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.     

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.     

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.     

Intrinsic activation of GABAA receptors suppresses epileptiform activity in the cerebral cortex of immature mice

Purpose: Activation of ionotropic γ‐aminobutyric acid type A (GABA_A) receptors induces in immature neocortical neurons a membrane depolarization that may contribute to the higher epilepsy susceptibility in newborns. To elucidate whether depolarizing GABAergic responses enhance or attenuate epileptiform activity in the immature neocortex, we investigated the effect of agonists, antagonists, and positive modulators of GABA_A receptors on epileptiform activity. Methods: We performed in vitro field potential recordings on isolated whole neocortex preparations and whole cell recordings of identified pyramidal neurons in 400‐μm slices of immature (postnatal day 1–7) mice. Epileptiform activity was induced by low Mg²⁺ solutions with or without 50–100 μm 4‐aminopyridine. Results: Bath application of GABA (3–100 μm , in the presence of tiagabine) attenuated epileptiform activity. The GABA transporter isoform 1 (GAT‐1) inhibitor tiagabine (30 μm ) and the GAT‐2/3 specific inhibitor SNAP 5114 (40 μm ) reduced the frequency of epileptiform activity. The benzodiazepines midazolam (0.2 μm ) and zolpidem (0.5 μm ) as well as the barbiturate phenobarbital (30 μm ) slightly attenuated epileptiform activity. Continuous bath application of the GABAergic antagonist gabazine (SR‐95531, 2–3 μm ) or picrotoxin (15 μm ) induced epileptiform discharges. Discussion: These results demonstrate, that (1) the activation or positive modulation of GABA_A receptors attenuates epileptiform activity, (2) GABA_A antagonists mediate a disinhibition, and (3) GABA uptake contributes to the regulation of extracellular GABA in immature neocortex. We conclude from these findings that a constant inhibition via GABA_A receptors is required to suppress epileptiform activity already in the immature neocortex.     

Reduced tonic inhibition in the dentate gyrus contributes to chronic stress‐induced impairments in learning and memory

It is well established that stress impacts the underlying processes of learning and memory. The effects of stress on memory are thought to involve, at least in part, effects on the hippocampus, which is particularly vulnerable to stress. Chronic stress induces hippocampal alterations, including but not limited to dendritic atrophy and decreased neurogenesis, which are thought to contribute to chronic stress‐induced hippocampal dysfunction and deficits in learning and memory. Changes in synaptic transmission, including changes in GABAergic inhibition, have been documented following chronic stress. Recently, our laboratory demonstrated shifts in E_GABA in CA1 pyramidal neurons following chronic stress, compromising GABAergic transmission and increasing excitability of these neurons. Interestingly, here we demonstrate that these alterations are unique to CA1 pyramidal neurons, since we do not observe shifts in E_GABA following chronic stress in dentate gyrus granule cells. Following chronic stress, there is a decrease in the expression of the GABA_A receptor (GABA_AR) δ subunit and tonic GABAergic inhibition in dentate gyrus granule cells, whereas there is an increase in the phasic component of GABAergic inhibition, evident by an increase in the peak amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the numerous changes observed in the hippocampus following stress, it is difficult to pinpoint the pertinent contributing pathophysiological factors. Here we directly assess the impact of a reduction in tonic GABAergic inhibition of dentate gyrus granule cells on learning and memory using a mouse model with a decrease in GABA_AR δ subunit expression specifically in dentate gyrus granule cells (Gabrd/Pomc mice). Reduced GABA_AR δ subunit expression and function in dentate gyrus granule cells is sufficient to induce deficits in learning and memory. Collectively, these findings suggest that the reduction in GABA_AR δ subunit‐mediated tonic inhibition in dentate gyrus granule cells contributes, at least in part, to deficits in learning and memory associated with chronic stress. These findings have significant implications regarding the pathophysiological mechanisms underlying impairments in learning and memory associated with stress and suggest a role for GABA_AR δ subunit containing receptors in dentate gyrus granule cells. © 2016 Wiley Periodicals, Inc.     

GABA-activated Chloride Currents of Postnatal Mouse Retinal Ganglion Cells are Blocked by Acetylcholine and Acetylcarnitine: How Specific are Ion Channels in Immature Neurons?

The goal of this study was to clarify pharmacological properties of GABA_A receptors in cells of the mouse retinal ganglion cell layer in situ. Spontaneous synaptic currents and responses to exogenous GABA were recorded from individual neurons in retinal whole mounts (postnatal days 1–3) or retinal stripe preparations (postnatal days 4–6). Drugs were applied by a fast local superfusion system. Current responses were measured with the patch-clamp technique in the whole-cell configuration. All cells responded to exogenous GABA (average EC₅₀ and Hill coefficient: 16.7 μM and 0.95 respectively) and generated GABAergic synaptic currents in response to elevated KCI. GABA-induced currents of retinal ganglion cells were blocked by bicuculline, picrotoxin and Zn²⁺, as well as strychnine, and increased by pentobarbital, clonazepam and 3α-hydroxy-5α-pregnan-20-one. In some retinal ganglion cells GABA caused an increase in the frequency of spontaneous synaptic currents, which points to a partially depolarizing action of this traditionally inhibitory neurotransmitter in the neural retina. Our major observation is that acetylcholine and acetylcarnitine blocked or reduced GABAergic inhibitory postsynaptic currents and responses to exogenous GABA. This effect was seen in only a fraction of retinal ganglion cells and occurred in both the undesensitized and the desensitized state of the GABA_A receptor. The block was voltage-independent and persisted during coapplication with the nicotinic and muscarinic acetylcholine receptor antagonists D-tubocurarine and atropine. In contrast to GABA-activated Cl⁻ currents, glycine-activated Ch currents remained unaffected by acetylcholine and acetylcarnitine. Acetylcarnitine had no effect on voltage-activated Ca²⁺ channel currents and glutamate-activated currents. Similar results were obtained in a dissociated cell culture preparation from the neonatal rat superior colliculus. In these cells acetylcholine induced a rightward shift in the dose - response curve for GABA. Taken together, these results indicate that acetylcholine and acetylcarnitine can act directly at the GABA_A binding site and thereby reduce the action of GABA in the immature retina.     

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.     

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.     

GABA induces GABAergic MSCs in cultured embryonic rat thalamic neurons

Application of 0.1–10 μM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 μM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl⁻ equilibrium potential. These MSCs were blocked by the GABA_A receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl⁻ channels activated pharmacologically by applying 1–10 μM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABA_A receptor agonist muscimol activated Cl⁻ current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl⁻ channels. The GABA_B receptor agonist (-)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca²⁺. Verapamil and Co²⁺, which block voltage-dependent calcium channels, completely blocked GABA-induced MSCs independent of their effects on the direct activation of a Cl⁻ current response. The results indicate that GABA can trigger GABAergic Cl⁻-dependent MSCs in a Ca_o²⁺-dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway. Synapse 25:15–23, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Development of Action Potential-dependent and Independent Spontaneous GABAA Receptor-mediated Currents in Granule Cells of Postnatal Rat Cerebellum

The postnatal development of spontaneous GABAergic transmission between cerebellar Golgi cells and granule cells was investigated with voltage-clamp recording from rat cerebellar slices, in symmetrical Cl^-conditions. Between postnatal days 7 and 14 (P7–14), bicuculline-and TTX (tetrodotoxin)-sensitive spontaneous inhibitory postsynaptic currents (sIPSCs), occurred at high frequency in 56% of granule cells. Between P10 and P14, sIPSCs were superimposed on tonic current of-12 ± 1.8 pA at -70 mV, that was accompanied by noise with variance of 17 ± 3 pA². Both the current and noise were inhibited by bicuculline. TTX blocked the bicuculline-sensitive current and noise by?60%. Between P18 and P25, sIPSCs were less frequent; all cells showed tonic, bicuculline-sensitive currents, but these were partially inhibited by TTX (?35%). Between P40 and P53, slPSCs were rare; tonic, bicuculline-sensitive currents and noise were greater in amplitude, with mean values of-17 pA and 22 pA² at-70 mV, they were present in all cells but they were not inhibited by TTX. Glycine receptor channels that were expressed in immature, but not adult cells, did not mediate spontaneous currents. Our results indicate that spontaneous transmission onto cerebellar granule cells in immature animals consists primarily of action potential-dependent, phasic release of vesicular GABA. This generates GABA_A receptor-mediated slPSCs. The effects of GABA transporter blockers suggest that it also produces the TTX-sensitive current-noise, as GABA spills out of synapses to activate extrasynaptic receptors or receptors in neighbouring synapses. In older animals, action potential-independent release of transmitter is predominant and results in tonic activation of GABA_A receptors. This does not appear to be spontaneous vesicular release of GABA. Neither does it appear to be reversed uptake of GABA, although further work is required to rule out these possibilities.     

Synchronous firing of inhibitory interneurons results in saturation of fast GABAA IPSC magnitude but not saturation of fast inhibitory efficacy in rat neocortical pyramidal cells

The kinetic properties of evoked fast inhibitory postsynaptic currents were examined to elucidate factors underlying the limit on the magnitude of fast inhibition in neocortex. Using whole-cell voltage-clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex, fast γ-aminobutyric acid-A (GABA_A)ergic inhibitory postsynaptic currents were selectively recorded by holding cells at potentials equal to excitatory postsynaptic current reversal (∼0 mV). As stimulus intensity was increased, the magnitude and duration of the fast inhibitory postsynaptic current increased. Over the range of stimuli applied (2–10 V), fast GABA_A-mediated inhibitory postsynaptic currents reached a maximum peak conductance of 25.9 ± 4.2 nS (range 10.5–41.2 nS) at intensities approximately 2-times threshold. As stimulus intensities were increased beyond this point of maximal conductance, the time constant of current decay increased as function of stimulus strength, while rise time remained unaffected. Exposure to nominally magnesium-free solutions did not affect maximal peak conductances of fast inhibitory postsynaptic currents, but did cause an increase in the time constants of current decay by 66.3 ± 23.6%, resulting in an 85.6 ± 24.6% increase in the total charge flux carried by single inhibitory postsynaptic currents. This effect may be due to prolonged activation of postsynaptic GABA_A receptors by excess GABA released in response to increased excitation. Exposure to the GABA uptake blocker, nipecotic acid, similarly prolonged current decay without affecting the maximal peak conductance. Our findings suggest that the limit on recruitment of evoked fast inhibition in neocortical layer V pyramidal cells arises from the saturation of postsynaptic GABA_A receptors. However, while there is a limit to the peak fast inhibitory postsynaptic conductance which can be recruited with increasing excitation, inhibitory strength may still be modulated by increasing charge flux through the prolongation of fast inhibition. Synapse 28:91–102, 1998. © 1998 Wiley-Liss, Inc.     

The Neuroactive Steroid 5α-Tetrahydrodeoxycorticosterone Increases GABAergic Postsynaptic Inhibition in Rat Neocortical Neurons in vitro

The neuroactive steroid 5α-pregnane-3α,21-diol-20-one (5α-tetrahydrodeoxycorticosterone; 5α-THDOC) has been shown to potentiate GABA-induced chloride currents in cell cultures and subcellular preparations. In this study, we recorded from pyramidal neurons in an in vitro slice preparation of the adult rat frontal neocortex using intracellular microelectrodes. 5α-THDOC (10 μM) increased and prolonged the inhibitory postsynaptic potential (IPSP). The mean maximal synaptic conductance of the early, GABA_A receptor-mediated, IPSP was enhanced to more than 700%, the one at the maximum of the late, partially GABA_A receptor-mediated, IPSP to approximately 400%. The progesterone/glucocorticoid receptor antagonist RU 38486 did not prevent the IPSP increase. At a concentration of 1 μM 5α-THDOC increased only the early IPSP to about 125%. Responses to the iontophoretically applied specific GABA_A receptor agonist muscimol but not to the specific GABA_B receptor agonist L-baclofen were enhanced by 5α-THDOC (10 μM). In the giga-seal whole-cell configuration when the GABA_B receptor-mediated IPSP component was absent due to intracellular perfusion, 5α-THDOC (10 μM) increased IPSPs to a similar extent as in the conventional microelectrode recordings. Excitatory postsynaptic potentials, resting membrane potential, input resistance and action potential amplitude were not affected by 5α-THDOC (10 μM). These data demonstrate that in neocortical tissue of the rat 5α-THDOC enhances GABAergic inhibition by interacting with postsynaptic GABA_A receptors while synaptic excitation and parameters of electric excitability remain unchanged.     

Tonically active NMDA receptors – a signalling mechanism critical for interneuronal excitability in the CA1 stratum radiatum

In contrast to tonic extrasynaptic γ‐aminobutyric acid (GABA)_A receptor‐mediated signalling, the physiological significance of tonic extrasynaptic N‐methyl‐d ‐aspartate (NMDA) receptor (NMDAR)‐mediated signalling remains uncertain. In this study, reversible open‐channel blockers of NMDARs, memantine and phencyclidine (PCP) were used as tools to examine tonic NMDAR‐mediated signalling in rat hippocampal slices. Memantine in concentrations up to 10 μm had no effect on synaptically evoked NMDAR‐mediated responses in pyramidal neurons or GABAergic interneurons. On the other hand, 10 μm memantine reduced tonic NMDAR‐mediated currents in GABAergic interneurons by approximately 50%. These tonic NMDAR‐mediated currents in interneurons contributed significantly to the excitability of the interneurons as 10 μm memantine reduced the disynaptic inhibitory postsynaptic current in pyramidal cells by about 50%. Moreover, 10 μm memantine, but also PCP in concentrations ≤ 1 μm , increased the magnitude of the population spike, likely because of disinhibition. The relatively higher impact of tonic NMDAR‐mediated signalling in interneurons was at least partly explained by the expression of GluN2D‐containing NMDARs, which was not observed in mature pyramidal cells. The current results are consistent with the idea that low doses of readily reversible NMDAR open‐channel blockers preferentially inhibit tonically active extrasynaptic NMDARs, and they suggest that tonically active NMDARs contribute more prominently to the intrinsic excitation in GABAergic interneurons than in pyramidal cells. It is proposed that this specific difference between interneurons and pyramidal cells can explain the disinhibition caused by the Alzheimer's disease medication memantine.     

Synaptic inhibition in the isolated respiratory network of neonatal rats

Gramicidin-perforated patch-clamp recording revealed phasic Cl^–-mediated hyperpolarizations in respiratory neurons of the brainstem–spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of γ-aminobutyric acid (GABA), i.e. GABA_A, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO₂/HCO₃^–-free solutions, but shifted from about ?65 mV to values more positive than ?20 mV upon dialysis of the cells with 144 instead of 4 mm Cl^–. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABA_B receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K⁺ channel-mediated hyperpolarization (reversal potential ?88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABA_A and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABA_B receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.     

Activity‐dependent intracellular chloride accumulation and diffusion controls GABAA receptor‐mediated synaptic transmission

In the CNS, prolonged activation of GABA_A receptors (GABA_ARs) has been shown to evoke biphasic postsynaptic responses, consisting of an initial hyperpolarization followed by a depolarization. A potential mechanism underlying the depolarization is an acute chloride (Cl^?) accumulation resulting in a shift of the GABA_A reversal potential (E_GABA). The amount of GABA‐evoked Cl^? accumulation and accompanying depolarization depends on presynaptic and postsynaptic properties of GABAergic transmission, as well as on cellular morphology and regulation of Cl^? intracellular concentration ([Cl^?]_i). To analyze the influence of these factors on the Cl^? and voltage behavior, we studied spatiotemporal dynamics of activity‐dependent [Cl^?]_i changes in multicompartmental models of hippocampal cells based on realistic morphological data. Simulated Cl^? influx through GABA_ARs was able to exceed physiological Cl^? extrusion rates thereby evoking HCO₃^? ‐dependent E_GABA shift and depolarizing responses. Depolarizations were observed in spite of GABA_A receptor desensitization. The amplitude of the depolarization was frequency‐dependent and determined by intracellular Cl^? accumulation. Changes in the dendritic diameter and in the speed of GABA clearance in the synaptic cleft were significant sources of depolarization variability. In morphologically reconstructed granule cells subjected to an intense GABAergic background activity, dendritic inhibition was more affected by accumulation of intracellular Cl^? than somatic inhibition. Interestingly, E_GABA changes induced by activation of a single dendritic synapse propagated beyond the site of Cl^? influx and affected neighboring synapses. The simulations suggest that E_GABA may differ even along a single dendrite supporting the idea that it is necessary to assign E_GABA to a given GABAergic input and not to a given neuron. © 2010 Wiley‐Liss, Inc.     

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.     

Hippocampal GABAergic dysfunction in a rat chronic mild stress model of depression

In major depression, one line of research indicates that a dysfunctional GABAergic inhibitory system is linked to the appearance of depressive symptoms. However, as the mechanistic details of such GABAergic deficit are largely unknown, we undertook a functional investigation of the GABAergic system in the rat chronic mild stress model of depression. Adult rats were exposed to an eight‐week long stress protocol leading to anhedonic‐like behavior. In hippocampal brain slices, phasic, and tonic GABA_A receptor‐mediated currents in dentate gyrus granule cells were examined using patch‐clamp recordings. In granule cells, the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was reduced to 41% in anhedonic‐like rats, which was associated with a reduced probability of evoked GABA release. Using immunohistochemical analysis, there was no change in the number of parvalbumin‐positive interneurons in the dentate gyrus. Notably, we observed a 60% increase in THIP‐activated tonic GABA_A mediated current in anhedonic‐like rats, suggesting an upregulation of extrasynaptic GABA_A receptors. Finally, five weeks treatment with the antidepressant escitalopram partially reversed the sIPSCs frequency. In summary, we have revealed a hippocampal dysfunction in the GABAergic system in the chronic mild stress model of depression in rats, caused by a reduction in action potential‐dependent GABA release. Since the function of the GABAergic system was improved by antidepressant treatment, in parallel with behavioral read outs, it suggests a role of the GABAergic system in the pathophysiology of depression. © 2010 Wiley‐Liss, Inc.     

Tonic GABA(A) receptor-mediated currents in human brain

GABA(A) receptors can mediate both phasic (synaptic) and tonic (extrasynaptic) forms of inhibition. It has been proposed that tonic inhibition plays a critical part in controlling neuronal and network excitability. Although tonic GABA(A) receptor-mediated currents have been well characterized in rodents, their existence in human tissue has yet to be demonstrated. Here we show that tonic currents can be recorded from human tissue obtained from patients undergoing temporal lobectomies. Tonic GABA(A) receptor-mediated currents were present in pyramidal cells and interneurons in layer V-VI of temporal neocortex and granule cells in the dentate gyrus. These tonic currents have cell type-specific pharmacologies, opening up the possibility of targeted therapeutics.     

Intracellular calcium stores modulate miniature GABA‐mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature γ-aminobutyric acid_A (GABA_A) receptor-mediated currents (in tetrodotoxin, 1 μm and kynurenic acid 1 mm ) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6–9) old rats. Switching from a Ca²⁺-containing to a nominally Ca²⁺-free medium (in which Ca²⁺ was substituted with Mg²⁺, in the presence or in the absence of 100 μm EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 μm over control) was 1.8 ± 0.5, (n = 9). In nominally Ca²⁺-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mm ), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mm ) reversibly reduced the amplitude of the current induced by GABA (100 μm ) indicating a clear postsynaptic effect on GABA_A receptor. Superfusion of ryanodine (30 μm ), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine–ryanodine-sensitive stores facilitates GABA release.     

Developmental Changes in Serotonergic Modulation of GABAergic Synaptic Transmission and Postsynaptic GABA<Subscript>A</Subscript> Receptor Composition in the Cerebellar Nuclei

Outputs from the cerebellar nuclei (CN) are important for generating and controlling movement. The activity of CN neurons is controlled not only by excitatory inputs from mossy and climbing fibers and by γ-aminobutyric acid (GABA)-based inhibitory transmission from Purkinje cells in the cerebellar cortex but is also modulated by inputs from other brain regions, including serotonergic fibers that originate in the dorsal raphe nuclei. We examined the modulatory effects of serotonin (5-HT) on GABAergic synapses during development, using rat cerebellar slices. As previously reported, 5-HT presynaptically decreased the amplitudes of stimulation-evoked inhibitory postsynaptic currents (IPSCs) in CN neurons, with this effect being stronger in slices from younger animals (postnatal days [P] 11–13) than in slices from older animals (P19–21). GABA release probabilities accordingly exhibited significant decreases from P11–13 to P19–21. Although there was a strong correlation between the GABA release probability and the magnitude of 5-HT-induced inhibition, manipulating the release probability by changing extracellular Ca²⁺ concentrations failed to control the extent of 5-HT-induced inhibition. We also found that the IPSCs exhibited slower kinetics at P11–13 than at P19–21. Pharmacological and molecular biological tests revealed that IPSC kinetics were largely determined by the prevalence of α₁ subunits within GABA_A receptors. In summary, pre- and postsynaptic developmental changes in serotonergic modulation and GABAergic synaptic transmission occur during the second to third postnatal weeks and may significantly contribute to the formation of normal adult cerebellar function.