GABAA Activation Research Articles

How progesterone stimulates cilia

Hormone Signaling The transport of eggs depends on the beating of the cilia of the cells lining the oviduct. Cilia beating is a Ca2+ signaling–dependent process. Jung et al. investigated how the hormone progesterone affects Ca2+ signaling in mouse ciliated oviduct cells. The hormone increased intracellular Ca2+ concentrations in a process requiring the cation channel TRPV4 and the stepwise activation of GABAA and GABAB receptors. These receptors associated with each other in response to progesterone and GABAergic agonists. Sci. Signal. 11 , eaam6558 (2018).

AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in sympathetic tone and reactivity and a persistent elevation in blood pressure during periods of normoxia, similar to that observed in patients with OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Our lab has previously reported that AT1aR activation influences GABAa mediated inhibition within the MnPO. Here we investigate the role of Angiotensin II (Ang II) signaling via the AT1a receptor in the MnPO and its influence on excitatory/inhibitory balance in rats subjected to CIH. Under isoflurane (2–3%) anesthesia, male Sprague‐Dawley rats (250–350 g) received microinfusions (0.4 μL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1a KD) or an AAV construct containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800–1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10×/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2–3%) and coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potential firing was recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Brief focal application of Ang II produced a transient increase in spontaneous action potential frequency of MnPO neurons from both CIH/Scr (n = 11) and normoxic/Scr (n = 9) rats. The Ang II dependent increase in activity was blocked in neurons from AT1a KD rats from both CIH (n = 12) and normoxic (n = 10) groups. The GABAa agonist muscimol decreased action potential activity (n = 10) of neurons from normoxic/Scr rats. This GABAa mediated inhibition was blunted in neurons from CIH/Scr (n = 13) and normoxic/AT1a KD (n = 10) rats. However, GABAa activation from neurons in the CIH/AT1a KD (n = 15) group produced an increase in spontaneous activity. The current study shows AT1aR KD mediated reduction in GABAa inhibition is exacerbated to the point where GABAa activation is excitatory following CIH. Future studies will address the influence of reduced AT1a signaling combined with reduced GABAa mediated inhibition on downstream targets of the MnPO in AT1aKD from both normoxic and CIH rats.Support or Funding InformationSupported by P01 HL088052This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased short-interval intracortical inhibition correlates with better pinch strength in patients with stroke and good motor recovery

BackgroundDeeper short-interval intracortical inhibition (SICI), a marker of GABAA activity, correlates with better motor performance in patients with moderate to severe hand impairments in the chronic phase after stroke. ObjectivesWe evaluated the correlation between SICI in the affected hemisphere and pinch force of the paretic hand in well-recovered patients. We also investigated the correlation between SICI and pinch force in controls. MethodsTwenty-two subjects were included in the study. SICI was measured with a paired-pulse paradigm. The correlation between lateral pinch strength and SICI was assessed with Spearman's rho. ResultsThere was a significant correlation (rho = 0.69, p = 0.014) between SICI and pinch strength in patients, but not in controls. SICI was significantly deeper in patients with greater hand weakness. ConclusionsThese preliminary findings suggest that decreased GABAA activity in M1AH correlates with better hand motor performance in well-recovered subjects with stroke in the chronic phase.

Regional difference in spontaneous firing inhibition by GABAA and GABAB receptors in nigral dopamine neurons.

GABAergic control over dopamine (DA) neurons in the substantia nigra is crucial for determining firing rates and patterns. Although GABA activates both GABAA and GABAB receptors distributed throughout the somatodendritic tree, it is currently unclear how regional GABA receptors in the soma and dendritic compartments regulate spontaneous firing. Therefore, the objective of this study was to determine actions of regional GABA receptors on spontaneous firing in acutely dissociated DA neurons from the rat using patch-clamp and local GABA-uncaging techniques. Agonists and antagonists experiments showed that activation of either GABAA receptors or GABAB receptors in DA neurons is enough to completely abolish spontaneous firing. Local GABA-uncaging along the somatodendritic tree revealed that activation of regional GABA receptors limited within the soma, proximal, or distal dendritic region, can completely suppress spontaneous firing. However, activation of either GABAA or GABAB receptor equally suppressed spontaneous firing in the soma, whereas GABAB receptor inhibited spontaneous firing more strongly than GABAA receptor in the proximal and distal dendrites. These regional differences of GABA signals between the soma and dendritic compartments could contribute to our understanding of many diverse and complex actions of GABA in midbrain DA neurons.

Transition from androgenic to neurosteroidal action of 5α-androstane-3α, 17β-diol through the type A γ-aminobutyric acid receptor in prostate cancer progression

Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABAAR. In the dual positive cancer cases, AKR1C3 and GABAAR subunit α1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces 5α-dihydrotesterone (DHT) to form 5α-androstane-3α, 17β-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABAAR in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABAAR-dependent, but are independent of the AR. In GABAAR-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABAAR antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABAAR activation and promoting PCa progression requires continued studies.

GABAA receptor: a unique modulator of excitability, Ca2+ signaling, and catecholamine release of rat chromaffin cells.

The role of gamma-aminobutyric acid (GABA) in adrenal medulla chromaffin cell (CC) function is just beginning to unfold. GABA is stored in catecholamine (CA)-containing dense core granules and is presumably released together with CA, ATP, and opioids in response to physiological stimuli, playing an autocrine-paracrine role on CCs. The reported paradoxical "dual action" of GABAA-R activation (enhancement of CA secretion and inhibition of synaptically evoked CA release) is only one aspect of GABA's multifaceted actions. In this review, we discuss recent physiological experiments on rat CCs in situ which suggest that GABA regulation of CC function may depend on the physiological context: During non-stressful conditions, GABAA-R activation by endogenous GABA tonically inhibits acetylcholine release from splanchnic nerve terminals and decreases spontaneous Ca2+ fluctuations in CCs, preventing unwanted CA secretion. During intense stress, splanchnic nerve terminals release acetylcholine, which depolarizes CCs and allows the Ca2+ influx that triggers the release of CA and GABA. With time, CA secretion declines, due to voltage-independent inhibition of Ca2+channels and desensitization of cholinergic nicotinic receptors. Nonetheless, acute activation of GABAA-R is depolarizing in about 50% of CCs, and thus GABA, acting as an autocrine/paracrine mediator, could help to maintain CA exocytosis under stress. GABAA-R activation is not excitatory in about half of CCs' population because it hyperpolarizes them or elicits no response. This percentage possibly varies, depending on functional demands, since GABAA-R-mediated actions are determined by the intracellular chloride concentration ([Cl-] i ) and therefore on the activity of cation-chloride co transporters, which is functionally regulated. These findings underscore a potential importance of a novel and complex GABA-mediated regulation of CC function and of CA secretion.

MPTP-Induced Dopamine Depletion in Basolateral Amygdala via Decrease of D2R Activation Suppresses GABAA Receptors Expression and LTD Induction Leading to Anxiety-Like Behaviors.

Anxiety disorders commonly occur in Parkinson’s disease. Using field potential recording and patch-clamp recording, we evaluated influence of MPTP-reduced dopaminergic afferent in basolateral amygdala (BLA), a main region for affective regulation, on excitatory–inhibitory circuits and synaptic plasticity. Field excitatory post-synaptic potential (fEPSP) slopes at external capsule-BLA synapses were increased in MPTP-mice with decreases in paired-pulse facilitation and long-term potentiation amplitude, which were corrected by bath-application of D2R agonist quinpirole or cannabinoid type 1 receptors agonist WIN55,212-2, but not D1R agonist SKF38393. Compared to single waveform fEPSP in control mice, a multi-spike waveform fEPSP was observed in MPTP-mice with prolongation of duration and an increase in paired-pulse inhibition, which were recovered by BLA-injection of quinpirole for 2 days rather than bath-application. Density of GABA-evoked current (IGABA) in BLA principal neurons and GABAAR-α2 subunit expression were reduced in MPTP-mice, which were recovered by administration of quinpirole. Decline of PKC phosphorylation in BLA of MPTP-mice was corrected by bath-application of quinpirole, but not SKF38393. In MPTP-mice, BLA-injection of quinpirole or PKC activator PMA could recover GABAAR expression, which was sensitive to PKC inhibitor GF109203X. The impairment of long-term depression (LTD) in MPTP-mice was rescued by bath-application of GABAAR agonist muscimol or BLA-injection of quinpirole and PMA. Finally, BLA-injection of muscimol, quinpirole or PMA relieved anxiety-like behaviors in MPTP-mice. The results indicate that the MPTP-induced dopamine depletion in BLA principal neurons through reducing D2R-mediated PKC phosphorylation suppresses GABAAR expression and activity, which impairs GABAAR-mediated inhibition and LTD induction leading to anxiety-like behaviors.

AT1aR dependent GABAa inhibition in the MnPO

The median preoptic nucleus (MnPO), located along the anterior wall of the third ventricle, receives input from other circumventricular organs (e.g. SFO and the OVLT) sensitive to circulating Angiotensin II (Ang II) and plasma Na+ concentrations suggesting an involvement in hydromineral balance and blood pressure regulation. Additionally, evidence suggest that although the MnPO is not directly sensitive to circulating Ang II, the SFO synthesizes Ang II and releases it on the MnPO as a neurotransmitter suggesting the role of the MnPO in hydromineral balance and blood pressure regulation is mediated in part by Ang II. The Ang II activation of AT1aR has also been shown to influence the function of GABAaRs though the mechanisms are still unclear. Here we investigate the role of Ang II signaling via the AT1a receptor in the MnPO and its influence on excitatory/inhibitory balance. Male Sprague‐Dawley rats (250–350g) received microinfusions (0.4 μL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (shAT1a) or an AAV construct containing the GFP reporter and a shRNA scramble (shScr) targeted to the MnPO. Two weeks following AAV injection, coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potential firing was recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Western blot and RT‐qPCR analyses of punch samples containing the MnPO were used to investigate the effect of AT1a KD on GABAa receptor subunits and KCC2 protein and mRNA expression. Brief focal application of Ang II produced a time dependent increase in action potential frequency of MnPO neurons (n = 12). The Ang II dependent increase in activity was blocked by bath application of the AT1aR antagonist Losartan (n = 12). Additionally, Ang II failed to alter firing rate of MnPO neurons in slices prepared from shAT1a injected rats (n = 12). In animals injected with the control vector, the GABAa agonist muscimol decreased action potential activity (n = 10). In rats that received microinjections of the shAT1a, muscimol failed to decrease action potential frequency (n = 10). In AT1a KD rats, RT‐qPCR analysis shows a reduction in AT1a and KCC2 mRNA but no reduction in GABAa Beta subunit mRNA in the MnPO. The current findings demonstrate that Ang II dependent increases in the excitability of MnPO neurons are mediated by AT1aRs. Moreover, AT1aRs are also necessary for the inhibitory effects of GABAaR activation by muscimol. The current study suggests the reduction in GABAa dependent inhibition following AT1a KD could be mediated by a down regulation of KCC2 and subsequent disruption of chloride homeostasis. Taken together, AT1aR in the MnPO contribute to a balance of excitatory and inhibitory activity in MnPO neurons. This balance could be important for the MnPO's role in the regulation of blood pressure and hydromineral balance. However, the signaling mechanisms underlying theses contributions of AT1aRs to MnPO activity remain to be determined.Support or Funding InformationSupported by P01 HL088052

Enhanced Thalamic Spillover Inhibition during Non-rapid-eye-movement Sleep Triggers an Electrocortical Signature of Anesthetic Hypnosis.

Alterations in thalamic γ-aminobutyric acid-mediated signaling are thought to underlie the increased frontal α-β frequency electrocortical activity that signals anesthetic-induced loss of consciousness with γ-aminobutyric acid receptor type A (GABAAR)-targeting general anesthetics. The general anesthetic etomidate elicits phasic extrasynaptic GABAAR activation ("spillover" inhibition) at thalamocortical neurons in vitro. We hypothesize that this action of etomidate at the thalamus is sufficient to trigger an increase in frontal α-β frequency electrocortical activity and that this effect of etomidate is fully recapitulated by enhanced thalamic spillover inhibition in vivo. We recorded electrocortical activity and sleep-wake behavior in freely behaving wild-type (n = 33) and extrasynaptic δ-subunit-containing GABAAR knockout mice (n = 9) during bilateral microperfusion of the thalamus with etomidate and/or other pharmacologic agents that influence GABAAR or T-type Ca channel activity. Microperfusion of etomidate into the thalamus elicited an increase in α-β frequency electrocortical activity that occurred only during non-rapid-eye-movement (REM) sleep (11.0 ± 11.8% and 16.0 ± 14.2% greater 8 to 12- and 12 to 30-Hz power, respectively; mean ± SD; both P < 0.031) and was not affected by blockade of thalamic T-type Ca channels. Etomidate at the thalamus also increased spindle-like oscillations during non-REM sleep (4.5 ± 2.4 spindle per minute with etomidate vs. 3.2 ± 1.7 at baseline; P = 0.002). These effects of etomidate were fully recapitulated by enhanced thalamic extrasynaptic GABAAR-mediated spillover inhibition. These findings identify how a prototypic GABAAR-targeting general anesthetic agent can elicit the characteristic brain wave pattern associated with anesthetic hypnosis when acting at the thalamus by promoting spillover inhibition and the necessity of a preexisting non-REM mode of activity in the thalamus to generate this effect.

Age-related changes in tonic activation of presynaptic versus extrasynaptic γ-amniobutyric acid type B receptors in rat medial prefrontal cortex

The present study examined the effect of age on both glutamatergic and γ-aminobutyric acid mediated (GABAergic) signaling in the rodent medial prefrontal cortex (mPFC), with an emphasis on revealing novel changes contributing to increased inhibition in age. Whole-cell patch clamp recordings were obtained from layer 2/3 mPFC pyramidal neurons in acute cortical slices prepared from either young (4 months) or aged (20–24 months) male F344 rats. Results indicated that GABAB receptors on GABAergic, but not on glutamatergic, inputs to layer 2/3 pyramidal cells are tonically activated by ambient GABA in young animals and further demonstrated that this form of tonic inhibition is significantly attenuated in aged mPFC. Moreover, concurrent with loss of tonic presynaptic GABAB autoreceptor activation, layer 2/3 pyramidal cells in aged mPFC are subjected to increased tonic activation of extrasynaptic GABAA and GABAB receptors. These data demonstrate a shift in the site of GABAB receptor-mediated inhibitory tone in the aged mPFC that clearly promotes increased inhibition of pyramidal cells in aged animals, and that may plausibly contribute to impaired executive function.

A role for inhibition in deafness-induced plasticity of the avian auditory brainstem

To better understand the effects of deafness on the brain, these experiments examine how disrupted balance between excitatory and inhibitory neurotransmission following the loss of excitatory input from the auditory nerve alters the central auditory system. In the avian cochlear nucleus, nucleus magnocellularis (NM), deprivation of excitatory input induced by deafness triggers neuronal death. While this neuronal death was previously accredited to the loss of excitatory drive, the present experiments examine an alternative hypothesis: that inhibitory input to NM, which may also be affected by deafness, contributes to neuronal death in NM. Using an in vitro slice preparation in which excitatory input from the auditory nerve is absent, we pharmacologically altered GABA receptor activation in NM, and assayed an early marker of neuronal health, antigenicity for the ribosomal antibody Y10B (Y10B-ir). We found that GABA decreases Y10B-ir, and that GABAA activation is necessary for the GABA-induced effect. We further found that endogenous GABAA activation similarly decreases Y10B-ir and this decrease requires extracellular Ca2+. Our results suggest that, in the absence of excitatory input, endogenous activation of ionotropic GABAA receptors is detrimental to NM neurons.

Evidence for increased motor cortical facilitation and decreased inhibition in atypical depression.

Major depressive disorder (MDD) is a clinically heterogeneous condition. However, the role of cortical glutamate and gamma-aminobutyric acid (GABA) receptor-mediated activity, implicated in MDD pathophysiology, has not been explored in different MDD subtypes. Our aim was to assess the atypical and melancholic depression subtypes regarding potential differences in GABA and glutamate receptor-mediated activity through established transcranial magnetic stimulation (TMS) neurophysiological measures from the motor cortex. We evaluated 81 subjects free of antidepressant medication, including 21 healthy controls and 20 patients with atypical, 20 with melancholic, and 20 with undifferentiated MDD. Single and paired-pulse TMS paradigms were used to evaluate intracortical facilitation (ICF), cortical silent period (CSP), and short intracortical inhibition (SICI), which index glutamate, GABAB receptor-, and GABAA receptor-mediated activity respectively. Patients with MDD demonstrated significantly decreased mean CSP values than healthy controls (Cohen's d = 0.22-0.3, P < 0.01 for all comparisons). Atypical depression presented a distinct cortical excitability pattern of decreased cortical inhibition and increased cortical facilitation, that is, an increased mean ICF and SICI ratios than other depression subtypes (d = 0.22-0.33, P < 0.01 for all comparisons). Different MDD subtypes may demonstrate different neurophysiology in relation to GABAA and glutamatergic activity. TMS as an investigational tool might be useful to distinguish between different MDD subtypes.

Sodium and water intake are not affected by GABAC receptor activation in the lateral parabrachial nucleus of sodium-depleted rats

The activation of GABAergic receptors, GABAA and GABAB, in the lateral parabrachial nucleus (LPBN) increases water and sodium intake in satiated and fluid-depleted rats. The present study investigated the presence of the GABAC receptor in the LPBN, its involvement in water and sodium intake, and its effects on cardiovascular parameters during the acute fluid depletion induced by furosemide combined with captopril (Furo/Cap). One group of male Wistar rats (290–300g) with bilateral stainless steel LPBN cannulas was used to test the effects of a GABAC receptor agonist and antagonist on the fluid intake and cardiovascular parameters. We investigated the effects of bilateral LPBN injections of trans-4-aminocrotonic acid (TACA) on the intake of water and 0.3M NaCl induced by acute fluid depletion (subcutaneous injection of Furo/Cap). c-Fos expression increased (P<0.05), suggesting LPBN neuronal activation. The injection of different doses of TACA (0.5, 2.0 and 160 nmol) in the LPBN did not change the sodium or water intake in Furo/Cap-treated rats (P>0.05). Treatment with the GABAC receptor antagonist (Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid sulfate (ZAPA, 10nmol) or with ZAPA (10nmol) plus TACA (160nmol) did not change the sodium or water intake compared with that for vehicle (saline) (P>0.05). Bilateral injections of the GABAC agonist in the LPBN of Furo/Cap-treated rats did not affect the mean arterial pressure (MAP) or heart rate (HR). The GABAC receptor expression in the LPBN was confirmed by the presence of a 50kDa band. Although LPBN neurons might express GABAC receptors, their activation produced no change in water and sodium intake or in the cardiovascular parameters in the acute fluid depletion rats. Therefore, the GABAC receptors in the LPBN might not interfere with fluid and blood pressure regulation.

17α-ethinyl estradiol attenuates depressive-like behavior through GABAA receptor activation/nitrergic pathway blockade in ovariectomized mice.

This study was performed to investigate the antidepressant-like effect of 17α-ethinyl estradiol (EE2) in ovariectomized (OVX) mice and the possible role of nitrergic and gamma aminobutyric acid (GABA)ergic pathways in this paradigm. Bilateral ovariectomy was performed in female mice, and different doses of EE2 were intraperitoneally injected either alone or combined with GABAA agonist, diazepam, GABAA antagonist, flumazenil, non-specific nitric oxide synthase (NOS) inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), specific nNOS inhibitor, 7-nitroindazole (7-NI), a nitric oxide (NO) precursor, L-arginine, and selective PDE5I, sildenafil. After locomotion assessment, immobility times were recorded in the forced swimming test (FST) and tail suspension test (TST). Moreover, hippocampal nitrite concentrations were measured in the examined groups. Ten days after ovariectomy, a significant prolonged immobility times were observed. EE2 (0.3 and 1μg/kg and 0.03, 0.1, and 1mg/kg) caused antidepressant-like activity in OVX mice in FST and TST. Diazepam (1 and 5mg/kg), L-NAME (30mg/kg), and 7-NI (100mg/kg) significantly reduced the immobility times. Co-administration of minimal and sub-effective doses of EE2 and diazepam (0.3μg/kg and 0.5mg/kg, respectively) exerted a significant antidepressant-like effect. The same effect was observed in combination of minimal and sub-effective doses of EE2 and either L-NAME or 7-NI. Moreover, combination of minimal and sub-effective doses of EE2, diazepam either L-NAME, or 7-NI emphasized the significant robust antidepressant-like activity. The study has demonstrated that lowest dose of EE2 exerts a significant antidepressant-like behavior. It is suggested that suppression of NO system, as well as GABAA activation, may be responsible for antidepressant-like activity of EE2 in OVX mice. Moreover, GABAA activation may inhibit nitrergic pathway.

New Findings on the Neurotransmitter Modulation of Defense in the Dorsal Periaqueductal Gray.

The dorsal periaqueductal gray (DPAG) has long been implicated in the pathophysiology of anxiety, particularly in panic disorder (PD). Evidence obtained with animal models indicates that different neurotransmitters/neuromodulators in this midbrain area are involved in the regulation of anxiety- (e.g. inhibitory avoidance) and panic- (e.g. escape) associated defensive behaviors. Earlier findings showed that activation of serotonin (5-HT) 1A and 2A receptors in the DPAG inhibits escape expression, a panicolytic-like effect. Recently gathered evidence shows that different classes of antipanic drugs, such as the selective serotonin reuptake inhibitor antidepressant fluoxetine or the benzodiazepine alprazolam, enhance the inhibitory action of 5-HT upon these receptors. They also show that opioidergic mechanisms, through the activation of μ-receptors, contribute to this process. As with 5-HT, activation of GABAA or GABAB receptors, or cannabinoid type 1receptors as well as the tropomyosin-related kinase B receptors by brain-derived neurotrophic factor in the DPAG also inhibits escape expression. There is evidence that chronic antidepressant treatment, besides facilitating 5-HT/opioid neurotransmission, also increases brain-derived neurotrophic factor levels in this area with an impact on its panicolytic effect. On the other hand, facilitation of corticotrophin releasing factor- or cholecystokinin-mediated neurotransmission in the DPAG, via CRF1 and CCK2 receptors, respectively, causes panicogenic-like effects with implications for the pathogenesis of PD. A better understanding of the neurochemical control of defense in the DPAG may foster the development of new strategies for pharmacological treatment of PD.

GABA transporters control GABAergic neurotransmission in the mouse subplate

The subplate is a transient layer between the cortical plate and intermediate zone in the developing cortex. Thalamo-cortical axons form temporary synapses on subplate neurons (SPns) before invading the cortical plate. Neuronal activity within the subplate is of critical importance for the development of neocortical circuits and architecture. Although both glutamatergic and GABAergic inputs on SPns were reported, short-term plasticity of GABAergic transmission has not been investigated yet. GABAergic postsynaptic currents (GPSCs) were recorded from SPns in coronal neocortical slices prepared from postnatal day 3–4 mice using whole-cell patch-clamp technique. Evoked GPSCs (eGPSCs) elicited by electrical paired-pulse stimulation demonstrated paired-pulse depression at all interstimulus intervals tested. Baclofen, a specific GABAB receptor (GABABR) agonist, reduced eGPSC amplitudes and increased paired-pulse ratio (PPR), suggesting presynaptic location of functional GABABRs. Baclofen-induced effects were alleviated by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), a selective GABABR blocker. Moreover, CGP55845 increased eGPSC amplitudes and decreased PPR even under control conditions, indicating that GABABRs are tonically activated by ambient GABA. Because extracellular GABA concentration is mainly regulated by GABA transporters (GATs), we asked whether GATs release GABA. 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid (NNC-711) (10μM), a selective GAT-1 blocker, increased eGPSC decay time, decreased eGPSC amplitudes and PPR. The two last effects but not the first one were blocked by CGP55845, indicating that GAT-1 blockade causes an elevation of extracellular GABA concentration and in turn activation of extrasynaptic GABAARs and presynaptic GABABRs. 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP-5114), a specific GAT-2/3 blocker, failed to affect eGPSC kinetics. However, in contrast to NNC-711 SNAP-5114 increased eGPSC amplitudes and decreased PPR. In the presence of SNAP-5114 CGP55845 did not influence GABAergic transmission, indicating that GABABRs are not activated any longer. We conclude that in the subplate GAT-2/3 operates in reverse mode. GABA released via GAT-2/3 activates presynaptic GABABRs on GABAergic synapses and tonically inhibits GABAergic inputs on SPns.

Propofol depresses cerebellar Purkinje cell activity via activation of GABAA and glycine receptors in vivo in mice

Propofol is an intravenous sedative-hypnotic agen, which causes rapid and reliable loss of consciousness. Under in vitro conditions, propofol activates GABAA and glycine receptors in spinal cord, hippocampus and hypothalamus neurons. However, the effects of propofol on the cerebellar neuronal activity under in vivo conditions are currently unclear. In the present study, we examined the effects of propofol on the spontaneous activity of Purkinje cells (PCs) in urethane-anesthetized mice by cell-attached recording and pharmacological methods. Our results showed that cerebellar surface perfusion of propofol (10–1000μM) induced depression of the PC simple spike (SS) firing rate in a dose-dependent manner, but without significantly changing the properties of complex spikes (CS). The IC50 of propofol for inhibiting SS firing of PCs was 144.5μM. Application of GABAA receptor antagonist, SR95531 (40μM) or GABAB receptor antagonist, saclofen (20μM), as well as glycine receptor antagonist, strychnine (10μM) alone failed to prevent the propofol-induced inhibition of PCs spontaneous activity. However, application the mixture of SR95531 (40μM) and strychnine (10μM) completely blocked the propofol-induced inhibition of PC SS firing. These data indicated that cerebellar surface application of propofol depressed PC SS firing rate via facilitation of GABAA and functional glycine receptors activity in adult cerebellar PCs under in vivo conditions. Our present results provide a new insight of the anesthetic action of propofol in cerebellar cortex, suggesting that propofol depresses the SS outputs of cerebellar PCs which is involved in both GABAA and glycine receptors activity.

Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation.

A developmental “switch” in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABAA activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABAA receptor activation in NKCC1-/- mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1-/- mice retained relatively normal responses to the GABAA agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO3 - mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

The reinforcing effects of ethanol within the nucleus accumbens shell involve activation of local GABA and serotonin receptors.

Ethanol is reinforcing within the nucleus accumbens shell (NACsh), but the underlying mechanisms remain unclear. Ethanol can potentiate the function of the GABAA, GABAB, and serotonin-3 (5-HT3) receptors. Therefore, the current study tested the hypothesis that activation of these receptors would be involved in the reinforcing effects of ethanol in the NACsh. An intracranial self-administration (ICSA) procedure was used to assess the reinforcing effects of ethanol in the NACsh of alcohol preferring (P) rats. The ICSA consisted of seven sessions: four sessions to establish 150 mg% ethanol self-infusion into the NACsh; sessions 5 and 6 with co-infusion of ethanol plus one concentration of the GABAA antagonist bicuculline (10 or 100 µM), the GABAB antagonist SCH 50911 (50, 75 or 100 µM), or the 5-HT3 receptor antagonist zacopride (10 or 100 µM); and session 7 with 150 mg% ethanol alone. All groups self-infused ethanol into the NACsh and readily discriminated the active from inactive lever during the acquisition sessions. Co-infusion of 100 µM, but not 10 µM, bicuculline or zacopride significantly decreased active responses during sessions 5 and 6. Co-infusion of 75 µM, but not 50 or 100 µM, SCH 50911 significantly attenuated responses for ethanol. Overall, the results suggest that the reinforcing effects of ethanol in the NACsh may be modulated by activation of local GABAA, GABAB and 5-HT3 receptors.

Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors.