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NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of gamma-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

In recent years, considerable progress has been achieved in deciphering the cellular and network functions of GABAergic transmission in the intact developing brain. First, in vivo studies in non-mammalian and mammalian species confirmed the long-held assumption that GABA acts as a mainly depolarizing neurotransmitter at early developmental stages. At the same time, GABAergic transmission was shown to spatiotemporally constrain spontaneous cortical activity, whereas firm evidence for GABAergic excitation in vivo is currently missing. Second, there is a growing body of evidence indicating that depolarizing GABA may contribute to the activity-dependent refinement of neural circuits. Third, alterations in GABA actions have been causally linked to developmental brain disorders and identified as potential targets of timed prophylactic interventions. In this article, we review these major recent findings and argue that both depolarizing and inhibitory GABA actions may be crucial for physiological brain maturation.

Striatal GABAergic signaling has been shown to be essential for basal ganglia output and proper motor performance. In the mouse neostriatum GABA transporter 1 (GAT-1) was previously found to assist in the clearance of GABA from the extracellular space and influence both phasic and tonic GABAergic inhibition of medium-sized striatal output neurons (SONs). It currently remains unknown whether GAT subtypes other than GAT-1 participate in the modulation of GABAergic transmission in this brain structure. In this study, we aimed at assessing the role of GAT-3 in the mouse neostriatum. To this end, we recorded GABAergic inhibitory postsynaptic currents (IPSCs) from SONs in brain slices at different developmental stages (postnatal days (P) 7-9, 12-14, and 28-34) using the whole-cell patch-clamp technique. When applied under control conditions, SNAP-5114 (40 μM), a specific GAT-3 blocker, did not affect miniature or evoked IPSCs (m/eIPSCs) and produced no significant effect on tonic GABAA receptor-mediated conductances in SONs. However, in the presence of NO-711 (10 μM), a specific GAT-1 blocker, SNAP-5114 reduced mIPSC frequencies without affecting mIPSC amplitudes or kinetics. In addition, SNAP-5114 reduced the mean amplitude of eIPSCs and increased the paired-pulse ratio. These effects were entirely abolished by CGP55845 (1 μM), a specific GABAB receptor blocker, indicating that they were mediated by presynaptic GABAB receptors. Similar results were obtained from all age groups. We conclude that GAT-3 is functionally expressed in the mouse neostriatum. Whereas an acute block of GAT-3 under resting conditions is fully compensated for by GAT-1, GAT-3 might provide an additional uptake capacity when neuronal activity and GABA release are increased. Synapse 63:921–929, 2009. © 2009 Wiley-Liss, Inc.

Fast and slowly rising inhibitory postsynaptic currents (IPSCs, IPSC F and IPSCs) in neocortical Cajal-Retzius cells are observed. In this study, zolpidem, a benzodiazepine agonist that specifically modulates γ-aminobutyric acid type A receptors (GABA A Rs) containing γ 2 subunit, was used to characterize GABA A RS mediating IPSC F and IPSCS. One-hundred-nanomolar zolpidem prolonged IPSC S , increased evoked IPSC S (elPSC s ) amplitude, and decreased paired-pulse ratio (PPR) of elPSCs. Two micromolar zolpidem prolonged both IPSC F and IPSC S , increased miniature IPSC F and elPSCp amplitudes, increased elPSCs amplitude but not miniature IPSCs amplitude, decreased PPR of elPSC s , but failed to affect PPR of elPSC F We conclude that IPSC F are mediated by α 2/3 -containing GABA A R S , which are not saturated by synaptic GABA release, whereas IPSCs are mediated by α 1 -containing and α 2/3 -containing GABA A R S , which are saturated by quantal GABA release.

Layer I of the developing cortex contains a dense GABAergic fiber plexus. These fibers provide excitatory inputs to Cajal-Retzius (CR) cells, the early born neurons in layer I. CR cells possess an extensive axonal projection and form synaptic contacts with excitatory, presumably pyramidal, neurons before birth. Interestingly, activity of CR cells declines during the first postnatal week, but mechanism(s) underlying this phenomenon is not yet known. Here we recorded inhibitory postsynaptic currents (IPSCs) in CR cells at postnatal day (P) 1-2 and P5-7. Blockade of adenosine A(1) receptors (A(1)Rs) increased the amplitude of evoked IPSCs (eIPSCs) and decreased paired-pulse ratio at P5-7 but not at P1-2. A(1)R activation decreased the mean eIPSC amplitude at P5-7, but failed to affect eIPSCs at P1-2. Ecto-adenosine triphosphatase (ATPase) inhibition completely abolished the A(1)R-mediated effects suggesting that extracellular ATP is the main source of adenosine. Because A(1)R blockade did not affect the median miniature IPSC amplitude, our results demonstrate that adenosine reduces gamma-aminiobutyric acid (GABA) release probability via presynaptic A(1)Rs at P5-7. As neuronal activity in layer I can depolarize pyramidal neurons influencing thereby glutamatergic synaptogenesis in the lower cortical layers, postnatal weakening of GABAergic transmission by adenosinergic system might reflect a developmental downregulation of this excitatory drive when glutamatergic synapses are formed.

A large body of evidence from in vitro studies suggests that GABA is depolarizing during early postnatal development. However, the mode of GABA action in the intact developing brain is unknown. Here we examine the in vivo effects of GABA in cells of the upper cortical plate using a combination of electrophysiological and Ca2+-imaging techniques. We report that at postnatal days (P) 3–4, GABA depolarizes the majority of immature neurons in the occipital cortex of anaesthetized mice. At the same time, GABA does not efficiently activate voltage-gated Ca2+ channels and fails to induce action potential firing. Blocking GABAA receptors disinhibits spontaneous network activity, whereas allosteric activation of GABAA receptors has the opposite effect. In summary, our data provide evidence that in vivo GABA acts as a depolarizing neurotransmitter imposing an inhibitory control on network activity in the neonatal (P3–4) neocortex. GABA depolarizes immature neurons in the central nervous system, yet the mode of GABA action in the developing brain is unknown. Here the authors demonstrate thatin vivoGABA acts as a depolarizing neurotransmitter imposing inhibitory control on network activity in the mouse postnatal day 3–4 neocortex.

The sulphydryl alkylating agent N-ethylmaleimide (NEM) has been often used as an uncoupler of pertussis toxin-sensitive G-proteins. However, the effects of NEM on gamma-aminobutyric acid (GABA)ergic synaptic transmission remain controversial. Using the whole-cell patch-clamp technique, GABA(A) receptor-mediated postsynaptic currents (IPSCs) have been recorded from Cajal-Retzius (CR) cells in layer I of the neonatal mouse visual cortex. NEM increased the frequencies of both spontaneous and miniature IPSCs (mIPSCs) without an effect on the median mIPSC amplitudes or mIPSC kinetics. The NEM actions on mIPSCs did not depend on the extracellular Ca(2+), Ca(2+) release from intracellular stores, adenylyl cyclase and protein kinase A activities. NEM increased the mean amplitudes of evoked IPSCs and strongly decreased the paired-pulse ratio. The size of the readily releasable pool of presynaptic vesicles (RRP) was estimated using a high-frequency stimulation protocol. The RRP size was not affected by NEM. In addition, NEM significantly decreased the latency between the stimulus and the onset of GABA release. These results suggest that NEM selectively increases GABA release probability. At postnatal day 2, mIPSCs were observed only in about 30% of CR cells. NEM application revealed, however, that more than 90% of CR cells receive GABAergic inputs. Therefore, NEM seems to be a useful tool to verify the existence of 'silent' GABAergic synapses.

Epileptic seizures rank among the most frequent neurologic symptoms during the neonatal period. Accumulating data from experimental animal studies and clinical trials in humans suggest that neonatal seizures could adversely affect normal brain development and result in long-term neurologic sequelae. Unfortunately, currently used anticonvulsive drugs are often ineffective in the neonatal period. One particularity of the immature neuronal network during neonatal development is that the neurotransmitter γ-aminobutyric acid (GABA) is mainly depolarizing, rather than hyperpolarizing as commonly observed in adults. This might, in part, explain not only the higher seizure propensity of the immature neuronal network, but also the limited anticonvulsive efficacy of GABA-enhancing drugs during early postnatal life. Accordingly, pharmacologic attenuation of GABAergic depolarization has been proposed as a strategy for neonatal seizure control. However, the underlying conjecture of a depolarizing mode of GABA action has been seriously challenged recently. In the present review, we will summarize the state of knowledge regarding GABAergic depolarization in early life and discuss how these data might impact a currently tested anticonvulsive strategy.

A large body of evidence suggests that the neurotransmitter GABA undergoes a developmental switch from being predominantly depolarizing–excitatory to predominantly hyperpolarizing–inhibitory. Recently published data, however, point to the possibility that the presumed depolarizing mode of GABA action during early development might represent an artifact due to an insufficient energy supply of thein vitropreparations used. Specifically, addition of the ketone bodydl-β-hydroxybutyrate (βHB) to the extracellular medium was shown to prevent GABA from exerting excitatory effects. Applying a complementary set of minimally invasive optical and electrophysiological techniques in brain slices from neonatal mice, we investigated the effects of βHB on GABA actions in immature cells of the upper cortical plate. Fluorescence imaging revealed that GABA-mediated somatic [Ca2+] transients, that required activation of GABAAreceptors and voltage-gated Ca2+channels, remained unaffected by βHB. Cell-attached current-clamp recordings showed that, in the presence of βHB, GABA still induced a membrane potential depolarization. To estimate membrane potential changes quantitatively, we used cell-attached recordings of voltage-gated potassium currents and demonstrated that the GABA-mediated depolarization was independent of supplementation of the extracellular solution with βHB. We conclude that,in vitro, GABA depolarizes immature cells of the upper cortical plate in the presence of the ketone body βHB. Our data thereby support the general concept of an excitatory-to-inhibitory switch of GABA action during early development.

GABAergic synapses on Cajal-Retzius neurons in layer I of the murine neocortex experience GABA(B) receptor (GABA(B)R)-mediated tonic inhibition. Extracellular GABA concentration ([GABA](o)) that determines the strength of GABA(B)R-mediated inhibition is controlled by GABA transporters (GATs). In this study, we hypothesized that the strength of presynaptic GABA(B)R activation reflects [GABA](o) in the vicinity of synaptic contacts. Slices obtained from two age groups were used, namely postnatal days (P)2-3 and P5-7. GABAergic postsynaptic currents (IPSCs) were recorded using the whole-cell patch-clamp technique. Minimal electrical stimulation in layer I was applied to elicit evoked IPSCs (eIPSCs) using a paired-pulse protocol. Three parameters were selected for comparison: the mean eIPSC amplitude, paired-pulse ratio, and failure rate. When GAT-1 and GAT-2/3 were blocked by NO-711 (10 microM) and SNAP-5114 (40 microM), respectively, no tonic GABA(B)R-mediated inhibition was observed. In order to restore the control levels of GABA(B)R-mediated inhibition, 250 and 125 nm exogenous GABA was required at P2-3 and P5-7, respectively. Addition of 3-mercaptopropionic acid, a glutamate decarboxylase inhibitor, did not significantly change the obtained values arguing against the suggestion that a mechanism different from GATs contributes to [GABA](o) control. We conclude that juxtasynaptic [GABA](o) is higher (about 250 nM) at P2-3 than at P5-7 (about 125 nM). As both radial cell migration and corticogenesis in general are strongly dependent on [GABA](o) and the formation of the last layer 2/3 is finished by P4 in rodents, the observed [GABA](o) reduction in layer I might reflect this crucial event in the cortical development.

Cajal-Retzius (CR) cells are principal cells of layer I in the developing neocortex. They are able to generate action potentials, make synaptic contacts in layer I and receive excitatory GABAergic inputs before birth. Although CR cells participate in neuronal network activity in layer I, the properties of their synaptic inputs are not yet characterized. We recorded miniature (mIPSCs) and evoked (eIPSCs) postsynaptic currents using the whole-cell patch-clamp technique. Most of CR cells displayed two types of mIPSCs, namely those with fast (mIPSC(F)) and slow (mIPSC(S)) rise kinetics. The mIPSC(F) mean amplitude was significantly larger than that of mIPSC(S), while their decay rates were not different. Peak-scaled non-stationary noise analysis revealed that mIPSC(S) and mIPSC(F) differed in their weighted single-channel conductance. In addition, zolpidem (100 nm), a modulator of alpha(1) subunit-containing GABA(A) receptors, selectively affected mIPSC(S) suggesting that different postsynaptic GABA(A) receptors mediate mIPSC(F) and mIPSC(S). eIPSCs also split into two populations with different rise kinetics. Fast eIPSCs (eIPSC(F)) displayed higher paired-pulse ratio (PPR) and lower GABA release probability than slowly rising eIPSCs (eIPSC(S)). As CGP55845, a GABA(B) receptor antagonist, eliminated the observed difference in PPR, the lower release probability at IPSC(F) connections probably reflects a stronger tonic GABA(B) receptor-mediated inhibition of IPSC(F) synapses. At low (0.1 Hz) stimulation frequency both inputs can effectively convert presynaptic action potentials into postsynaptic ones; however, only IPSC(F) connections reliably transfer the presynaptic activity patterns at higher stimulation rates. Thus, CR cells receive two GABAergic inputs, which differ in the quantal amplitude, the probability of GABA release and the frequency dependence of signal transfer.

At early stages of brain development, GABA plays a dual role. It fulfills important trophic functions and provides a major excitatory drive for the immature neuronal network. Here, we investigated whether GABA itself can limit the strength of excitatory GABAergic synapses on Cajal-Retzius (CR) cells in sagittal slices from the mouse visual cortex. (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), a specific GABABreceptor (GABABR) blocker, increased the frequency of spontaneous Ca2+transients and spontaneous and miniature IPSCs (mIPSCs) but did not affect mIPSC amplitudes or kinetics.CGP55845significantly increased evoked IPSC (eIPSC) amplitudes and decreased the paired-pulse ratio (PPR). Baclofen, a specific GABABR agonist, produced opposite effects. The size of the readily releasable pool was not affected by these GABABR modulators. The sameCGP55845actions were observed at physiological temperatures, but they were abolished after glutamate decarboxylase block with 3-mercaptopropionic acid (3-MP). These results indicate that presynaptic GABABRs dynamically regulate GABA release probability. SNAP-5114, a specific GABA transporter-2/3 (GAT-2/3) blocker, enhanced mIPSC frequencies, decreased PPR, and increased eIPSC amplitudes without changing eIPSC kinetics. These effects were blocked byCGP55845and 3-MP. NO-711, a specific GAT-1 blocker, prolonged eIPSC decay and decreased eIPSC/mIPSC amplitudes. These NO-711-mediated effects were not sensitive toCGP55845and 3-MP. We conclude that the strength of GABAergic inputs to CR cells is constrained by GABABRs that are persistently activated by ambient GABA. The latter is also provided by GAT-2/3 operating in the reversed mode. Presynaptic GAT-1 functions in the uptake mode and possibly provides GABA for presynaptic vesicle filling.