However, it is difficult to draw conclusions about the functional consequences of subthreshold GABAergic depolarizations, since GABAergic membrane shunting and additional effects on voltage-dependent ion channels or action potential threshold must be considered. To systematically investigate factors that determine the GABAergic effect on neuronal excitability we performed whole cell patch-clamp recordings from Cajal-Retzius cells in immature rat neocortex, using [Cl Ϫ ] i between 10 and 50 mM. The effect of focal GABA application was quantified by measuring various parameters of GABAergic responses including the shift in minimal threshold current (rheobase). The rheobase shift was correlated with other parameters of the GABAergic responses by multiple linear regression analyses with a set of simple mathematical models. Our experiments demonstrate that focal GABA application induces heterogeneous rheobase shifts in Cajal-Retzius cells that could not be predicted reliably from [Cl Ϫ ] i or the GABAergic membrane depolarization. Implementation of a linear mathematical model, which takes the GABAergic membrane conductance and the difference between action potential threshold and GABA reversal potential into account, resulted in a close correlation between calculated and experimentally obtained rheobase shifts. Addition of a linear term proportional to the GABAergic membrane depolarization improved the accuracy of correlation. The main advantage of using multiple linear regression with simple models is that direction and strength of GABAergic excitability shifts can be analyzed by using only measured parameters of GABAergic responses and with minimal a priori information about cellular parameters. cortical development; shunting inhibition; intracellular chloride concentration; rheobase GABA IS THE MAIN INHIBITORY neurotransmitter in the adult nervous system and mediates its effect via ionotropic GABA A or GABA C receptors and metabotropic GABA B receptors (Emson

To unravel the functional implications of activity-dependent Cl- changes during early stages of neuronal development, we determined which changes in the GABA reversal potential (E (GABA)) and GABAergic rheobase shifts were induced by episodes of GABA(A) receptor activation using gramicidin-perforated patch-clamp recordings from Cajal-Retzius cells in tangential cortical slices of newborn mice. Under this condition, focal application of the GABA(A) agonist muscimol (10 μM) depolarized the membrane by 15 ± 0.8 mV (n = 35). Such subthreshold GABAergic depolarizations considerably reduced the rheobase, corresponding to an excitatory action. After repetitive focal muscimol applications (50 pulses at 0.5 Hz) a significant reduction of E (GABA) and an attenuation of the excitatory GABAergic rheobase shift were observed, while the GABAergic membrane conductance and the absolute value of the rheobase were unaltered after the muscimol pulses. Bath application of 100 μM carbachol induced bursts of spontaneous GABAergic postsynaptic potentials. Both, E (GABA) and the excitatory GABAergic rheobase shift was significantly reduced after such barrage of carbachol-induced GABAergic postsynaptic potentials, while neither the GABAergic membrane conductance nor the absolute value of the rheobase was affected under this condition. Both results indicate that GABAergic activity itself can limit the excitatory effects of GABA(A) receptor activation, which supports the hypothesis that the low capacity of the Cl- homeostasis in immature neurons could be a substrate for synaptic scaling and homeostatic plasticity.

Neuroligin-4 (Nlgn4) is a cell adhesion protein that regulates synapse organization and function. Mutations in human NLGN4 are among the causes of autism spectrum disorders. In mouse, Nlgn4 knockout (KO) perturbs GABAergic synaptic transmission and oscillatory activity in hippocampus, and causes social interaction deficits. The complex profile of cellular and circuit changes that are caused by Nlgn4-KO is still only partly understood. Using Nlgn4-KO mice, we found that Nlgn4-KO increases the power in the alpha frequency band of spontaneous network activity in the barrel cortex under urethane anesthesia in vivo. Nlgn4-KO did not affect single-whisker-induced local field potentials, but suppressed the late evoked multiunit activity in vivo. Although Nlgn4-KO did not affect evoked EPSCs in layer 4 (L4) spiny stellate cells in acute thalamocortical slices elicited by electrical stimulation of thalamocortical inputs, it caused a lower frequency of both miniature (m) IPSCs and mEPSCs, and a decrease in the number of readily releasable vesicles at GABAergic and glutamatergic connections, weakening both excitatory and inhibitory transmission. However, Nlgn4 deficit strongly suppresses glutamatergic activity, shifting the excitation-inhibition balance to inhibition. We conclude that Nlgn4-KO does not influence the incoming whisker-mediated sensory information to the barrel cortex, but modifies intracortical information processing.

SUMMARYPurpose: Despite the consistent observation that c-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 lM) and picrotoxin (100 lM) induced epileptiform discharges, whereas activation of GABA A receptors attenuated epileptiform discharges. Under low-Mg 2+ conditions, 100 nM gabazine and 1 lM picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole-cell patchclamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1-lM 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.

Key pointsr Taurine has a pro-and anticonvulsive effect on the immature hippocampus, depending on the dose.r The taurine effect is mediated by GABA A and glycine receptors. r The taurine effect can be partially mimicked by glycine. r Inhibition of glycine receptors has a weak proconvulsive effect on the immature hippocampus. r We conclude that an endogenous activation of glycine receptors by glycine or taurine contributed to the control of neuronal excitability in the immature hippocampus.Abstract While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 μM taurine or 10 μM glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μM 4-aminopyridine in low Mg 2+ solution. This proconvulsive effect was prevented by 3 μM strychnine or after incubation with the loop diuretic bumetanide (10 μM), suggesting that it required glycine receptors and an active NKCC1-dependent Cl − accumulation. Application of higher doses of taurine (ࣙ1 mM) or glycine (100 μM) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABA A receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μM) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro-and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.R. Chen and A. Okabe contributed equally to this publication.