Werner Kilb

Cl− uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1

GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl−]i in immature neocortical neurones causes GABAA receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl− accumulation remain controversial. Therefore, the GABA reversal potential (EGABA) or [Cl−]i in early postnatal rat neocortical neurones was measured by the gramicidin‐perforated patch‐clamp method, and the relative expression levels of the cation−Cl− cotransporter mRNAs (in the same cells) were examined by semiquantitative single‐cell multiplex RT‐PCR to look for statistical correlations with [Cl−]i. The mRNA expression levels were positively (the Cl− accumulating Na+,K+−2Cl− cotransporter NKCC1) or negatively (the Cl− extruding K+−Cl− cotransporter KCC2) correlated with [Cl−]i. NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl−]i and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on EGABA were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA‐mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex.

Rapid developmental switch in the mechanisms driving early cortical columnar networks

The immature cerebral cortex self-organizes into local neuronal clusters long before it is activated by patterned sensory inputs. In the cortical anlage of newborn mammals, neurons coassemble through electrical or chemical synapses either spontaneously or by activation of transmitter-gated receptors. The neuronal network and the cellular mechanisms underlying this cortical self-organization process during early development are not completely understood. Here we show in an intact in vitro preparation of the immature mouse cerebral cortex that neurons are functionally coupled in local clusters by means of propagating network oscillations in the beta frequency range. In the newborn mouse, this activity requires an intact subplate and is strongly synchronized within a cortical column by gap junctions. With the developmental disappearance of the subplate at the end of the first postnatal week, activation of NMDA (N-methyl-d-aspartate) receptors in the immature cortical network is essential to generate this columnar activity pattern. Our findings show that during a brief developmental period the cortical network switches from a subplate-driven, gap-junction-coupled syncytium to a synaptic network acting through NMDA receptors to generate synchronized oscillatory activity, which may function as an early functional template for the development of the cortical columnar architecture.

Kinetic Properties of Cl− Uptake Mediated by Na+-Dependent K+-2Cl− Cotransport in Immature Rat Neocortical Neurons

GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity. Although it is well accepted that depolarizing GABA actions are caused by an elevated intracellular Cl− concentration ([Cl−]i), the mechanisms of Cl− accumulation in immature neurons are still a matter of debate. Using patch-clamp, microfluorimetric, immunohistochemical, and molecular biological approaches, we studied the mechanism of Cl− uptake in Cajal-Retzius (CR) cells of immature [postnatal day 0 (P0) to P3] rat neocortex. Gramicidin-perforated patch-clamp and 6-methoxy-N-ethylquinolinium-microfluorimetric measurements revealed a steady-state [Cl−]i of ∼30 mm that was reduced to values close to passive distribution by bumetanide or Na+-free solutions, suggesting a participation of Na+-K+-2Cl− cotransport isoform 1 (NKCC1) in maintaining elevated [Cl−]i. Expression of NKCC1 was found in CR cells on the mRNA and protein levels. To determine the contribution of NKCC1 to [Cl−]i homeostasis in detail, Cl− uptake rates were analyzed after artificial [Cl−]i depletion. Active Cl− uptake was relatively slow (47.2 ± 5.0 μm/s) and was abolished by bumetanide or Na+-free solution. Accordingly, whole-cell patch-clamp recordings revealed a low Cl− conductance in CR cells. The low capacity of NKCC1-mediated Cl− uptake was sufficient to maintain excitatory GABAergic membrane responses, however, only at low stimulation frequencies. In summary, our results demonstrate that NKCC1 is abundant in CR cells of immature rat neocortex and that the slow Cl− uptake mediated by this transporter is sufficient to maintain high [Cl−]i required to render GABA responses excitatory.

Thalamic Network Oscillations Synchronize Ontogenetic Columns in the Newborn Rat Barrel Cortex

Neocortical areas are organized in columns, which form the basic structural and functional modules of intracortical information processing. Using voltage-sensitive dye imaging and simultaneous multi-channel extracellular recordings in the barrel cortex of newborn rats in vivo, we found that spontaneously occurring and whisker stimulation-induced gamma bursts followed by longer lasting spindle bursts were topographically organized in functional cortical columns already at the day of birth. Gamma bursts synchronized a cortical network of 300-400 µm in diameter and were coherent with gamma activity recorded simultaneously in the thalamic ventral posterior medial (VPM) nucleus. Cortical gamma bursts could be elicited by focal electrical stimulation of the VPM. Whisker stimulation-induced spindle and gamma bursts and the majority of spontaneously occurring events were profoundly reduced by the local inactivation of the VPM, indicating that the thalamus is important to generate these activity patterns. Furthermore, inactivation of the barrel cortex with lidocaine reduced the gamma activity in the thalamus, suggesting that a cortico-thalamic feedback loop modulates this early thalamic network activity.

Electrical activity patterns and the functional maturation of the neocortex.

At the earliest developmental stages, sensory neocortical areas in various species reveal distinct patterns of spontaneous neuronal network activity. These activity patterns either propagate over large neocortical areas or synchronize local neuronal ensembles. In vitro and in vivo experiments indicate that these spontaneous activity patterns are generated from neuronal networks in the cerebral cortex, in subcortical structures or in the sensory periphery (retina, cochlea, whiskers). At early stages spontaneous periphery‐driven and also sensory evoked activity is relayed to the developing cerebral cortex via the thalamus and the neocortical subplate, which amplifies the afferent sensory input. These early local and large‐scale neuronal activity patterns influence a variety of developmental processes during corticogenesis, such as neurogenesis, apoptosis, neuronal migration, differentiation and network formation. The experimental data also indicate that disturbances in early neuronal patterns may have an impact on the development of cortical layers, columns and networks. In this article we review our current knowledge on the origin of early electrical activity patterns in neocortical sensory areas and their functional implications on shaping developing cortical networks.

Model-specific effects of bumetanide on epileptiform activity in the in-vitro intact hippocampus of the newborn mouse

The immature brain has a higher susceptibility to develop seizures, which often respond poorly to classical pharmacological treatment. It has been recently suggested that bumetanide, which blocks Na(+)-dependent K(+)-Cl(-)-cotransporter isoform 1 (NKCC1) and thus attenuates depolarizing GABAergic responses, could soothe epileptiform activity in immature nervous systems. To evaluate whether bumetanide consistently attenuates epileptiform activity, we investigated the effect of 10 microM bumetanide in five different in-vitro epilepsy models using field potential recordings in the CA3 region of intact mouse hippocampal preparations at postnatal day 4-7. Bumetanide reduced amplitude and frequency of ictal-like events (ILE) induced by 8.5 mM K(+), but it increased the frequency of ILE induced by 1 microM kainate. Inhibition of ligand-gated Cl(-) channels by 10 microM gabazine and 30 microM strychnine induced interictal activity (IA) that was only marginally affected by bumetanide. Removal of extracellular Mg(2+) induced both ILE and IA. Bumetanide had no effect on these ILE but enhanced the IA. Low-Mg(2+) solution containing 20 microM 4-AP induced late-recurrent discharges, which were slightly attenuated by bumetanide. In summary, our results demonstrate that bumetanide exerts diverse effects in different in-vitro epilepsy models.

Development of the GABAergic System from Birth to Adolescence

The neurotransmitter GABA (γ-aminobutyric acid), acting via inotropic GABAA and metabotropic GABAB receptors, plays an essential role in a variety of distinct neuronal processes, including regulation of neuronal excitability, determination of temporal aspects of spike trains, control of the size and propagation of neuronal assemblies, generation of oscillatory activity, and neuronal plasticity. Although the developmental switch between excitatory and inhibitory GABAA receptor–mediated responses is widely appreciated, the fact that the postnatal maturation of the GABAergic system lasts until late adolescence is not so persuasively promoted. This review summarizes recent knowledge of the maturation of various aspects of the GABAergic systems, like functional expression of GABA synthesizing/degrading enzymes and transporters, density of GABAergic synapses, GABAergic projection patterns, GABA receptor subunit composition, and properties of GABAergic interneurons, with an emphasis on the late developmental alterations. In addition, some aspects of the development of mental capabilities during adolescence and their relation the delayed maturation of the GABAergic system are presented.

Cellular physiology of the neonatal rat cerebral cortex: Intrinsic membrane properties, sodium and calcium currents

The cellular physiology of the primary somatosensory cortex was studied in postnatal day (P) 0 to P5 rats using whole‐cell patch‐clamp recordings. Visually identified Cajal‐Retzius, subplate, bifurcated pyramidal, and immature, putatively migrating neurons showed resting membrane potentials between –44 and –50 mV and TTX‐sensitive action potentials. Immature pyramidal neurons with the smallest surface area (∼1,600 μm2) revealed the largest input resistance (∼1.8 GΩ), and subplate cells with the largest surface area (∼6,200 μm2) showed an input resistance of ∼1 GΩ. Ontogenetically older Cajal‐Retzius and subplate cells revealed shorter and larger action potentials compared to bifurcated and immature pyramidal neurons. Whereas Cajal‐Retzius and subplate cells responded to injection of depolarizing current pulses with a repetitive nonadapting and fast spiking firing pattern, immature pyramidal neurons showed strong adaptation. Subplate cells revealed the fastest action potentials, largest sodium current amplitude (–714 pA), and highest sodium current density (–38 μA/cm2), enabling these cells to transmit afferent activity faithfully to postsynaptic neurons. Whereas all cell types expressed a high‐voltage‐activated (HVA) calcium current, none of them showed a significant low‐voltage‐activated calcium current. The largest peak (–25.5 μA/cm2) and steady‐state (–7.6 μA/cm2) HVA calcium current density could be observed in immature presumed migrating neurons. In contrast, Cajal‐Retzius and subplate neurons showed a significantly lower peak (−4.9 μA/cm2) and steady‐state (<−3.3 μA/cm2) HVA calcium current density. Whereas a large HVA calcium current may promote neuronal migration of immature neurons, low intracellular calcium levels may provoke apoptosis in Cajal‐Retzius and subplate cells. J. Neurosci. Res. 62:574–584, 2000.

Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex

Due to their unique structural and functional properties, subplate cells are ideally suited to function as important amplifying units within the developing neocortical circuit. Subplate neurons have extensive dendritic and axonal ramifications and relatively mature functional properties, i.e. their action potential firing can exceed frequencies of 40 Hz. At earliest stages of corticogenesis subplate cells receive functional synaptic inputs from the thalamus and from other cortical and non-cortical sources. Glutamatergic and depolarizing GABAergic inputs arise from cortical neurons and neuromodulatory inputs arise from the basal forebrain and other sources. Activation of postsynaptic metabotropic receptors, i.e. muscarinic receptors, elicits in subplate neurons oscillatory burst discharges which are transmitted via electrical and chemical synapses to neighbouring subplate cells and to immature neurons in the cortical plate. The tonic non-synaptic release of GABA from GABAergic subplate cells facilitates the generation of burst discharges. These cellular bursts are amplified by prominent gap junction coupling in the subplate and cortical plate, thereby eliciting 10–20 Hz oscillations in a local columnar network. Thus, we propose that neuronal networks are organized at earliest stages in a gap junction coupled columnar syncytium. We postulate that the subplate does not only serve as a transient relay station for afferent inputs, but rather as an active element amplifying the afferent and intracortical activity.

Neuronal precursor‐specific activity of a human doublecortin regulatory sequence

The doublecortin (DCX) gene encodes a 40‐kDa microtubule‐associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5‐kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III β‐tubulin isotype and microtubule‐associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5‐kb region demonstrated the relevance of specific regions containing transcription factor‐binding sites. Moreover, application of neurogenesis‐related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct.