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

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

Inhibition of thalamic excitability by 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol: a selective role for δ-GABAA receptors

The sedative and hypnotic agent 4,5,6,7‐tetrahydroisoxazolo[4,5‐c]pyridine‐3‐ol (THIP) is a GABA_A receptor (GABA_AR) agonist that preferentially activates δ‐subunit‐containing GABA_ARs (δ‐GABA_ARs). To clarify the role of δ‐GABA_ARs in mediating the sedative actions of THIP, we utilized mice lacking the α₁‐ or δ‐subunit in a combined electrophysiological and behavioural analysis. Whole‐cell patch‐clamp recordings were obtained from ventrobasal thalamic nucleus (VB) neurones at a holding potential of ?60 mV. Application of bicuculline to wild‐type (WT) VB neurones revealed a GABA_AR‐mediated tonic current of 92 ± 19 pA, which was greatly reduced (13 ± 5 pA) for VB neurones of δ^0/0 mice. Deletion of the δ‐ but not the α₁‐subunit dramatically reduced the THIP (1 μm )‐induced inward current in these neurones (WT, ?309 ± 23 pA; δ^0/0, ?18 ± 3 pA; α₁^0/0, ?377 ± 45 pA). Furthermore, THIP selectively decreased the excitability of WT and α₁^0/0 but not δ^0/0 VB neurones. THIP did not affect the properties of miniature inhibitory post‐synaptic currents in any of the genotypes. No differences in rotarod performance and locomotor activity were observed across the three genotypes. In WT mice, performance of these behaviours was impaired by THIP in a dose‐dependent manner. The effect of THIP on rotarod performance was blunted for δ^0/0 but not α₁^0/0 mice. We previously reported that deletion of the α₁‐subunit abolished synaptic GABA_A responses of VB neurones. Therefore, collectively, these findings suggest that extrasynaptic δ‐GABA_ARs vs. synaptic α₁‐subunit‐containing GABA_ARs of thalamocortical neurones represent an important molecular target underpinning the sedative actions of THIP.     

Attenuated Benzodiazepine‐Sensitive Tonic GABAA Currents of Supraoptic Magnocellular Neuroendocrine Cells in 24‐h Water‐Deprived Rats

In supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs), γ‐GABA, via activation of GABA_A receptors (GABA_ARs), mediates persistent tonic inhibitory currents (I_tonic), as well as conventional inhibitory postsynaptic currents (IPSCs, I_phasic). In the present study, we examined the functional significance of I_tonic in SON MNCs challenged by 24‐h water deprivation (24WD). Although the main characteristics of spontaneous IPSCs were similar in 24WD compared to euhydrated (EU) rats, I_tonic, measured by bicuculline (BIC)‐induced I_holding shifts, was significantly smaller in 24WD compared to EU rats (P < 0.05). Propofol and diazepam prolonged IPSC decay time to a similar extent in both groups but induced less I_tonic in 24WD compared to EU rats, suggesting a selective decrease in GABA_A receptors mediating I_tonic over I_phasic in 24WD rats. THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol), a preferential δ subunit agonist, and L‐655,708, a GABA_A receptor α₅ subunit selective imidazobenzodiazepine, caused a significantly smaller inward and outward shift in I_holding, respectively, in 24WD compared to EU rats (P < 0.05 in both cases), suggesting an overall decrease in the α₅ subunit‐containing GABA_ARs and the δ subunit‐containing receptors mediating I_tonic in 24WD animals. Consistent with a decrease in 24WD I_tonic, bath application of GABA induced significantly less inhibition of the neuronal firing activity in 24WD compared to EU SON MNCs (P < 0.05). Taken together, the results of the present study indicate a selective decrease in GABA_ARs functions mediating I_tonic as opposed to those mediating I_phasic in SON MNCs, demonstrating the functional significance of I_tonic with respect to increasing neuronal excitability and hormone secretion in 24WD rats.     

GABAB receptor‐dependent modulation of network activity in the rat prefrontal cortex in vitro

GABA (γ‐aminobutyric acid) can mediate inhibition via pre‐ and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA_B receptor‐dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage‐clamp experiments the presence of a tonic GABA_B receptor‐dependent outward current in these neurons. This GABA_Bergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA_B current was similar at different temperatures. In addition to the tonic GABA_B current, we found presynaptic GABA_B effects, GABA_B‐mediated inhibitory postsynaptic currents and tonic GABA_A currents. Second, we performed current‐clamp experiments to evaluate the functional impact of GABA_B receptor‐mediated inhibition in the mPFC. Activating or inactivating GABA_B receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input–output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca²⁺‐imaging that GABA_B receptor‐mediated tonic inhibition is capable of regulating network activity. Blocking GABA_B receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA_B receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.     

Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABAA receptors

The cytotoxic action of the γ-isomer of hexachlorocyclohexane (γ-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 μM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA_A receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 μM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA_A antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA_A receptor. When 100 μM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 μM lindane. The GABA_B receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA_A receptor. J. Neurosci. Res. 52:276–285, 1998. © 1998 Wiley-Liss, Inc.     

Phasic GABAA -receptor activation is required to suppress epileptiform activity in the CA3 region of the immature rat hippocampus

Purpose: Despite the consistent observation that γ‐aminobutyric acid A (GABA_A) receptors mediate excitatory responses at perinatal stages, the role of the GABAergic system in the generation of neonatal epileptiform activity remains controversial. Therefore, we analyzed whether tonic and phasic GABAergic transmission had differential effects on neuronal excitability during early development. Methods: We performed whole cell patch‐clamp and field potential recordings in the CA3 region of hippocampal slices from immature (postnatal day 4–7) rats to analyze the effect of specific antagonists and modulators of tonic and phasic GABAergic components on neuronal excitability. Key Findings: The GABAergic antagonists gabazine (3 μm ) and picrotoxin (100 μm ) induced epileptiform discharges, whereas activation of GABA_A receptors attenuated epileptiform discharges. Under low‐Mg²⁺ conditions, 100 nm gabazine and 1 μm picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole‐cell patch‐clamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1‐μm 4,5,6,7‐tetrahydroisoxaz‐olo[5,4‐c]‐pyridin‐3‐ol (THIP) induced a tonic current of ?12 ± 2.5 pA (n = 6) and provoked epileptiform discharges in 57% (n = 21) of the slices. Significance: We conclude from these results that in the early postnatal rat hippocampus a constant phasic synaptic activity is required to control excitability and prevent epileptiform activity, whereas tonic GABAergic currents can mediate excitatory responses. Pharmacologic intervention at comparable human developmental stages should consider these ambivalent GABAergic actions.     

Fast Ca2+ responses in astrocyte end‐feet and neurovascular coupling in mice

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity‐dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca²⁺ changes in astrocytes versus neurons. Here, we used two‐photon microscopy to assess Ca²⁺ changes in neuropil, astrocyte processes, and astrocyte end‐feet in response to whisker pad stimulation in mice. We also developed a pixel‐based analysis to improve the detection of rapid Ca²⁺ signals in the subcellular compartments of astrocytes. Fast Ca²⁺ responses were observed using both chemical and genetically encoded Ca²⁺ indicators in astrocyte end‐feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine‐hydrogen‐maleate (MK801) attenuated fast Ca²⁺ responses in the neuropil and astrocyte processes, but not in astrocyte end‐feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP), an agonist for the extrasynaptic GABA_A receptor (GABA_AR), increased CBF responses and the fast Ca²⁺ response in astrocyte end‐feet but did not affect Ca²⁺ responses in astrocyte processes and neuropil. These results suggest that fast Ca²⁺ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca²⁺ responses in astrocyte end‐feet are unaffected by MK801 but increase via GABA_AR‐dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca²⁺ increases in end‐feet adjust CBF during synaptic activity.     

Activation of extrasynaptic GABAA receptors inhibits cyclothiazide-induced epileptiform activity in hippocampal CA1 neurons

Extrasynaptic GABA_A receptors (GABA_ARs)-mediated tonic inhibition is reported to involve in the pathogenesis of epilepsy. In this study, we used cyclothiazide (CTZ)-induced in vitro brain slice seizure model to explore the effect of selective activation of extrasynaptic GABA_ARs by 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-ol (THIP) on the CTZ-induced epileptiform activity in hippocampal neurons. Perfusion with CTZ dose-dependently induced multiple epileptiform peaks of evoked population spikes (PSs) in CA1 pyramidal neurons, and treatment with THIP (5 μmol/L) significantly reduced the multiple PS peaks induced by CTZ stimulation. Western blot showed that the δ-subunit of the GABA_AR, an extrasynaptic specific GABA_AR subunit, was also significantly down-regulated in the cell membrane 2 h after CTZ treatment. Our results suggest that the CTZ-induced epileptiform activity in hippocampal CA1 neurons is suppressed by the activation of extrasynaptic GABA_ARs, and further support the hypothesis that tonic inhibition mediated by extrasynaptic GABA_ARs plays a prominent role in seizure generation.     

Dopamine depletion induces neuron‐specific alterations of GABAergic transmission in the mouse striatum

Lack of dopamine (DA) in the striatum and the consequential dysregulation of thalamocortical circuits are major causes of motor impairments in Parkinson's disease. The striatum receives multiple cortical and subcortical afferents. Its role in movement control and motor skills learning is regulated by DA from the nigrostriatal pathway. In Parkinson's disease, DA loss affects striatal network activity and induces a functional imbalance of its output pathways, impairing thalamocortical function. Striatal projection neurons are GABAergic and form two functionally antagonistic pathways: the direct pathway, originating from DA receptor type 1‐expressing medium spiny neurons (D₁R‐MSN), and the indirect pathway, from D₂R‐MSN. Here, we investigated whether DA depletion in mouse striatum also affects GABAergic function. We recorded GABAergic miniature IPSCs (mIPSC) and tonic inhibition from D₁R‐ and D₂R‐MSN and used immunohistochemical labeling to study GABA_AR function and subcellular distribution in DA‐depleted and control mice. We observed slower decay kinetics and increased tonic inhibition in D₁R‐MSN, while D₂R‐MSN had increased mIPSC frequency after DA depletion. Perisomatic synapses containing the GABA_AR subunits α₁ or α₂ were not affected, but there was a strong decrease in non‐synaptic GABA_ARs containing these subunits, suggesting altered receptor trafficking. To broaden these findings, we also investigated GABA_ARs in GABAergic and cholinergic interneurons and found cell type‐specific alterations in receptor distribution, likely reflecting changes in connectivity. Our results reveal that chronic DA depletion alters striatal GABAergic transmission, thereby affecting cellular and circuit activity. These alterations either result from pathological changes or represent a compensatory mechanism to counteract imbalance of output pathways.     

The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABAA receptor‐mediated inhibition

Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described ‘spillover’ mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ‐aminobutyric acid (GABA)_A receptors (GABA_ARs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABA_ARs to the thalamic effects of etomidate, we performed whole‐cell recordings from mouse TC neurons lacking synaptic (α1^0/0) or extrasynaptic (δ^0/0) GABA_ARs. Etomidate (3 μm ) significantly inhibited action‐potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABA_ARs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ‐GABA_ARs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate‐induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states.     

Subpopulations of neurons in the rat neostriatum display GABABR1 receptor immunoreactivity

Immunoreactivity for gamma aminobutyric acid BR1 receptor (GABA_BR1) was detected in the neuropilar elements as well as in the perikarya of neurons in the neostriatum. Many of the GABA_BR1-immunoreactive perikarya were medium-sized with a thin rim of cytoplasm. They resembled the morphology of medium spiny neurons, the projection neurons of the neostriatum. In addition, some GABA_BR1-immunoreactive neurons were densely labeled and were of medium to large in size. These neurons were characterized by double immunofluorescence using their neurochemicals as markers. Over 90% of the parvalbumin- and choline acetyltransferase-immunoreactive neurons and about 80% of the nitric oxide synthase-immunoreactive neurons displayed GABA_BR1 immunoreactivity. The present results show for the first time that the major four subpopulations of striatal neurons express GABA_BR1 receptor and may have a functional implication in the GABA neurotransmission in the microcircuitry of the neostriatum.     

GABA transporter subtype 1 and GABA transporter subtype 3 modulate glutamatergic transmission via activation of presynaptic GABA(B) receptors in the rat globus pallidus

The intra‐pallidal application of γ‐aminobutyric acid (GABA) transporter subtype 1 (GAT‐1) or GABA transporter subtype 3 (GAT‐3) transporter blockers [1‐(4,4‐diphenyl‐3‐butenyl)‐3‐piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1‐[2‐[tris(4‐methoxyphenyl)methoxy]ethyl]‐(S)‐3‐piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA_B heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole‐cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA_A receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA_B receptor blockade by the intra‐cellular application of N‐(2,6‐dimethylphenylcarbamoylmethyl)‐triethylammonium bromide (OX314), bath application of SKF 89976A (10 μm ) or SNAP 5114 (10 μm ) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)‐3‐[[(1S)‐1‐(3,4‐dichlorophenyl)ethyl]amino‐2‐hydroxypropyl](phenylmethyl)phosphinic acid, a GABA_B receptor antagonist. The paired‐pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA_B heteroreceptors following GAT‐1 or GAT‐3 blockade. In conclusion, our findings demonstrate that presynaptic GABA_B heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced by GAT inactivation, thereby suggesting that GAT blockade represents a potential mechanism by which overactive subthalamopallidal activity may be reduced in parkinsonism.     

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

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

Tonic inhibition of striatal dopamine transmission: effects of benzodiazepine and GABAA receptor antagonists on extracellular dopamine levels

At present, it is unclear whether ligands which bind at the benzodiazepine/GABA receptor complex play a tonic modulatory role with regard to striatal dopamine (DA) transmission. The present study was designed to examine the effects of Ro15-1788, a benzodiazepine (BZ) receptor antagonist, and SR 95531, a GABA_A receptor antagonist, on striatal extracellular DA (DA_[e]) concentrations in anesthetized and awake rats using the technique of in vivo microdialysis. Local administration of Ro15-1788 resulted in a dose-dependent increase in DA_[e] in both anesthetized and awake animals. The Ro15-1788-induced increase in DA_[e] was blocked by coadministration of the BZ agonist diazepam, as well as GABA. Local administration of SR 95531 also resulted in a dose-dependent alteration in striatal DA levels in both anesthetized and awake animals. The SR 95531-induced increase in DA was blocked by coadministration of GABA. The results suggest that GABA may play a tonic inhibitory role with regard to striatal DA transmission.     

γ‐Aminobutyric acid‐ρ expression in ependymal glial cells of the mouse cerebellum

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ‐aminobutyric acid type A (GABA_A) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA‐mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13–PN35). These currents were blocked by TPMPA (a highly specific GABA_Aρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA_A receptors). Pentobarbital failed to modulate GABA_A‐mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT‐PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA_A receptor‐mediated responses that are unique among glial cells of the brain. © 2013 Wiley Periodicals, Inc.     

Circadian pacemaker neurons of the Madeira cockroach are inhibited and activated by GABAA and GABAB receptors

GABA is the most abundant neurotransmitter in the circadian pacemaker circuits of mammals and insects. In the Madeira cockroach the accessory medulla (AME) in the brain′s optic lobes is the circadian clock that orchestrates rest‐activity rhythms in synchrony with light dark cycles. Three prominent GABAergic tracts connect the AME to termination sites of compound eye photoreceptors in the lamina and medulla. Parallel GABAergic light entrainment pathways were suggested to either advance or delay the clock for adjustment to changing photoperiods. In agreement with this hypothesis GABA activated or inhibited AME clock neurons, allowing for the distinction of three different GABA response types. Here, we examined which GABA receptors are responsible for these response types. We found that both ionotropic GABA_A receptors and metabotropic GABA_B receptors were expressed in AME clock cells. Via different signalling pathways, either one of them could account for all three GABA response types. The muscimol‐dependently activated GABA_A receptor formed a chloride channel, while the SKF 97541‐dependently activated GABA_B receptor signalled via G‐proteins, apparently targeting potassium channels. Expression of chloride exporters or importers determined whether GABA_A receptor activation hyper‐ or depolarized AME neurons. For GABA_B receptor responses second messenger gated channels present in the clock cells appeared to decide about the polarity of the GABA response. In summary, circadian clock neurons co‐expressed inhibitory and/or excitatory GABA_A and GABA_B receptors in various combinations, while cotransporter expression and the set of second messenger gated ion channels present allowed for distinct signalling in different clock neurons.     

Robust tonic GABA currents can inhibit cell firing in mouse newborn neocortical pyramidal cells

Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl^? reversal potential (E_Cl). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA_A receptors (GABA_ARs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2–4] then decreases dramatically by the second postnatal week (P7–10 and P30–40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA_AR α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 μM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E_Cl recorded from P2–5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability.     

Tonic GABAergic control of mouse dentate granule cells during postnatal development

The dentate gyrus is the main hippocampal input structure receiving strong excitatory cortical afferents via the perforant path. Therefore, inhibition at this ‘hippocampal gate’ is important, particularly during postnatal development, when the hippocampal network is prone to seizures. The present study describes the development of tonic GABAergic inhibition in mouse dentate gyrus. A prominent tonic GABAergic component was already present at early postnatal stages (postnatal day 3), in contrast to the slowly developing phasic postsynaptic GABAergic currents. Tonic currents were mediated by GABA_A receptors containing α₅‐ and δ‐subunits, which are sensitive to low ambient GABA concentrations. The extracellular GABA level was determined by synaptic GABA release and GABA uptake via the GABA transporter 1. The contribution of these main regulatory components was surprisingly stable during postnatal granule cell maturation. Throughout postnatal development, tonic GABAergic signals were inhibitory. They increased the action potential threshold of granule cells and reduced network excitability, starting as early as postnatal day 3. Thus, tonic inhibition is already functional at early developmental stages and plays a key role in regulating the excitation/inhibition balance of both the adult and the maturing dentate gyrus.     

Tonic GABAA Receptor-Mediated Currents of Human Cortical GABAergic Interneurons Vary Amongst Cell Types

Persistent anion conductances through GABA_A receptors (GABA_ARs) are important modulators of neuronal excitability. However, it is currently unknown how the amplitudes of these currents vary among different cell types in the human neocortex, particularly among diverse GABAergic interneurons. We have recorded 101 interneurons in and near layer 1 from cortical tissue surgically resected from both male and female patients, visualized 84 of them and measured tonic GABA_AR currents in 48 cells with an intracellular [Cl^–] of 65 mm and in the presence of 5 μm GABA. We compare these tonic currents among five groups of interneurons divided by firing properties and four types of interneuron defined by axonal distributions; rosehip, neurogliaform, stalked-bouton, layer 2–3 innervating and a pool of other cells. Interestingly, the rosehip cell, a type of interneuron only described thus far in human tissue, and layer 2–3 innervating cells exhibit larger tonic currents than other layer 1 interneurons, such as neurogliaform and stalked-bouton cells; the latter two groups showing no difference. The positive allosteric modulators of GABA_ARs allopregnanolone and DS2 also induced larger current shifts in the rosehip and layer 2–3 innervating cells, consistent with higher expression of the δ subunit of the GABA_AR in these neurons. We have also examined how patient parameters, such as age, seizures, type of cancer and anticonvulsant treatment may alter tonic inhibitory currents in human neurons. The cell type-specific differences in tonic inhibitory currents could potentially be used to selectively modulate cortical circuitry.SIGNIFICANCE STATEMENT Tonic currents through GABA_A receptors (GABA_ARs) are a potential therapeutic target for a number of neurologic and psychiatric conditions. Here, we show that these currents in human cerebral cortical GABAergic neurons display cell type-specific differences in their amplitudes which implies differential modulation of their excitability. Additionally, we examine whether the amplitudes of the tonic currents measured in our study show any differences between patient populations, finding some evidence that age, seizures, type of cancer, and anticonvulsant treatment may alter tonic inhibition in human tissue. These results advance our understanding of how pathology affects neuronal excitability and could potentially be used to selectively modulate cortical circuitry.