Activity-dependent regulation of the BAX/BCL-2 pathway protects cortical neurons from apoptotic death during early development.

During early brain development, homeostatic removal of cortical neurons is crucial and requires multiple control mechanisms. We investigated in the cerebral cortex of mice whether the BAX/BCL-2 pathway, an important regulator of apoptosis, is part of this machinery and how electrical activity might serve as a set point of regulation. Activity is known to be a pro-survival factor; however, how this effect is translated into enhanced survival chances on a neuronal level is not fully understood. In this study, we show that caspase activity is highest at the neonatal stage, while developmental cell death peaks at the end of the first postnatal week. During the first postnatal week, upregulation of BAX is accompanied by downregulation of BCL-2 protein, resulting in a high BAX/BCL-2 ratio when neuronal death rates are high. In cultured neurons, pharmacological blockade of activity leads to an acute upregulation of Bax, while elevated activity results in a lasting increase of BCL-2 expression. Spontaneously active neurons not only exhibit lower Bax levels than inactive neurons but also show almost exclusively BCL-2 expression. Disinhibition of network activity prevents the death of neurons overexpressing activated CASP3. This neuroprotective effect is not the result of reduced caspase activity but is associated with a downregulation of the BAX/BCL-2 ratio. Notably, increasing neuronal activity has a similar, non-additive effect as the blockade of BAX. Conclusively, high electrical activity modulates BAX/BCL-2 expression and leads to higher tolerance to CASP3 activity, increases survival, and presumably promotes non-apoptotic CASP3 functions in developing neurons.

Chromatin compaction precedes apoptosis in developing neurons.

While major changes in cellular morphology during apoptosis have been well described, the subcellular changes in nuclear architecture involved in this process remain poorly understood. Imaging of nucleosomes in cortical neurons in vitro before and during apoptosis revealed that chromatin compaction precedes the activation of caspase-3 and nucleus shrinkage. While this early chromatin compaction remained unaffected by pharmacological blockade of the final execution of apoptosis through caspase-3 inhibition, interfering with the chromatin dynamics by modulation of actomyosin activity prevented apoptosis, but resulted in necrotic-like cell death instead. With super-resolution imaging at different phases of apoptosis in vitro and in vivo, we demonstrate that chromatin compaction occurs progressively and can be classified into five stages. In conclusion, we show that compaction of chromatin in the neuronal nucleus precedes apoptosis execution. These early changes in chromatin structure critically affect apoptotic cell death and are not part of the final execution of the apoptotic process in developing cortical neurons.

Spontaneous Activity Predicts Survival of Developing Cortical Neurons.

Spontaneous activity plays a crucial role in brain development by coordinating the integration of immature neurons into emerging cortical networks. High levels and complex patterns of spontaneous activity are generally associated with low rates of apoptosis in the cortex. However, whether spontaneous activity patterns directly encode for survival of individual cortical neurons during development remains an open question. Here, we longitudinally investigated spontaneous activity and apoptosis in developing cortical cultures, combining extracellular electrophysiology with calcium imaging. These experiments demonstrated that the early occurrence of calcium transients was strongly linked to neuronal survival. Silent neurons exhibited a higher probability of cell death, whereas high frequency spiking and burst behavior were almost exclusively detected in surviving neurons. In local neuronal clusters, activity of neighboring neurons exerted a pro-survival effect, whereas on the functional level, networks with a high modular topology were associated with lower cell death rates. Using machine learning algorithms, cell fate of individual neurons was predictable through the integration of spontaneous activity features. Our results indicate that high frequency spiking activity constrains apoptosis in single neurons through sustained calcium rises and thereby consolidates networks in which a high modular topology is reached during early development.

Optogenetically Controlled Activity Pattern Determines Survival Rate of Developing Neocortical Neurons.

A substantial proportion of neurons undergoes programmed cell death (apoptosis) during early development. This process is attenuated by increased levels of neuronal activity and enhanced by suppression of activity. To uncover whether the mere level of activity or also the temporal structure of electrical activity affects neuronal death rates, we optogenetically controlled spontaneous activity of synaptically-isolated neurons in developing cortical cultures. Our results demonstrate that action potential firing of primary cortical neurons promotes neuronal survival throughout development. Chronic patterned optogenetic stimulation allowed to effectively modulate the firing pattern of single neurons in the absence of synaptic inputs while maintaining stable overall activity levels. Replacing the burst firing pattern with a non-physiological, single pulse pattern significantly increased cell death rates as compared to physiological burst stimulation. Furthermore, physiological burst stimulation led to an elevated peak in intracellular calcium and an increase in the expression level of classical activity-dependent targets but also decreased Bax/BCL-2 expression ratio and reduced caspase 3/7 activity. In summary, these results demonstrate at the single-cell level that the temporal pattern of action potentials is critical for neuronal survival versus cell death fate during cortical development, besides the pro-survival effect of action potential firing per se.

Rapid nucleus-scale reorganization of chromatin in neurons enables transcriptional adaptation for memory consolidation.

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals.

Gabaergic Interneurons in Early Brain Development: Conducting and Orchestrated by Cortical Network Activity.

Throughout early phases of brain development, the two main neural signaling mechanisms-excitation and inhibition-are dynamically sculpted in the neocortex to establish primary functions. Despite its relatively late formation and persistent developmental changes, the GABAergic system promotes the ordered shaping of neuronal circuits at the structural and functional levels. Within this frame, interneurons participate first in spontaneous and later in sensory-evoked activity patterns that precede cortical functions of the mature brain. Upon their subcortical generation, interneurons in the embryonic brain must first orderly migrate to and settle in respective target layers before they can actively engage in cortical network activity. During this process, changes at the molecular and synaptic level of interneurons allow not only their coordinated formation but also the pruning of connections as well as excitatory and inhibitory synapses. At the postsynaptic site, the shift of GABAergic signaling from an excitatory towards an inhibitory response is required to enable synchronization within cortical networks. Concomitantly, the progressive specification of different interneuron subtypes endows the neocortex with distinct local cortical circuits and region-specific modulation of neuronal firing. Finally, the apoptotic process further refines neuronal populations by constantly maintaining a controlled ratio of inhibitory and excitatory neurons. Interestingly, many of these fundamental and complex processes are influenced-if not directly controlled-by electrical activity. Interneurons on the subcellular, cellular, and network level are affected by high frequency patterns, such as spindle burst and gamma oscillations in rodents and delta brushes in humans. Conversely, the maturation of interneuron structure and function on each of these scales feeds back and contributes to the generation of cortical activity patterns that are essential for the proper peri- and postnatal development. Overall, a more precise description of the conducting role of interneurons in terms of how they contribute to specific activity patterns-as well as how specific activity patterns impinge on their maturation as orchestra members-will lead to a better understanding of the physiological and pathophysiological development and function of the nervous system.

Coincident Activation of Glutamate Receptors Enhances GABAA Receptor-Induced Ionic Plasticity of the Intracellular Cl--Concentration in Dissociated Neuronal Cultures.

Massive activation of γ-amino butyric acid A (GABAA) receptors during pathophysiological activity induces an increase in the intracellular Cl--concentration ([Cl-]i), which is sufficient to render GABAergic responses excitatory. However, to what extent physiological levels of GABAergic activity can influence [Cl-]i is not known. Aim of the present study is to reveal whether moderate activation of GABAA receptors mediates functionally relevant [Cl-]i changes and whether these changes can be augmented by coincident glutamatergic activity. To address these questions, we used whole-cell patch-clamp recordings from cultured cortical neurons [at days in vitro (DIV) 6-22] to determine changes in the GABA reversal potential (EGABA) induced by short bursts of GABAergic and/or synchronized glutamatergic stimulation. These experiments revealed that pressure-application of 10 short muscimol pulses at 10 Hz induced voltage-dependent [Cl-]i changes. Under current-clamp conditions this muscimol burst induced a [Cl-]i increase of 3.1 ± 0.4 mM (n = 27), which was significantly enhanced to 4.6 ± 0.5 mM (n = 27) when glutamate was applied synchronously with the muscimol pulses. The muscimol-induced [Cl-]i increase significantly attenuated the inhibitory effect of GABA, as determined by the GABAergic rheobase shift. The synchronous coapplication of glutamate pulses had no additional effect on the attenuation of GABAergic inhibition, despite the larger [Cl-]i transients under these conditions. In summary, these results indicate that moderate GABAergic activity can induce functionally relevant [Cl-]i transients, which were enhanced by coincident glutamate pulses. This ionic plasticity of [Cl-]i may contribute to short-term plasticity of the GABAergic system.

Modulation of Neocortical Development by Early Neuronal Activity: Physiology and Pathophysiology.

Animal and human studies revealed that patterned neuronal activity is an inherent feature of developing nervous systems. This review summarizes our current knowledge about the mechanisms generating early electrical activity patterns and their impact on structural and functional development of the cerebral cortex. All neocortical areas display distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, intermittent spontaneous activity is synchronized within small neuronal networks, becoming more complex with further development. This transition is accompanied by a gradual shift from electrical to chemical synaptic transmission, with a particular role of non-synaptic tonic currents before the onset of phasic synaptic activity. In this review article we first describe functional impacts of classical neurotransmitters (GABA, glutamate) and modulatory systems (e.g., acetylcholine, ACh) on early neuronal activities in the neocortex with special emphasis on electrical synapses, nonsynaptic and synaptic currents. Early neuronal activity influences probably all developmental processes and is crucial for the proper formation of neuronal circuits. In the second part of our review, we illustrate how specific activity patterns might interfere with distinct neurodevelopmental processes like proliferation, migration, axonal and dendritic sprouting, synapse formation and neurotransmitter specification. Finally, we present evidence that transient alterations in neuronal activity during restricted perinatal periods can lead to persistent changes in functional connectivity and therefore might underlie the manifestation of neurological and neuropsychiatric diseases.

Electrical activity controls area-specific expression of neuronal apoptosis in the mouse developing cerebral cortex.

Programmed cell death widely but heterogeneously affects the developing brain, causing the loss of up to 50% of neurons in rodents. However, whether this heterogeneity originates from neuronal identity and/or network-dependent processes is unknown. Here, we report that the primary motor cortex (M1) and primary somatosensory cortex (S1), two adjacent but functionally distinct areas, display striking differences in density of apoptotic neurons during the early postnatal period. These differences in rate of apoptosis negatively correlate with region-dependent levels of activity. Disrupting this activity either pharmacologically or by electrical stimulation alters the spatial pattern of apoptosis and sensory deprivation leads to exacerbated amounts of apoptotic neurons in the corresponding functional area of the neocortex. Thus, our data demonstrate that spontaneous and periphery-driven activity patterns are important for the structural and functional maturation of the neocortex by refining the final number of cortical neurons in a region-dependent manner.

Homeostatic interplay between electrical activity and neuronal apoptosis in the developing neocortex.

An intriguing feature of nervous system development in most animal species is that the initial number of generated neurons is higher than the number of neurons incorporated into mature circuits. A substantial portion of neurons is indeed eliminated via apoptosis during a short time window - in rodents the first two postnatal weeks. While it is well established that neurotrophic factors play a central role in controlling neuronal survival and apoptosis in the peripheral nervous system (PNS), the situation is less clear in the central nervous system (CNS). In postnatal rodent neocortex, the peak of apoptosis coincides with the occurrence of spontaneous, synchronous activity patterns. In this article, we review recent results that demonstrate the important role of electrical activity for neuronal survival in the neocortex, describe the role of Ca2+ and neurotrophic factors in translating electrical activity into pro-survival signals, and finally discuss the clinical impact of the tight relation between electrical activity and neuronal survival versus apoptosis.

NKCC1-Mediated GABAergic Signaling Promotes Postnatal Cell Death in Neocortical Cajal-Retzius Cells.

During early development, a substantial proportion of central neurons undergoes programmed cell death. This activity-dependent process is essential for the proper structural and functional development of the brain. To uncover cell type-specific differences in the regulation of neuronal survival versus apoptosis, we studied activity-regulated cell death in Cajal-Retzius neurons (CRNs) and the overall neuronal population in the developing mouse cerebral cortex. CRNs in the upper neocortical layer represent an early-born neuronal population, which is important for cortical development and largely disappears by apoptosis during neonatal stages. In contrast to the overall neuronal population, activity blockade with tetrodotoxin improved survival of CRNs in culture. Activation of GABAA receptors also blocked spontaneous activity and caused overall cell death including apoptosis of CRNs. Blockade of the Na-K-Cl transporter NKCC1 in vitro or its genetic deletion in vivo rescued CRNs from apoptosis. This effect was mediated by blockade of the p75NTR receptor signaling pathway. In summary, we discovered a novel developmental death pathway mediated by NKCC1, via GABAA receptor-mediated membrane depolarization and p75NTR signaling in CRNs. This pathway controls apoptosis of CRNs and may be critically involved in neurodevelopmental disorders such as autism and schizophrenia.

Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion.

We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl-/HCO3- exchanger pendrin and the Na+-driven Cl-/2HCO3- exchanger (NDCBE) in ß-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na+ homeostasis and provide evidence that the Na+/Cl- cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K+ concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+-activated K+ channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients.

Spontaneous Neuronal Activity in Developing Neocortical Networks: From Single Cells to Large-Scale Interactions.

Neuronal activity has been shown to be essential for the proper formation of neuronal circuits, affecting developmental processes like neurogenesis, migration, programmed cell death, cellular differentiation, formation of local and long-range axonal connections, synaptic plasticity or myelination. Accordingly, neocortical areas reveal distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, when immature neurons start to develop voltage-dependent channels, spontaneous activity is highly synchronized within small neuronal networks and governed by electrical synaptic transmission. Subsequently, spontaneous activity patterns become more complex, involve larger networks and propagate over several neocortical areas. The developmental shift from local to large-scale network activity is accompanied by a gradual shift from electrical to chemical synaptic transmission with an initial excitatory action of chloride-gated channels activated by GABA, glycine and taurine. Transient neuronal populations in the subplate (SP) support temporary circuits that play an important role in tuning early neocortical activity and the formation of mature neuronal networks. Thus, early spontaneous activity patterns control the formation of developing networks in sensory cortices, and disturbances of these activity patterns may lead to long-lasting neuronal deficits.

Disruption of Slc4a10 augments neuronal excitability and modulates synaptic short-term plasticity.

Slc4a10 is a Na(+)-coupled Cl(-)-HCO3 (-) exchanger, which is expressed in principal and inhibitory neurons as well as in choroid plexus epithelial cells of the brain. Slc4a10 knockout (KO) mice have collapsed brain ventricles and display an increased seizure threshold, while heterozygous deletions in man have been associated with idiopathic epilepsy and other neurological symptoms. To further characterize the role of Slc4a10 for network excitability, we compared input-output relations as well as short and long term changes of evoked field potentials in Slc4a10 KO and wildtype (WT) mice. While responses of CA1 pyramidal neurons to stimulation of Schaffer collaterals were increased in Slc4a10 KO mice, evoked field potentials did not differ between genotypes in the stratum radiatum or the neocortical areas analyzed. Paired pulse facilitation was diminished in the hippocampus upon disruption of Slc4a10. In the neocortex paired pulse depression was increased. Though short term plasticity is modulated via Slc4a10, long term potentiation appears independent of Slc4a10. Our data support that Slc4a10 dampens neuronal excitability and thus sheds light on the pathophysiology of SLC4A10 associated pathologies.

Early GABAergic circuitry in the cerebral cortex.

In the cerebral cortex GABAergic signaling plays an important role in regulating early developmental processes, for example, neurogenesis, migration and differentiation. Transient cell populations, namely Cajal-Retzius in the marginal zone and thalamic input receiving subplate neurons, are integrated as active elements in transitory GABAergic circuits. Although immature pyramidal neurons receive GABAergic synaptic inputs already at fetal stages, they are integrated into functional GABAergic circuits only several days later. In consequence, GABAergic synaptic transmission has only a minor influence on spontaneous network activity during early corticogenesis. Concurrent with the gradual developmental shift of GABA action from excitatory to inhibitory and the maturation of cortical synaptic connections, GABA becomes more important in synchronizing neuronal network activity.

Comparison of spike parameters from optically identified GABAergic and glutamatergic neurons in sparse cortical cultures.

Primary neuronal cultures share many typical features with the in vivo situation, including similarities in distinct electrical activity patterns and synaptic network interactions. Here, we use multi-electrode array (MEA) recordings from spontaneously active cultures of wildtype and glutamic acid decarboxylase 67 (GAD67)-green fluorescent protein (GFP) transgenic mice to evaluate which spike parameters differ between GABAergic interneurons and principal, putatively glutamatergic neurons. To analyze this question we combine MEA recordings with optical imaging in sparse cortical cultures to assign individual spikes to visually-identified single neurons. In our culture system, excitatory and inhibitory neurons are present at a similar ratio as described in vivo, and spike waveform characteristics and firing patterns are fully developed after 2 weeks in vitro. Spike amplitude, but not other spike waveform parameters, correlated with the distance between the recording electrode and the location of the assigned neuron's soma. Cluster analysis of spike waveform properties revealed no particular cell population that may be assigned to putative inhibitory or excitatory neurons. Moreover, experiments in primary cultures from transgenic GAD67-GFP mice, which allow optical identification of GABAergic interneurons and thus unambiguous assignment of extracellular signals, did not reveal any significant difference in spike timing and spike waveform parameters between inhibitory and excitatory neurons. Despite of our detailed characterization of spike waveform and temporal spiking properties we could not identify an unequivocal electrical parameter to discriminate between individual excitatory and inhibitory neurons in vitro. Our data suggest that under in vitro conditions cellular classifications of single neurons on the basis of their extracellular firing properties should be treated with caution.

Minireview: pH and synaptic transmission.

As a general rule a rise in pH increases neuronal activity, whereas it is dampened by a fall of pH. Neuronal activity per se also challenges pH homeostasis by the increase of metabolic acid equivalents. Moreover, the negative membrane potential of neurons promotes the intracellular accumulation of protons. Synaptic key players such as glutamate receptors or voltage-gated calcium channels show strong pH dependence and effects of pH gradients on synaptic processes are well known. However, the processes and mechanisms that allow controlling the pH in synaptic structures and how these mechanisms contribute to normal synaptic function are only beginning to be resolved.

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I.

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.