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Spontaneous correlated activity is a universal hallmark of immature neural circuits. However, the cellular dynamics and intrinsic mechanisms underlying network burstiness in the intact developing brain are largely unknown. Here, we use two-photon Ca2+ imaging to comprehensively map the developmental trajectories of spontaneous network activity in the hippocampal area CA1 of mice in vivo. We unexpectedly find that network burstiness peaks after the developmental emergence of effective synaptic inhibition in the second postnatal week. We demonstrate that the enhanced network burstiness reflects an increased functional coupling of individual neurons to local population activity. However, pairwise neuronal correlations are low, and network bursts (NBs) recruit CA1 pyramidal cells in a virtually random manner. Using a dynamic systems modeling approach, we reconcile these experimental findings and identify network bi-stability as a potential regime underlying network burstiness at this age. Our analyses reveal an important role of synaptic input characteristics and network instability dynamics for NB generation. Collectively, our data suggest a mechanism, whereby developing CA1 performs extensive input-discrimination learning prior to the onset of environmental exploration.

Spontaneous correlated activity is a universal hallmark of immature neural circuits. However, the cellular dynamics and intrinsic mechanisms underlying network burstiness in the intact developing brain are largely unknown. Here, we use two-photon Ca2+ imaging to comprehensively map the developmental trajectories of spontaneous network activity in hippocampal area CA1 in vivo. We unexpectedly find that network burstiness peaks after the developmental emergence of effective synaptic inhibition in the second postnatal week. We demonstrate that the enhanced network burstiness reflects an increased functional coupling of individual neurons to local population activity. However, pairwise neuronal correlations are low, and network bursts (NBs) recruit CA1 pyramidal cells in a virtually random manner. Using a dynamic systems modeling approach, we reconcile these experimental findings and identify network bi-stability as a potential regime underlying network burstiness at this age. Our analyses reveal an important role of synaptic input characteristics and network instability dynamics for NB generation. Collectively, our data suggest a mechanism, whereby developing CA1 performs extensive input-discrimination learning prior to the onset of environmental exploration.

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.

GABAergic signaling provides inhibitory stabilization and spatiotemporally coordinates the firing of recurrently connected excitatory neurons in mature cortical circuits. Inhibition thus enables self-generated neuronal activity patterns that underlie various aspects of sensation and cognition. In this review, we aim to provide a conceptual framework describing how and when GABA-releasing interneurons acquire their network functions during development. Focusing on the developing visual neocortex and hippocampus in mice and rats in vivo, we hypothesize that at the onset of patterned activity, glutamatergic neurons are stable by themselves and inhibitory stabilization is not yet functional. We review important milestones in the development of GABAergic signaling and illustrate how the cell-type-specific strengthening of synaptic inhibition toward eye opening shapes cortical network dynamics and allows the developing cortex to progressively disengage from extra-cortical synaptic drive. We translate this framework to human cortical development and discuss clinical implications for the treatment of neonatal seizures.

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 GABA actions 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 depolarizing GABA in the hippocampus in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Neuronal network activity in the developing brain is generated in a discontinuous manner. In the visual cortex during the period of physiological blindness of immaturity, this activity mainly comprises retinally triggered spindle bursts or Ca2 + clusters thought to contribute to the activity-dependent construction of cortical circuits. In spite of potentially important developmental functions, the spatial structure of these activity patterns remains largely unclear. In order to address this issue, we here used three-dimensional two-photon Ca2 + imaging in the visual cortex of neonatal mice at postnatal days (P) 3–4 in vivo. Large-scale voxel imaging covering a cortical depth of 200 μm revealed that Ca2 + clusters, identified as spindle bursts in simultaneous extracellular recordings, recruit cortical glutamatergic neurons of the upper cortical plate (CP) in a column-like manner. Specifically, the majority of Ca2 + clusters exhibit prominent horizontal confinement and high intra-cluster density of activation involving the entire depth of the upper CP. Moreover, using simultaneous Ca2 + imaging from hundreds of neurons at single-cellular resolution, we demonstrate that the degree of neuronal co-activation within Ca2 + clusters displays substantial heterogeneity. We further provide evidence that co-activated cells within Ca2 + clusters are spatially distributed in a non-stochastic manner. In summary, our data support the conclusion that dense coding in the form of column-like Ca2 + clusters is a characteristic property of network activity in the developing visual neocortex. Such knowledge is expected to be relevant for a refined understanding of how specific spatiotemporal characteristics of early network activity instruct the development of cortical circuits.

Summary: Synchronized activity is a universal characteristic of immature neural circuits that is essential for their developmental refinement and strongly depends on GABAergic neurotransmission. A major subpopulation of GABA-releasing interneurons (INs) expresses somatostatin (SOM) and proved critical for rhythm generation in adulthood. Here, we report a mechanism whereby SOM INs promote neuronal synchrony in the neonatal CA1 region. Combining imaging and electrophysiological approaches, we demonstrate that SOM INs and pyramidal cells (PCs) coactivate during spontaneous activity. Bidirectional optogenetic manipulations reveal excitatory GABAergic outputs to PCs that evoke correlated network events in an NKCC1-dependent manner and contribute to spontaneous synchrony. Using a dynamic systems modeling approach, we show that SOM INs affect network dynamics through a modulation of network instability and amplification threshold. Our study identifies a network function of SOM INs with implications for the activity-dependent construction of developing brain circuits. : Developing neural circuits generate burst-like spontaneous activity. Flossmann et al. report a mechanism whereby somatostatin interneurons promote neuronal synchrony in the neonatal hippocampus. This action depends on depolarizing GABAergic output synapses and requires chloride uptake via NKCC1. Their findings have implications for the activity-dependent refinement of developing brain circuits. Keywords: GABA, interneurons, development, hippocampus, optogenetics

Calcium imaging provides an indirect observation of the underlying neural dynamics and enables the functional analysis of neuronal populations. However, the recorded fluorescence traces are temporally smeared, thus making the reconstruction of exact spiking activity challenging. Most of the established methods to tackle this issue are limited in dealing with issues such as the variability in the kinetics of fluorescence transients, fast processing of long-term data, high firing rates, and measurement noise. We propose a novel, heuristic reconstruction method to overcome these limitations. By using both synthetic and experimental data, we demonstrate the four main features of this method: 1) it accurately reconstructs both isolated spikes and within-burst spikes, and the spike count per fluorescence transient, from a given noisy fluorescence trace; 2) it performs the reconstruction of a trace extracted from 1,000,000 frames in less than 2 s; 3) it adapts to transients with different rise and decay kinetics or amplitudes, both within and across single neurons; and 4) it has only one key parameter, which we will show can be set in a nearly automatic way to an approximately optimal value. Furthermore, we demonstrate the ability of the method to effectively correct for fast and rather complex, slowly varying drifts as frequently observed in in vivo data. NEW & NOTEWORTHY Reconstruction of spiking activities from calcium imaging data remains challenging. Most of the established reconstruction methods not only have limitations in adapting to systematic variations in the data and fast processing of large amounts of data, but their results also depend on the user’s experience. To overcome these limitations, we present a novel, heuristic model-free-type method that enables an ultra-fast, accurate, near-automatic reconstruction from data recorded under a wide range of experimental conditions.

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.

Diverse neuropsychiatric diseases were recently linked to specific anti-neuronal autoantibodies targeting synaptic proteins. Symptoms can range from epileptic seizures to cognitive impairment to movement disorders, commonly responding to treatment with immunotherapy. Several of these autoantibodies target inhibitory synapses that use GABA or glycine as neurotransmitters. Despite their relatively low abundance, inhibitory neurons are extraordinarily diverse in anatomical, electrophysiological and molecular terms, reflecting the variable clinical phenotypes of affected patients. Indeed, data on the antibody effects in neuronal cultures or animals models suggest that most of these antibodies are directly pathogenic by down-regulating synaptic proteins, activating complement or antagonizing ligand binding. The present review summarizes the current achievements in the field of humoral autoimmunity related to inhibitory networks, state-of-the-art diagnostics and clinical characterization of patients. In many instances, the phenotypic spectrum of patients with GABA receptor, glycine receptor, amphiphysin or GAD65 antibodies mirror the experimental findings, suggesting that ongoing work will markedly contribute to the better understanding of pathophysiology in this exciting patient group and might pave the way for disease-specific immunotherapy.

Summary AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.

γ-aminobutyric acid (GABA) mediates synaptic inhibition in the adult brain, but acts as a predominantly depolarizing and partially excitatory neurotransmitter in immature neurons. Recent

Zusammenfassung:Während γ-Aminobuttersäure (GABA) im adulten Gehirn synaptische Hemmung vermittelt, wirkt es auf unreife Nervenzellen zumeist depolarisierend und teilweise erregend.

The development of genetically modified mice in which subpopulations of cortical neurons are labelled by fluorescent proteins has greatly facilitated single-cellular imaging and electrophysiology studies in vitro and in vivo. However, the parallel visualization of both inhibitory and excitatory neocortical neurons remains problematic. We here provide an alternative approach to identify GABAergic neurons in the context of in vivo calcium imaging. The method relies on the Emx1(IREScre) recombinase driven expression of a red fluorescent protein in excitatory neurons and glia. We quantitatively examined the upper layers of the visual neocortex in vivo and found that due to pronounced neuropil staining Emx1(IREScre)-negative and Emx1(IREScre)-positive neurons can be reliably differentiated based on negative and positive contrast, respectively. Immunohistochemical analyses confirmed that the entire population of GABAergic interneurons is represented by Emx1(IREScre)-negative cells. The potential usefulness of the method is exemplified by calcium imaging of sensory-evoked responses in the primary visual cortex. We conclude that the proposed method extends the repertoire of strategies aimed at discriminating two major neocortical neuron populations in situ.

Summary 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.

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.

GABAergic medium-sized striatal output neurons (SONs) provide the principal output for the neostriatum. In vitro and in vivo data indicate that spike discharge of SONs is tightly controlled by effective synaptic inhibition. Although phasic GABAergic transmission critically depends on ambient GABA levels, the role of GABA transporters (GATs) in neostriatal GABAergic synaptic transmission is largely unknown. In the present study we aimed at elucidating the role of GAT-1 in the developing mouse neostriatum (postnatal day (P) 7–34). We recorded GABAergic postsynaptic currents (PSCs) using the whole-cell patch-clamp technique. Based on the effects of NO-711, a specific GAT-1 blocker, we demonstrate that GAT-1 is operative at this age and influences GABAergic synaptic transmission by presynaptic and postsynaptic mechanisms. Presynaptic GABABR-mediated suppression of GABA release was found to be functional at all ages tested; however, there was no evidence for persistent GABABR activity under control conditions, unless GAT-1 was blocked (P12–34). In addition, whereas no tonic GABAAR-mediated conductances were detected in SONs until P14, application of a specific GABAAR antagonist caused distinct tonic outward currents later in development (P19–34). In the presence of NO-711, tonic GABAAR-mediated currents were also observed at P7–14 and were dramatically increased at more mature stages. Furthermore, GAT-1 block reduced the median amplitude of GABAergic miniature PSCs indicating a decrease in quantal size. We conclude that in the murine neostriatum GAT-1 operates in a net uptake mode. It prevents the persistent activation of presynaptic GABABRs (P12–34) and prevents (P7–14) or reduces (P19–34) tonic postsynaptic GABAAR activity.

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.

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.

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.

Two-photon laser-scanning microscopy enables to record neuronal network activity in three-dimensional space while maintaining single-cellular resolution. One of the proposed approaches combines galvanometric x-y scanning with piezo-driven objective movements and employs hardware feedback signals for position monitoring. However, readily applicable methods to quantify the accuracy of those feedback signals are currently lacking. Here we provide techniques based on contact-free laser reflection and laser triangulation for the quantification of positioning accuracy of each spatial axis. We found that the lateral feedback signals are sufficiently accurate (defined as

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.

Cajal-Retzius (CR) cells have been demonstrated to fulfil an important secretory function in the developing neocortex. On the other hand, the contribution of CR cell electrical activity during cortex development is still unclear. Using the whole-cell patch-clamp technique, we studied low-voltage-activated (LVA) Ca(2+) channels in CR cells in the layer I of the mouse visual cortex. CR cells were found to display a transient Ca(2+) current (I(T)) in response to a depolarization step from -100 mV to -40 mV. I(T) showed: (i) typical for LVA Ca(2+) channels voltage dependence of activation (half-activation at -55 mV) and inactivation (half-inactivation at -76 mV); (ii) fast activation and inactivation kinetics, with time constants of 1.4 and 28 ms, respectively, at -40 mV; (iii) fast recovery from steady-state inactivation (time constant: 290 ms); (iv) a complete block by 1 microm mibefradil; and (v) a partial block (to 55%) by 100 microm Ni(2+). The density of I(T) dramatically decreased between postnatal day (P) 1 and P9. Immunostaining demonstrated the presence and postnatal downregulation of the alpha(1G)-subunit of LVA Ca(2+) channels in CR cells. Experiments performed in the current-clamp mode revealed that mibefradil delayed an action potential generation in response to a suprathreshold depolarizing current at P1, but not at P8-9. We suggest that LVA Ca(2+) channels might influence CR cell excitability during the first postnatal week and thereby contribute to the shaping of synaptic connectivity in the neocortical layer I.

Background Delayed cerebral vasospasm is the most common cause of mortality and severe neurological impairment in patients who survive subarachnoid hemorrhage. Despite improvements in the field of diagnostic imaging, options for prevention and medical treatment—primarily with the calcium channel antagonist nimodipine or hemodynamic manipulations—are insufficient. Previous studies have suggested that heme and bilirubin oxidation end products, originating from degraded hemoglobin around ruptured blood vessels, are involved in the development of vasospasm by inhibiting large conductance BK C a potassium channels in vascular smooth muscle cells. In this study, we identify individual heme degradation products regulating arteriolar diameter in dependence of BK C a channel activity. Methods and Results Using differential interference contrast video microscopy in acute brain slices, we determined diameter changes of intracerebral arterioles in mouse visual cortex. In preconstricted vessels, the specific BK C a channel blockers paxilline and iberiotoxin as well as iron‐containing hemin caused vasoconstriction. In addition, the bilirubin oxidation end product Z ‐ BOX A showed a stronger vasoconstrictive potency than its regio‐isomer Z ‐ BOX B. Importantly, Z ‐ BOX A had the same vasoconstrictive effect, independent of its origin from oxidative degradation or chemical synthesis. Finally, in slices of Slo1‐deficient knockout mice, paxilline and Z ‐ BOX A remained ineffective in changing arteriole diameter. Conclusions We identified individual components of the oxidative bilirubin degradation that led to vasoconstriction of cerebral arterioles. The vasoconstrictive effect of Z ‐ BOX A and Z ‐ BOX B was mediated by BK C a channel activity that might represent a signaling pathway in the occurrence of delayed cerebral vasospasm in subarachnoid hemorrhage patients.

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.

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.