Spontaneous GABAergic Postsynaptic Currents Research Articles

Imbalance between the function of Na+-K+-2Cl and K+-Cl impairs Cl- homeostasis in human focal cortical dysplasia.

Altered expression patterns of Na+-K+-2Cl- (NKCC1) and K+-Cl- (KCC2) co-transporters have been implicated in the pathogenesis of epilepsy. Here, we assessed the effects of imbalanced NKCC1 and KCC2 on γ-aminobutyric acidergic (GABAergic) neurotransmission in certain brain regions involved in human focal cortical dysplasia (FCD). We sought to map a micro-macro neuronal network to better understand the epileptogenesis mechanism. In patients with FCD, we resected cortical tissue from the seizure the onset zone (SOZ) and the non-seizure onset zone (non-SOZ) inside the epileptogenic zone (EZ). Additionally, we resected non-epileptic neocortical tissue from the patients with mesial temporal lobe epilepsy (MTLE) as control. All of tissues were analyzed using perforated patch recordings. NKCC1 and KCC2 co-transporters expression and distribution were analyzed by immunohistochemistry and western blotting. Results revealed that depolarized GABAergic signals were observed in pyramidal neurons in the SOZ and non-SOZ groups compared with the control group. The total number of pyramidal neurons showing GABAergic spontaneous postsynaptic currents was 11/14, 7/17, and 0/12 in the SOZ, non-SOZ, and control groups, respectively. The depolarizing GABAergic response was significantly dampened by the specific NKCC1 inhibitor bumetanide (BUM). Patients with FCD exhibited higher expression and internalized distribution of KCC2, particularly in the SOZ group. Our results provide evidence of a potential neurocircuit underpinning SOZ epileptogenesis and non-SOZ seizure susceptibility. Imbalanced function of NKCC1 and KCC2 may affect chloride ion homeostasis in neurons and alter GABAergic inhibitory action, thereby contributing to epileptogenesis in FCDs. Maintaining chloride ion homeostasis in the neurons may represent a new avenue for the development of novel anti-seizure medications (ASMs).

Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21–23days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4–5days. By 35–38days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4–5days and in ~80% by 21–23days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics.

Dysfunctional inhibitory mechanisms in locus coeruleus neurons of the wistar kyoto rat.

Background:The noradrenergic nucleus locus coeruleus (LC) has functional relevance in several psychopathologies such as stress, anxiety, and depression. In addition to glutamatergic and GABAergic synaptic inputs, the activation of somatodendritic α2-adrenoceptors is the main responsible for LC activity regulation. The Wistar Kyoto (WKY) rat exhibits depressive- and anxiety-like behaviors and hyperresponse to stressors. Thus, the goal of the present study was to investigate in vitro the sensitivity of α2-adrenoceptors, as well as the glutamatergic and GABAergic synaptic activity on LC neurons of the WKY strain.Methods:For that purpose patch-clamp whole-cell recordings were done in LC slices.Results:The α2-adrenoceptors of LC neurons from WKY rats were less sensitive to the effect induced by the agonist UK 14 304 as compared to that recorded in the Wistar (Wis) control strain. In addition, the GABAergic input to LC neurons of WKY rats was significantly modified compared to that in Wis rats, since the amplitude of spontaneous GABAergic postsynaptic currents was reduced and the half-width increased. On the contrary, no significant alterations were detected regarding glutamatergic input to LC neurons between rat strains.Conclusions:These results point out that in WKY rats the inhibitory control exerted by α2-adrenoceptors and GABAergic input onto LC neurons is dysregulated. Overall, this study supports in this animal model the hypothesis that claims an imbalance between the glutamatergic-GABAergic systems as a key factor in the pathophysiology of depression.

GABAergic transmission to kisspeptin neurons is differentially regulated by time of day and estradiol in female mice.

Gonadotropin-releasing hormone (GnRH) secretion is regulated by estradiol feedback. This feedback switches from negative to positive in females; this switch depends on time of day in many species. Estradiol feedback is likely conveyed via afferents. Kisspeptin neurons of the arcuate nucleus and anteroventral-periventricular region (AVPV) may differentially regulate GnRH neurons during negative and positive feedback, respectively. We tested estradiol and time of day regulation of GABAergic transmission and postsynaptic response to GABA in these two populations using transgenic mice with GFP-identified kisspeptin neurons. Ovariectomized (OVX) mice treated or not with estradiol (E) were studied in the AM (negative feedback) or PM (positive feedback). GABAA receptor reversal potential was unaffected by time of day or estradiol. GABA depolarized the membrane potential of arcuate neurons from OVX+E mice; this response was blunted in cells from OVX mice. GABA hyperpolarized AVPV kisspeptin neurons, except in the OVX PM group in which GABA did not alter membrane potential attributable to a PM hyperpolarization of baseline membrane potential. In both kisspeptin neuron populations from OVX mice, the frequency of GABAergic spontaneous postsynaptic currents was increased in the PM; this increase was blunted by estradiol. In arcuate, but not AVPV, kisspeptin neurons, estradiol reduced miniature postsynaptic current amplitude independent of time of day. Using nonstationary fluctuation analysis and diazepam to manipulate GABAA receptor apparent affinity, the decrease in arcuate miniature postsynaptic current amplitude was attributed to decreased number of receptors bound by GABA. Time of day and estradiol feedback thus both target presynaptic and postsynaptic mechanisms to differentially regulate kisspeptin neurons via GABAergic transmission.

Inhibition of different GABA transporter systems is required to attenuate epileptiform activity in the CA3 region of the immature rat hippocampus

GABA transporters (GATs) are an essential element of the GABAergic system, which regulate excitability in the central nervous system and are thus used as targets for anticonvulsive therapy. However, in the immature nervous system the functions of the GABAergic system and the expression profile of GATs are distinct from the adult situation, obscuring to predict how different GAT isoforms influence epileptiform activity. Therefore we analyzed the effects of subtype specific GAT inhibitors on repetitive epileptiform discharges using field potential and whole-cell patch-clamp recordings in the CA3 region of hippocampal slices of immature (postnatal days 4-7) rats. These experiments revealed that inhibition of GAT-1 with either tiagabine (30 μM) or NO-711 (10 μM) exhibited only a minor anticonvulsive effect on repetitive epileptiform discharges. Blockade of GAT-2/3 with SNAP-5114 (40 μM) had no anticonvulsive effect, but significantly prolonged the decay of spontaneous GABAergic postsynaptic currents. In contrast, the combined application of 10 μM NO-711 and 40 μM SNAP-5114 blocked epileptiform activity in 33% of all slices and reduced the occurrence of epileptiform discharges by 54% in the remaining slices. In addition, the input resistance decreased by 10.5 ± 1.0% under this condition. These results indicate that both GAT-1 and GAT-2/3 are functional in the immature hippocampus and that only the combined inhibition of GAT 1-3 is sufficient to promote a considerable anticonvulsive effect. We conclude from these results that both GAT-1 and GAT-2/3 act synergistically to regulate the excitability in the immature hippocampus.

Glutamate transporters and presynaptic metabotropic glutamate receptors protect neocortical Cajal–Retzius cells against over-excitation

Cajal-Retzius (CR) cells, early generated neurons in the marginal zone of developing neocortex, are reported to be highly vulnerable to excitotoxic damage. Because extracellular glutamate concentration in the central nervous system is mainly controlled by glutamate transporters (EAATs), we studied the effects of EAAT blockade on CR cells. DL: -TBOA, a specific EAAT antagonist, induced NMDA receptor-dependent bursting discharges in layer 2/3 pyramidal neurons, indicating that EAATs operate in the uptake mode and their blockade leads to elevation of extracellular glutamate concentration. In CR cells, however, DL: -TBOA failed to change either the membrane resistance or holding current, and moreover, it reduced the frequency of spontaneous GABAergic postsynaptic currents. DL: -TBOA decreased the mean amplitude and increased paired-pulse ratio of evoked GABAergic postsynaptic currents, indicating the presynaptic locus of its action. Indeed, LY379268, a specific agonist of group II metabotropic glutamate receptors (mGluR-II), mimicked the DL: -TBOA-mediated effects, and LY341495, an unspecific mGluR antagonist, eliminated the DL: -TBOA-induced effects. As dihydrokainic acid, a specific EAAT2 blocker, failed to affect evoked GABAergic postsynaptic currents, whereas TFB-TBOA, a selective blocker of EAAT1 and EAAT2, produced effects similar to that of DL: -TBOA, extracellular glutamate concentration in the marginal zone is mainly controlled by EAAT1 (GLAST). Thus, even though CR cells are highly vulnerable to excitotoxic damage, a number of mechanisms serve to protect them against excessive extracellular glutamate concentration including a lack of functional glutamatergic synapses, Mg(2+) blockade of NMDA receptors, and presynaptic mGluRs that inhibit transmission at GABAergic synapses.

Presynaptic muscarinic receptor subtypes involved in the enhancement of spontaneous GABAergic postsynaptic currents in hippocampal neurons

We investigated the effects of muscarinic acetylcholine receptor (mAChR) activation on GABAergic synaptic transmission in rat hippocampal neurons. Current-clamp recordings revealed that methacholine produced membrane depolarization and action potential firing. Methacholine augmented the bicuculline-sensitive and GABA(A) -mediated frequency of spontaneous inhibitory postsynaptic currents (sIPSCs); the action of methacholine had a slow onset and longer duration. The increase in methacholine-evoked sIPSCs was completely inhibited by atropine and was insensitive to glutamatergic receptor blockers. Interestingly, methacholine action was not inhibited by intracellular perfusion with GDP-β-S, suggesting that muscarinic effects on membrane excitability and sIPSC frequency are mainly presynaptic. McN-A-343 and pirenzepine, selective agonist and antagonist of the m1 mAChR subtype, respectively, neither enhanced sIPSCs nor inhibited the methacholine effect. However, the m3-m5 mAChR antagonist 4-DAMP, and the m2-m4 mAChR antagonist himbacine inhibited the methacholine effect. U73122, an IP(3) production inhibitor, and 2APB, an IP(3) receptor blocker, drastically decreased the methacholine effect. Recording of miniature events revealed that besides the effect exerted by methacholine on membrane firing properties and sIPSC frequency, muscarinic receptors also enhanced the frequency of mIPSCs with no effect on their amplitude, possibly modulating the molecular machinery subserving vesicle docking and fusion and suggesting a tight colocalization at the active zone of the presynaptic terminals. These data strongly suggest that by activating presynaptic m2, m3, m4 and m5 mAChRs, methacholine can increase membrane excitability and enhance efficiency in the GABA release machinery, perhaps through a mechanism involving the release of calcium from the endoplasmic reticulum.

Estradiol Induces Diurnal Shifts in GABA Transmission to Gonadotropin-Releasing Hormone Neurons to Provide a Neural Signal for Ovulation

Ovulation is initiated by a surge of gonadotropin-releasing hormone (GnRH) secretion by the brain. GnRH is normally under negative feedback control by ovarian steroids. During sustained exposure to estradiol in the late follicular phase of the reproductive cycle, however, the feedback action of this steroid switches to positive, inducing the surge. Here, we used an established ovariectomized, estradiol-treated (OVX+E) mouse model exhibiting daily surges to investigate the neurobiological mechanisms underlying this switch. Specifically, we examined changes in GABA transmission to GnRH neurons, which can be excited by GABA(A) receptor activation. Spontaneous GABAergic postsynaptic currents (PSCs) were recorded in GnRH neurons from OVX+E and OVX mice in coronal and sagittal slices. There were no diurnal changes in PSC frequency in cells from OVX mice in either slice orientation. In OVX+E cells in both orientations, PSC frequency was low during negative feedback but increased at surge onset. During the surge peak, this increase subsided in coronal slices but persisted in sagittal slices. Comparison of PSCs before and during tetrodotoxin (TTX) treatment showed TTX decreased PSC frequency in OVX+E cells in sagittal slices, but not coronal slices. This indicates estradiol acts on multiple GABAergic afferent populations to increase transmission through both activity-dependent and -independent mechanisms. Estradiol also increased PSC amplitude during the surge. Estradiol and the diurnal cycle thus interact to induce shifts in both GABA transmission and postsynaptic response that would produce appropriate changes in GnRH neuron firing activity and hormone release.

Reactive Oxygen Species Mediate the Potentiating Effects of ATP on GABAergic Synaptic Transmission in the Immature Hippocampus

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABA(A)-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.

Tilting the Balance

Cortical function depends on the proper balance between excitation and inhibition: If the balance tilts toward excitation, a seizure may occur, whereas if the balance shifts too far toward inhibition, network activity may shut down. GABA (γ-aminobutyric acid) is the major inhibitory transmitter in the adult brain; however, GABA is excitatory during early development, leading Bernard et al. to investigate the possible inhibitory role of endocannabinoids in regulating network activity in the developing brain. After using immunocytochemistry to localize cannabinoid type 1 (CB1) receptors to the terminals of hippocampal GABAergic interneurons, the authors combined electrophysiology with pharmacological analysis to show that endocannabinoids released from pyramidal cells and interneurons in rat pup hippocampal slices could act through CB1 receptors to inhibit GABA release. Pharmacological analysis in the presence of glutamate antagonists indicated that CB1 receptors were tonically activated in hippocampal slices and that CB1 blockade enhanced the amplitude of spontaneous GABAergic postsynaptic currents and stimulated the appearance of spontaneous epileptiform discharges, whereas endocannabinoid accumulation decreased the spontaneous firing rate of interneurons. In the absence of glutamatergic antagonists, CB1 blockade stimulated network activity and CB1 stimulation inhibited network activity. A CB1 antagonist triggered epileptic discharges in 5-day-old rat pups in vivo, whereas pharmacological activation of CB1 receptors decreased spontaneous neuronal activity. Thus, the authors conclude that endocannabinoid signaling acts to regulate network activity in the developing brain and that its disruption could have major functional consequences. C. Bernard, M. Milh, Y. M. Morozov, Y. Ben-Ari, T. F. Freund, H. Gozlan, Altering cannabinoid signaling during development disrupts neuronal activity. Proc. Natl. Acad. Sci. U.S.A. 102 , 9388-9393 (2005). [Abstract] [Full Text]

6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) increases GABA A receptor-mediated spontaneous postsynaptic currents in the dentate granule cells of rat hippocampal slices

6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) is widely used as an antagonist on non-NMDA glutamate receptors. However, several studies have shown that CNQX increases the spontaneous inhibitory postsynaptic current frequency at hippocampal pyramidal neurons and cerebellar granule cells. Dentate granule cells are known to be another distinctive type of principal neurons in hippocampus, and receive dense synaptic input from hilar interneurons. Thus, we examined the effects of CNQX on the dentate granule cells and hilar interneurons with whole-cell recording. CNQX increased the frequency of GABAergic spontaneous postsynaptic currents (sPSCs) on the granule cells, and increased the resting potential and the action potential frequency of the hilar interneurons. These increases were not observed with other glutamate receptor antagonists. The increases in sPSC frequency may be caused by the depolarization and the action potentials of the interneurons.

Metabolic Regulation of Fertility through Presynaptic and Postsynaptic Signaling to Gonadotropin-Releasing Hormone Neurons

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction and are inhibited by negative energy balance. In normal adults, these neurons maintain elevated intracellular chloride so that GABA(A) receptor activation is excitatory. We hypothesized that fasting alters homeostatic mechanisms to eliminate excitatory responses to GABA but rejected this hypothesis when brief, local GABA application elicited action currents in GnRH neurons from fed and fasted mice. This response was specific to GABA(A) receptors, because glycine elicited no response. We next found that fasting reduced the frequency of spontaneous GABAergic postsynaptic currents (PSCs) and that this was reversed by in vivo treatment with leptin during the fast. In the presence of tetrodotoxin to minimize presynaptic actions, leptin also potentiated the postsynaptic response of these cells to GABA(A) receptor activation. Postsynaptic effects of leptin on GABAergic miniature PSCs were eliminated by inhibiting JAK2/3 (Janus kinase), the tyrosine kinase through which leptin receptors signal. In all experiments, elimination of PSCs at ECl or by treatment with the GABAA receptor antagonist bicuculline confirmed that PSCs were specifically mediated by GABA(A) receptor chloride channels. These data demonstrate that fasting and leptin act presynaptically and postsynaptically to alter GABAergic drive to GnRH neurons, providing evidence for GABAergic communication of metabolic cues to GnRH neurons, and suggest the possibility for functional leptin receptors on GnRH neurons. They further demonstrate cytokine modulation of the postsynaptic response to GABA in mammals, which may be important to central neural regulation in both healthy and diseased states.

In vivo evidence for an activity-independent maturation of AMPA/NMDA signaling in the developing hippocampus

Correlated pre- and postsynaptic activity is thought to promote maturation of excitatory synapses in the developing brain by directing AMPA receptors to pure NMDA synapses. However, this hypothesis has not been tested in vivo. Here, we have performed such test by inhibiting correlated neural activity in vivo using a single injection of tetanus toxin into the rat hippocampal CA1 area at postnatal day 1. When examined in the acute slice preparation (1–7 days post-injection), there was a strong reduction, down to 20% of control level, in the frequency of glutamatergic and GABAergic spontaneous postsynaptic currents (sPSCs). This activity deprivation led to a growth retardation of CA1 pyramidal neurons and to markedly faster decay kinetics of NMDA sPCSs. However, it did not alter the relationship between AMPA and NMDA sPSCs with respect to either their frequency or amplitude. Thus, although critical for certain aspects of neuronal development, correlated neural activity in the neonatal hippocampus does not seem to promote incorporation of AMPA receptors at pure NMDA synapses.

Muscarinic acetylcholine receptors potentiate the GABAergic transmission in the developing rat inferior colliculus

Postsynaptic currents (PSCs) were recorded using the patch-clamp technique in neurons of the rat inferior colliculus (IC) to investigate the muscarinic modulation of the GABAergic transmission. In the presence of strychnine (0.5 μM) and kynurenic acid (1 mM), spontaneous GABAergic PSCs were observed in all IC neurons investigated. Muscarine (10 μM) greatly potentiated the frequency of these GABAergic PSCs (618% of the control). 4-DAMP (50 nM), a M3 receptor preferring antagonist, greatly blocked the muscarine-evoked PSC frequency increase. The muscarinic antagonists telenzepine (50 nM; M1 preferring), methoctramine (10 μM; M2 preferring), and himbazine (10 μM; M4 preferring), and the nicotinic antagonist mecamylamine (10 μM) did not significantly affect the muscarine effect. These findings indicate that the muscarinic modulation of the GABAergic transmission is primarily mediated by M3 receptors, while M1-, M2- and M4- and nicotinic receptors do not participate substantially. In the presence of tetrodotoxin (0.5 μM), muscarine failed to increase the PSC frequency indicating that its effect depended on the generation of spikes. We suggest that GABAergic interneurones express M3 receptors at some distance from the terminal. Their activation excites GABAergic interneurones, thereby enhancing GABA release in the IC. The muscarinic modulation of the GABAergic transmission may play an important role in the maturation of inhibitory synapses in the developing IC.

GABA Receptors Containing Rdl Subunits Mediate Fast Inhibitory Synaptic Transmission inDrosophilaNeurons

GABAergic inhibition in Drosophila, as in other insects and mammals, is important for regulation of activity in the CNS. However, the functional properties of synaptic GABA receptors in Drosophila have not been described. Here, we report that spontaneous GABAergic postsynaptic currents (sPSCs) in cultured embryonic Drosophila neurons are mediated by picrotoxin-sensitive chloride-conducting receptors. A rapid increase in spontaneous firing in response to bath application of picrotoxin demonstrates that these GABA receptors mediate inhibition in the neuronal networks formed in culture. Many of the spontaneous GABAergic synaptic currents are sodium action potential independent [miniature IPSCs (mIPSCs)] but are regulated by external calcium levels. The large variation in mIPSC frequency, amplitude, and kinetics properties between neurons suggests heterogeneity in GABA receptor number, location, and/or subtype. A decrease in the mean mIPSC decay time constant between 2 and 5 d, in the absence of a correlated change in rise time, demonstrates that the functional properties of the synaptic GABA receptors are regulated during maturation in vitro. Finally, neurons from the GABA receptor subunit mutant Rdl exhibit reduced sensitivity to picrotoxin blockade of the mIPSCs and resistance to picrotoxin-induced increases in spontaneous firing frequency. This demonstrates that Rdl containing GABA receptors play a direct role in mediating synaptic inhibition in Drosophila neural circuits formed in culture.

Excitatory actions of GABA increase BDNF expression via a MAPK-CREB-dependent mechanism--a positive feedback circuit in developing neurons.

During early neuronal development, GABA functions as an excitatory neurotransmitter, triggering membrane depolarization, action potentials, and the opening of plasma membrane Ca(2+) channels. These excitatory actions of GABA lead to a number of changes in neuronal structure and function. Although the effects of GABA on membrane biophysics during early development have been well documented, little work has been done to examine the possible mechanisms underlying GABA-regulated plastic changes in the developing brain. This study focuses on GABA-regulated kinase activity and transcriptional control. We utilized a combination of Western blotting and immunocytochemical techniques to examine two potential downstream pathways regulated by GABA excitation: the p42/44 mitogen-activated protein kinase (MAPK) cascade and the transcription factor cyclic AMP response element binding protein (CREB). During early development of cultured hypothalamic neurons (5 days in vitro), stimulation with GABA triggered activation of the MAPK cascade and phosphorylation of CREB at Ser 133. These effects were mediated by the GABA(A) receptor, since administration of the GABA(A) receptor-specific agonist muscimol (50 microM) triggered pathway activation, and pretreatment with the GABA(A)-receptor specific antagonist bicuculline (20 microM) blocked pathway activation. Immunocytochemistry revealed a spatial and temporal correlation between activation of the MAPK cascade and CREB phosphorylation. Pretreatment with the MAPK/ERK kinase (MEK) inhibitor U0126 (10 microM) attenuated CREB phosphorylation, indicating that the MAPK pathway regulates that activation state of CREB. In contrast to the excitatory effects observed during early development, in more mature neurons, GABA functions as an inhibitory transmitter. Consistent with this observation, GABA(A) receptor activation did not stimulate MAPK cascade activation or CREB phosphorylation in mature cultures (18 days in vitro). To determine whether GABA(A) receptor activation during early development stimulates gene expression, we examined the inducible expression of the neurotrophin brain-derived neurotrophic factor (BDNF). Both GABA and muscimol stimulated BDNF expression, and pretreatment with U0126 attenuated GABA-induced BDNF expression. Whole cell electrophysiological recording was used to assess the effects of BDNF on GABA release. BDNF (100 ng/ml) dramatically increased the frequency of excitatory GABAergic spontaneous postsynaptic currents. Together, these data suggest a positive excitatory feedback loop between GABA and BDNF expression during early development, where GABA facilitates BDNF expression, and BDNF facilitates the synaptic release of GABA. Signaling via the MAPK cascade and the transcription factor CREB appear to play a substantial role in this process.

Distinct muscarinic receptors enhance spontaneous GABA release and inhibit electrically evoked GABAergic synaptic transmission in the chick lateral spiriform nucleus

The effects of muscarinic agonists on GABAergic synaptic transmission were examined using whole-cell patch-clamp recording in chick brain slices containing the lateral spiriform nucleus. Bath application of muscarine (10 microM) both increased the frequency of spontaneous GABAergic postsynaptic currents and reduced the amplitude of evoked GABAergic polysynaptic postsynaptic currents elicited by focal afferent fiber electrical stimulation. Both of these muscarinic actions were reversible and dose-dependent. Two M(1) antagonists, telenzepine and pirenzipine, and to a lesser extent the M(2) antagonist methoctramine, protected against muscarine's inhibition of the evoked polysynaptic currents. Other M(2) antagonists (tripitramine and gallamine) as well as the M(3) antagonist 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride) and an M(4) antagonist (tropicamide) provided little or no protection against muscarine in this assay. In contrast, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, tropicamide and telenzepine, but not pirenzepine, methoctramine, tripitramine and gallamine, blocked muscarine's enhancement of spontaneous GABAergic currents. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-m-chlorocarbanilate chloride] and CDD-0097 (5-propargyloxycarbonyl-1,4,5,6-tetrahydropyrimidine hydrochloride), two M(1) agonists, mimicked muscarine's inhibition of the evoked polysynaptic GABAergic currents but did not mimic muscarine's enhancement of spontaneous GABAergic currents. Both actions of muscarine persisted when slices were pretreated with pertussis toxin or N-ethylmaleimide, which inactivate G-proteins coupled to M(2) and M(4) receptors while leaving G-proteins coupled to M(1), M(3) and M(5) receptors intact. Muscarine had no significant effect on the amplitude of the direct postsynaptic current elicited by exogenous GABA in the presence of tetrodotoxin. The results demonstrate that distinct muscarinic receptors oppositely modulate GABAergic transmission in the lateral spiriform nucleus. The receptor mediating the inhibition of evoked GABAergic polysynaptic currents is pharmacologically similar to an M(1) receptor, while the enhancement of spontaneous GABAergic currents appears to be mediated by an M(3) receptor.

Spontaneous GABAergic postsynaptic currents in Cajal–Retzius cells in neonatal rat cerebral cortex

Cajal-Retzius cells are among the first neurons appearing during corticogenesis, and play an important role in the establishment of cortical lamination. The variety of neurotransmitter receptors recently found on these cells imply that they are integrated in the neonatal cortical network. To investigate the presence and properties of spontaneous synaptic activity we performed whole-cell patch-clamp recordings from visually identified and biocytin-labelled Cajal-Retzius cells in a tangential slice preparation of neonatal rat cerebral cortex (postnatal days P0-P5). Spontaneous postsynaptic currents (sPSCs) could be observed in about 23% of the cells using a pipette solution containing 136 m M Cl-. The sPSCs occurred at a low frequency (0.07 +/- 0.07 Hz, n = 42 cells), had an average amplitude of 24.3 +/- 12.4 pA (n = 415 events) and could not be divided in subpopulations according to their amplitude distribution or kinetic properties. The sPSCs were blocked by the GABAA antagonist bicuculline (100 microM), while the glutamatergic antagonists (+/-)-2-amino-5-phosphonopentatonic acid (APV, 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), as well as tetrodotoxin (1-2 microM), a blocker of voltage-gated sodium-currents, had no significant effect on sPSCs. The incidence rate of sPSCs declined within the age of the rats and no sPSCs could be observed after P4. These results suggest that Cajal-Retzius cells transiently receive action potential-independent and GABA(A) receptor-mediated spontaneous synaptic input, which may contribute to the refinement of cortical circuits.

Activation of metabotropic glutamate receptors inhibits GABAergic transmission in the rat subfornical organ

Glutamate is known to increase neuronal excitability in the subfornical organ, a circumventricular organ devoid of the blood–brain barrier. To understand the synaptic mechanism of neuronal excitation by glutamate in this nucleus, we examined the effects of glutamate on GABAergic spontaneous inhibitory postsynaptic currents recorded from subfornical organ neurons in the rat brain slice. The baseline frequency, amplitude and decay time-constant of such spontaneous synaptic currents were 5.60 Hz, 119 pA and 17.3 ms, respectively. Glutamate (10–1000 μM) selectively inhibited the frequency of spontaneous GABAergic inhibitory postsynaptic currents (half-maximal effective concentration=47 μM) with little effects on their amplitudes and decay time constants. The inhibitory effect of glutamate on the frequency of spontaneous GABAergic postsynaptic currents was not blocked by tetrodotoxin (1 μM), or by the antagonists of ionotropic glutamate receptors. In contrast, such inhibitory effect of glutamate was mimicked by general or group II selective metabotropic glutamate receptor agonists such as DCGIV (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (half-maximal effective concentration=112 nM), but not by the agonists for group I or group III metabotropic glutamate receptors. Under current clamp mode, glutamate reduced the frequencies of spontaneous inhibitory postsynaptic potentials and action potentials in subfornical organ neurons. Our data indicate that glutamate decreases the frequency of spontaneous inhibitory postsynaptic currents by acting on the group II metabotropic glutamate receptors on axonal terminals in the subfornical organ. From these results we suggest that the glutamate-induced modulation of tonic GABAergic inhibitory synaptic activity can influence the excitability of subfornical organ neurons.

The role of GABAergic inputs for coincidence detection in the neurones of nucleus laminaris of the chick.

1. Synaptic inputs to nucleus laminaris (NL) neurones were studied in a brainstem slice preparation of chick embryos (E15-20) using the whole-cell patch clamp technique. NL neurones are third order auditory neurones and are proposed to behave as coincidence detectors concerned with interaural timing discrimination. 2. Under voltage clamp conditions, electrical stimuli applied to either ventral or dorsal dendritic layers evoked EPSCs. These fast currents decayed with a time constant of 1.1 ms near the resting potential, reversed close to 0 mV, and were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 microM) or 6, 7-dinitro-quinoxaline-2,3-dione (DNQX, 20 microM). Coincident or near coincident stimulation of the ventral and the dorsal dendritic layers increased the probability of action potential generation (response probability). 3. In the presence of CNQX (40 microM) other postsynaptic currents (PSCs) were observed, which reversed close to the equilibrium potential for chloride (ECl), and were reversibly blocked by bicuculline (20 microM) and, therefore, were mediated by GABAA receptors. Spontaneous GABAergic PSCs were inward going near the resting membrane potential immediately after starting whole-cell recording with a low Cl- (5 mM, ECl = -90 mV) pipette medium, but became outward-going with time. This indicates that GABAergic inputs may generate depolarizing potentials in intact NL neurones. 4. Local GABA (10 microM) application reduced both the EPSP and EPSC amplitude and shortened the EPSP decay time constant (from 5.3 to 2. 1 ms), while the EPSC decay time constant was not affected (from 1.3 to 1.2 ms). These GABA effects were mostly due to the shunting conductance of the postsynaptic GABAA receptors. 5. Depolarizing current injections combined with electrical stimuli to a unilateral axon bundle simulated bilateral synaptic inputs. Response probability increased with decreased interstimulus intervals, while local GABA (10 microM) application to the soma narrowed the time dependence of the response probability. 6. These results suggest that GABAergic inputs to NL neurones may serve to improve coincidence detection of the bilateral excitatory inputs through an increase in membrane conductance.