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.     

Unique Actions of GABA Arising from Cytoplasmic Chloride Microdomains

Developmental, cellular, and subcellular variations in the direction of neuronal Cl^– currents elicited by GABA_A receptor activation have been frequently reported. We found a corresponding variance in the GABA_A receptor reversal potential (E_GABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl^–]_i) in individual dendrites. We determined [Cl^–]_i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl^–-sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl^–-sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of E_GABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl^–]_i were highly correlated. [Cl^–]_i microdomains persisted after pharmacological inhibition of cation–chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl^–]_i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl^– microdomains that modify the impact of GABAergic inputs.SIGNIFICANCE STATEMENT Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABA_A response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABA_A reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.     

Dynamic regulation of GABAA receptors at synaptic sites

γ-Aminobutyric acid type A receptors (GABA_ARs) mediate fast synaptic inhibition in brain and spinal cord. They are ligand-gated ion channels composed of numerous distinct subunit combinations. For efficient synaptic transmission, GABA_ARs need to be localized to and anchored at postsynaptic sites in precise apposition to presynaptic nerve terminals that release the neurotransmitter GABA. Neurons therefore require distinct mechanisms to regulate intracellular vesicular protein traffic, plasma membrane insertion, synaptic clustering and turnover of GABA_ARs. The GABA_A receptor-associated protein GABARAP interacts with the γ2 subunit of GABA_ARs and displays high homology to proteins involved in membrane fusion underlying Golgi transport and autophagic processes. The binding of GABARAP with NSF, microtubules and gephyrin together with its localization at intracellular membranes suggests a role in GABA_AR targeting and/or degradation. Growth factor tyrosine kinase receptor activation is involved in the control of GABA_AR levels at the plasma membrane. In particular insulin recruits GABA_ARs to the cell surface. Furthermore, the regulation of GABA_AR surface half-life can also be the consequence of negative modulation at the proteasome level. Plic-1, a ubiquitin-like protein binds to both the proteasome and GABA_ARs and the Plic1-GABA_AR interaction is important for the maintenance of GABA-activated current amplitudes. At synaptic sites, GABA_ARs are clustered via gephyrin-dependent and gephyrin-independent mechanisms and may subsequently become internalized via clathrin-mediated endocytosis underlying receptor recycling or degradation processes. This article discusses these recent data in the field of GABA_AR dynamics.     

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.     

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.     

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABA_A receptors (GABA_ARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABA_ARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg⁻¹ day⁻¹, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABA_ARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl⁻ currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC₅₀ or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn²⁺ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn²⁺ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABA_AR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABA_AR development across the brain, although the mechanism(s) underlying this action remains to be determined.     

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.     

Nucleus-specific modulation of phasic and tonic inhibition by endogenous neurosteroidogenesis in the murine thalamus

Neurosteroids are potent allosteric modulators of GABA_A receptors (GABA_ARs). Although the effects of exogenous neurosteroids on GABA_AR function are well documented, less is known about effects of neurosteroids produced by local endogenous biosynthesis. The neurosteroidogenic enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase are expressed in two nuclei of somatosensory thalamus, the thalamic reticular nucleus (nRT) and ventrobasal nucleus (VB). Here, the effects of acute blockade of neurosteroidogenesis by the 5α-reductase inhibitor finasteride on phasic and tonic GABA_AR-mediated currents were examined in nRT and VB of mice. In nRT, finasteride altered the decay and amplitude, but not the frequency, of phasic currents, with no effect on tonic inhibition. In VB neurons, by contrast, finasteride reduced both the size and frequency of phasic currents, and also reduced the degree of tonic inhibition. These studies thus provide novel evidence for endogenous modulation of GABA_AR function by 5α-reduced neurosteroids in the mature thalamus.     

GABA(A) receptor-mediated excitation in dissociated neurons from human hypothalamic hamartomas

The cellular mechanisms underlying intrinsic epileptogenesis in human hypothalamic hamartoma (HH) are unknown. We previously reported that HH tissue is composed predominantly of GABAergic neurons, but how GABAergic-neuron-rich HH tissue is intrinsically epileptogenic is unclear. Here, we tested the hypotheses that some HH neurons exhibit immature features and that GABA excites these neurons via activation of GABA_A receptors (GABA_ARs). Gramicidin-perforated and cell-attached patch-clamp recordings were performed using freshly-dissociated HH neurons to evaluate GABA_AR-mediated currents, Cl⁻ equilibrium potentials, and intracellular Cl⁻ concentrations. Single-cell RT-PCR and immunocytochemical techniques were used to examine cation-Cl⁻ co-transporter (NKCC1 and KCC2) gene and KCC2 protein expression and molecular markers of maturation. From a total of 93 acutely-dissociated HH neurons from 34 patients, 76% were small (soma: 6–9 μm) and 24% were large (soma: > 20 µm) in size. Under gramicidin-perforated patch recording conditions, GABA_AR activation depolarized/excited large but hyperpolarized/inhibited small HH neurons in most cases. Compared to small HH neurons, large HH neurons exhibited more positive Cl⁻ equilibrium potentials, higher intracellular Cl⁻ concentrations, lower KCC2 expression, and an immature phenotype, consistent with GABA_AR-mediated excitation. Taken collectively, we provide novel evidence for and mechanistic insights into HH epileptogenicity: GABA_AR-mediated excitation.     

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.     

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.     

Chronic neurosteroid treatment attenuates single cell GABAA response and its potentiation by modulators in cortical neurons

In previous studies we have observed that chronic neurosteroid 5α-pregnan-3α-ol-20-one (5α3α) treatment produced downregulation of the GABA_A receptors, heterologous uncoupling, and decreased heterologous efficacy at the GABA_A receptor complex in cultured mammalian cortical neurons. In this study, using whole cell recording, we examined the consequence of chronic 5α3α (1 μM; 5 days) treatment on GABA-induced currents in isolated cortical neurons. We observed that the GABA current was decreased by 78% after 5 days treatment of cortical cells with 1 μM; 5α3α. We also observed decreased pentobarbital, and 5α3α potentiation of GABA currents after chronic 5α3α treatment. These findings support the notion that GABA response, and its potentiation by pentobarbital, and neurosteroid, 5α3α, are attenuated after chronic 5α 3α treatment.     

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.     

GABAA receptor subunit composition and competition at synapses are tuned by GABAB receptor activity

GABA_BRs have a well-established role in controlling neuronal excitability and presynaptic neurotransmitter release. We examined the role of GABA_BR activity in modulating the number and lateral diffusion of GABA_ARs at inhibitory synapses. Changes in diffusion of GABA_ARs at synapses were observed when subunit heterogeneity was taken into account. While α1-GABA_ARs were unaffected, α2- and α5-GABA_ARs showed inverse changes in enrichment and diffusion. The intracellular TM3-4 loop of α2 was sufficient to observe the changes in diffusion by GABA_BR activity, whereas the loop of α5 was not. The opposing effect on α2- and α5-GABA_ARs was caused by a competition between GABA_ARs for binding slots at synapses. Receptor immobilization by cross-linking revealed that α5-GABA_AR trapping at synapses is regulated by modulation of α2-GABA_AR mobility. Finally, PKC activity was determined to be part of the signaling pathway through which GABA_BR activity modulates α2-GABA_AR diffusion at synapses. These results outline a novel mechanism for tuning inhibitory transmission in a subunit-specific manner, and for the first time describe competition between GABA_ARs with different subunit compositions for binding slots at synapses.     

Experimental early‐life febrile seizures induce changes in GABAAR‐mediated neurotransmission in the dentate gyrus

Purpose: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ‐aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA_AR function in dentate granule cells. Methods: We applied an immature rat model of hyperthermia (HT)–induced FS. GABA_AR‐mediated neurotransmission was studied using whole‐cell patch‐clamp recordings from dentate granule neurons in hippocampal slices within 6–9 days post‐HT. Key Findings: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole‐cell GABA responses revealed a doubled GABA_AR sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole‐cell GABA responses showed similar kinetics in postsynaptic GABA_ARs of HT and NT rats. quantitative real‐time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA_AR subunit expression, which was most pronounced for the α3 subunit. Significance: The data support the hypothesis that FS persistently alter neuronal excitability.     

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.     

Cell type-specific distribution of GABAA receptor subtypes in the mouse dorsal striatum

The striatum is the main input nucleus of the basal ganglia, mediating motor and cognitive functions. Striatal projection neurons are GABAergic medium spiny neurons (MSN), expressing either the dopamine receptor type 1 (D₁-R MSN) and forming the direct, movement-promoting pathway, or dopamine receptor type 2 (D₂-R MSN), forming the indirect movement-suppressing pathway. Locally, activity and synchronization of MSN are modulated by several subtypes of GABAergic and cholinergic interneurons. Overall, GABAergic circuits in the striatum remain poorly characterized, and little is known about the intrastriatal connectivity of interneurons and the distribution of GABA_A receptor (GABA_AR) subtypes, distinguished by their subunit composition, in striatal synapses. Here, by using immunofluorescence in mouse tissue, we investigated the distribution of GABA_ARs containing the α₁, α₂, or α₃ subunit in perisomatic synapses of striatal MSN and interneurons, as well as the innervation pattern of D₁R- and D₂R-MSN soma and axonal initial segment (AIS) by GABAergic and cholinergic interneurons. Our results show that perisomatic GABAergic synapses of D₁R- and D₂R-MSN contain the GABA_AR α₁ and/or α₂ subunits, but not the α₃ subunit; D₂R-MSN have significantly more α₁-GABA_ARs on their soma than D₁R-MSN. Further, interneurons have few perisomatic synapses containing α₂-GABA_ARs, whereas α₃-GABA_ARs (along with the α₁-GABA_ARs) are abundant in perisomatic synapses of CCK⁺, NPY⁺/SOM⁺, and vAChT⁺ interneurons. Each MSN and interneuron population analyzed received a distinct pattern of GABAergic and cholinergic innervation, complementing this postsynaptic heterogeneity. In conclusion, intra-striatal GABAergic circuits are distinguished by cell-type specific innervation patterns, differential expression and postsynaptic targeting of GABA_AR subtypes.     

Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development

The volatile anesthetic sevoflurane, which is widely used in pediatric surgery, has proposed effects on GABA_A receptor‐mediated extrasynaptic tonic inhibition. In the developing striatum, medium‐sized spiny projection neurons have tonic GABA currents, which function in the excitatory/inhibitory balance and maturation of striatal neural circuits. In this study, we examined the effects of sevoflurane on the tonic GABA currents of medium spiny neurons in developing striatal slices. Sevoflurane strongly increased GABA_A receptor‐mediated tonic conductance at postnatal days 3–35. The antagonist of the GABA transporter‐1, 1‐[2‐[[(diphenylmethylene)imino]oxy]ethyl]‐1,2,5,6‐tetrahydro‐3‐pyridinecarboxylic acid hydrochloride further increased tonic GABA conductance during the application of sevoflurane, thereby increasing the total magnitude of tonic currents. Both GABA (5 μm ) and 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol hydrochloride, the δ‐subunit‐containing GABA_A receptor agonist, induced tonic GABA currents in medium spiny neurons but not in cholinergic neurons. However, sevoflurane additively potentiated the tonic GABA currents in both cells. Interestingly, 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol hydrochloride‐sensitive neurons made a large current response to sevoflurane, indicating the contribution of the δ‐subunit on sevoflurane‐enhanced tonic GABA currents. Our findings suggest that sevoflurane can affect the tone of tonic GABA inhibition in a developing striatal neural network.     

A single dose of lorazepam reduces paired‐pulse suppression of median nerve evoked somatosensory evoked potentials

Paired‐pulse behaviour in the somatosensory cortex is an approach to obtain insights into cortical processing modes and to obtain markers of changes of cortical excitability attributable to learning or pathological states. Numerous studies have demonstrated suppression of the response to the stimulus that follows a first one after a short interval, but the underlying mechanisms remain elusive, although there is agreement that GABAergic mechanisms seem to play a crucial role. We therefore aimed to explore the influence of the GABA_A agonist lorazepam on paired‐pulse somatosensory evoked potentials (SEPs). We recorded and analysed SEPs after paired median nerve stimulation in healthy individuals before and after they had received a single dose of 2.5 mg of lorazepam as compared with a control group receiving placebo. Paired‐pulse suppression was expressed as a ratio of the amplitudes of the second and the first peaks. We found that, after lorazepam application, paired‐pulse suppression of the cortical N20 component remained unchanged, but suppression of the N20–P25 complex was significantly reduced, indicative of GABAergic involvement in intracortical processing. Our data suggest that lorazepam most likely enhances inhibition within the cortical network of interneurons responsible for creating paired‐pulse suppression, leading to reduced inhibitory drive with a subsequently reduced amount of suppression. The results provide further evidence that GABA_A‐mediated mechanisms are involved in the generation of median nerve evoked paired‐pulse suppression.     

Differential intracellular regulation of cortical GABAA and spinal glycine receptors in cultured neurons

Using patch-clamp techniques we studied several aspects of intracellular GABA_A and glycine Cl⁻ current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl⁻ current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 μM PMA reduced the amplitude of the GABA_A and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 μM okadaic acid, a phospatase inhibitor, induced potentiation in both currents. The amplitude of GABA_A and glycine currents recorded with 1 mM internal CaCl₂ and 10 mM EGTA (10 nM free Ca²⁺) decayed by less than 30% of control. Increasing the CaCl₂ concentration to 10 mM (34 μM free Ca²⁺) induced a transient potentiation of the GABA_A current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca²⁺ concentration. Activation of G proteins with internal FAl⁻₄ induced an inhibition of the GABA_A current, but potentiated the amplitude of the strychnine-sensitive Cl⁻ current. These results indicate that GABA_A and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca²⁺. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.