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.

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.

Allopregnanolone augments epileptiform activity of an in-vitro mouse hippocampal preparation in the first postnatal week.

In the immature brain the neurotransmitter γ-amino butyric acid (GABA) mediates a membrane depolarization and can contribute to both, inhibition and excitation. Therefore the consequences of a positive modulation of GABA(A) receptors by neurosteroids on epileptiform activity are hard to predict. In order to analyze whether neurosteroids attenuate or exaggerate epileptiform activity in the immature brain, we investigated the effect of the neurosteroid allopregnanolone on epileptiform activity in an in-toto hippocampus preparation of early postnatal mice (postnatal days 4-7) using field potential recordings. These in-vitro experiments revealed that 0.5 µmol/L allopregnanolone had no effect on ictal-like epileptiform activity, but increased the occurrence of interictal epileptiform events. The allopregnanolone-induced enhancement of interictal epileptiform activity could be blocked by a selective inhibition of synaptic GABAA receptors. In contrast, allopregnanolone had no effect on interictal epileptiform activity upon enhanced extrasynaptic GABAergic activity. Patch-clamp experiments demonstrated that allopregnanolone prolonged the decay of GABAergic postsynaptic currents, but had no effect on tonic GABAergic currents. We conclude from these results that allopregnanolone can enhance excitability in the immature hippocampus viaprolonged synaptic GABAergic currents. This potential effect of neurosteroids on brain excitability should be considered if they are applied as anticonvulsants to premature or early postnatal babies.