ3 subunit subtype increases the maximal efficacy of neuroactive steroids compared to other γ subunit subtypes. These results suggest that the heteromeric assembly of different GABA_A receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA_A receptors that in turn produce distinctly different modulatory interactions between neuroactive steroids acting at the GABA_A receptor.     

Bihemispheric reduction of GABAA receptor binding following focal cortical photothrombotic lesions in the rat brain

Focal brain lesions may lead to neuronal dysfunctions in remote (exofocal) brain regions. In the present study, focal lesions were induced in the hindlimb representation area of the parietal cerebral cortex in rats using the technique of photothrombosis. Photothrombosis occurs after illumination of the brain through the intact skull following intravenous injection of the photosensitive dye Rose Bengal. This resulted in cortical lesions with a diameter of about 2 mm. Quantitative receptor autoradiography was used to study alterations in the density of []muscimol binding sites to GABA_A receptors seven days after lesion induction. A reduced GABA_A receptor binding (−13 to −27% of the control value) was found in layers II and III of remote exofocal regions in the ipsi- and contralateral cortex. The reduction was consistently more intense in the ipsilateral cortical areas than in those of the contralateral hemisphere. Using extracellular recordings, significant correlations between GABA_A receptor binding and paired pulse inhibition could be demonstrated. The present investigation demonstrates that focal brain lesions cause a widespread, functionally effective down-regulation of GABA_A receptors. These postlesional changes may result from lesion-induced alterations in cortical connectivity.     

Prolonged exposure to hypobaric hypoxia transiently reduces GABAA receptor number in mice cerebral cortex

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day×21 days) applied to adult male mice on GABA_A receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [³H]GABA specifically bound to GABA_A receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (B_max control, 4.70±0.19, hypoxic, 3.33±0.10 pmol/mg protein), with no effect on GABA binding sites affinity (K_d control: 159.3±13.3 nM, hypoxic: 164.2±15.1 nM). Decreased B_max values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA_A receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5α-pregnan-3α-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA_A receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.     

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABA_A and GABA_B receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABA_A receptor binding without significantly altering GABA_B receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABA_A, but not GABA_B receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABA_A receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABA_B receptors located on non-cholinergic neuronal elements in the striatum.     

Developmental changes of GABAA receptor-chloride channels in rat Meynert neurons

The developmental changes of GABA_A receptors were investigated in Meynert neurons freshly dissociated from day 0, 2 week-, and 6 month-old rats using both nystatin and gramicidin perforated patch recording modes under voltage-clamp conditions. The age-related changes in the current amplitude and threshold concentration in the concentration–response relationships for GABA indicated the developmental alteration of the GABA_A receptor subunits and the channel density. The GABA-induced

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measured by the gramicidin perforated patch mode shifted to more negative with development. The decay time constant of GABAergic inhibitory postsynaptic spontaneous currents (sIPSCs) in the synaptic active zone accelerated with aging. The GABA-induced currents were potentiated in a concentration dependent manner in the presence of benzodiazepine (BZP) agonists, diazepam (DZP) and zolpidem (ZPM). The potentiation rate of DZP on the GABA_A response decreased with aging, but not in the case of ZPM, which demonstrated a stronger action in the aging rat neurons. These results suggested that the GABA_A receptor·Cl⁻ channel complexes may thus change both the assembly and interaction of subunits as well as their functional roles with aging.     

The expression of GABAA receptor subunits in the substantia nigra is developmentally regulated and region-specific

The substantia nigra pars reticulata (SNR) controls the spread of seizures. GABA_Aergic drug (agonist or antagonist) infusions into the SNR have age-specific and site-specific effects on flurothyl-induced seizures. Developmental and cell-specific regulation of GABA_A receptor subunit expression may be responsible for these specific effects. To test this hypothesis,in situ hybridization was used to examine regional expression of α1 and γ2L GABA_A receptor subunit mRNAs in the SNR during development. Distinct temporal and spatial patterns of expression were observed. In rats at postnatal days (PN) 21–60, fewer neurons were labeled with probes directed to α1 and γ2L subunits in SNR_anterior compared with SNR_posterior. In addition, neurons in SNR_anterior contained higher amounts of hybridization grains than in SNR_posterior. In PN 15 rats, the labeling of neurons was relatively diffuse throughout the anterior and posterior SNR regions with moderate amounts of hybridization grains for both subunits. The finding of age-related differential distribution of α1 and γ2L subunit mRNAs in the SNR suggests that GABA_A receptor heterogeneity may play a role in the age-specific and site-specific effects of GABA_Aergic agents on seizures in the SNR.

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Sommario La sostanza nera pars reticulata (SNR) controlla la propagazione delle crisi. Infusioni di GABA agonisti o antagonisti nella SNR hanno effetti specifici in rapporto all’età e alla sede sulle crisi indotte con flurothyl, per la probabile influenza di fattori regolatori dell’espressione di subunità del receptore GABA_A specificamente legati allo sviluppo e al tipo cellulare. L’ipotesi è stata verificata ana-lizzando l’ espressione regionale del mRNA delle subunità recettoriali GABA_A α1 e γ2L durante lo sviluppo. Diverse modalità di espressione temporale e spaziale sono state osservate. Nel ratto ad una età postnatale (PN) di 21–60 giorni, il numero di neuroni marcati con sonde dirette alle subunità α1 e γ2L è risultato inferiore rispetto alla posteriore. Inoltre, i neuroni di SNR anteriore contenevano una quantità più elevata di granuli di ibridizzazione rispetto a quelli di SNR posteriore. In ratti PN 15, invece, la marcatura di neuroni è risultata relativamente diffusa nell’ambito delle regioni anteriori e posteriori di SNR con una quantità moderata di granuli di ibridizzazione per entrambe le subunità. Il rilievo di una distribuzione differenziale età dipendente dell’mRNA delle subunità α1 e γ2L nella SNR suggerisce che un’eterogeneità recettoriale possa giocare uno specifico ruolo nel determinare gli effetti età e sito-specifici sulle crisi di agenti GABAergici iniettati nella SNR.

     

Opioid peptides modulate GABAA receptor responses in neurons of bullfrog dorsal root ganglia

Effects of enkephalin and selective opioid-receptor agonists on GABA-induced current were examined in dissociated neurons of bullfrog dorsal root ganglia (DRG) by using whole-cell patch-clamp method. Leucine-(Leu)-enkephalin and methionine-(Met)-enkephalin depressed GABA_A receptor-mediated currents. DPDPE, DAMGO and dynorphin-A (Dyn-A) also depressed the inward current produced by GABA: the order of agonist potency was DPDPE ≥ DAMGO> Dyn-A. Naloxone blocked the inhibitory effects of ekephalins and other opioid agonists on the GABA current. Naltrindole (NTI), a δ-receptor antagonist, prevented the DPDPE-induced depression of the GABA current. β-Funaltrexamine (β-FNA), a μ-receptor antagonist, reduced the DAMGO-induced depression of GABA currents. Nor-binaltorphimine (nor-BNI), a κ-receptor antagonist, reduced the effects of Dyn-A in depressing the GABA current The results suggest that enkephalin down-regulates GABA_A receptor function through mainly δ- and μ-opioid receptors in bullfrog DRG neurons.     

Persistent reduction of GABAA receptor-mediated inhibition in rat hippocampus after chronic intermittent ethanol treatment

GABA_A receptor-mediated function was studied in rats treated with chronic intermittent ethanol (CIE). Rats were given 60 doses of 6 g/kg ethanol every 24 h by gastric intubation, with repeated intoxicating and withdrawal episodes leading to a kindling-like increase in seizure susceptibility (Kokka et al., Alcohol: Clin. Exp. Res., 17 (1993) 525-531). Efflux of ³⁶Cl⁻, evoked by application of muscimol, a measure of GABAA receptor function, was examined in 300 μm slices obtained from frontal, parietal, and temporal cortex, hippocampus, and inferior colliculus, one day after the last administration of ethanol. Compared to controls, the ³⁶Cl⁻ efflux in hippocampal slices of CIE rats was significantly reduced by 29%, while there were no changes in the other brain regions studied. In hippocampal slices, paired-pulse inhibition in CAI pyramidal neurons, measured extracellularly using homosynaptic orthodromic stimulation at an interval of 10 ms, was significantly reduced in CIE rats. A significant decrease by 40% both at 2 and 40 days after 60 doses of ethanol was found, implying a persistent decrease in GABA_A receptor-mediated inhibition in CIE rats. These reductions in paired-pulse inhibition are consistent with the decrease in the pentylenetetrazol (PTZ) seizure threshold which was previously observed in CIE rats. Therefore, we suggest that this reduction of GABA_A receptor-mediated inhibition contributes to the persistent increase in seizure susceptibility of CIE rats.     

Glucocorticoid modulates the sensitivity of the GABAA receptor on primary afferent neurons of bullfrogs

With intracellular and voltage-clamp recording techniques, we have demonstrated that the glucocorticoids, prednisolone and hydrocortisone at a concentration of 5 microM to 1 mM, reversibly depressed gamma-aminobutyric acid (GABA)-induced responses on primary afferent neurons of bullfrogs. An analysis with dose-response curves revealed that the glucocorticoids decreased the sensitivity of the GABAA receptor in a non-competitive manner. We suggest that glucocorticoids act as an antagonist of the GABAA receptor on primary afferent neurons, probably by reducing the number of functional GABAA receptor ionic channel complexes.