Frequency- and spike-timing-dependent mitochondrial Ca2+ signaling regulates the metabolic rate and synaptic efficacy in cortical neurons.

Mitochondrial activity is crucial for the plasticity of central synapses, but how the firing pattern of pre- and postsynaptic neurons affects the mitochondria remains elusive. We recorded changes in the fluorescence of cytosolic and mitochondrial Ca2+ indicators in cell bodies, axons, and dendrites of cortical pyramidal neurons in mouse brain slices while evoking pre- and postsynaptic spikes. Postsynaptic spike firing elicited fast mitochondrial Ca2+ responses that were about threefold larger in the somas and apical dendrites than in basal dendrites and axons. The amplitude of these responses and metabolic activity were extremely sensitive to the firing frequency. Furthermore, while an EPSP alone caused no detectable Ca2+ elevation in the dendritic mitochondria, the coincidence of EPSP with a backpropagating spike produced prominent, highly localized mitochondrial Ca2+ hotspots. Our results indicate that mitochondria decode the spike firing frequency and the Hebbian temporal coincidences into the Ca2+ signals, which are further translated into the metabolic output and most probably lead to long-term changes in synaptic efficacy.

Subcellular distribution of persistent sodium conductance in cortical pyramidal neurons.

Cortical pyramidal neurons possess a persistent Na+ current (INaP) which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, INaP is active at subthreshold voltages and therefore plays an important role in neuronal input-output processing. The subcellular distribution of channels responsible for INaP and the mechanisms which render them persistent are not known. Using high-speed fluorescence Na+ imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the INaP elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na+ conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying INaP was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by about 10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most INaP originates in the AIS. Finally, we show that endogenous polyamines constrain INaP availability in both somato-dendritic and axonal compartments of non-dialyzed cortical neurons.SIGNIFICANCE STATEMENT:The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (INaP) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na+ imaging and electrical recordings, we reconstructed the INaP in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages INaP originates predominately from the axon, due to the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains INaP availability in all compartments of non-dialyzed cortical neurons.

Aberrant activity of mitochondrial NCLX is linked to impaired synaptic transmission and is associated with mental retardation.

Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes.

Dynamic Gain Analysis Reveals Encoding Deficiencies in Cortical Neurons That Recover from Hypoxia-Induced Spreading Depolarizations.

Cortical regions that are damaged by insults, such as ischemia, hypoxia, and trauma, frequently generate spreading depolarization (SD). At the neuronal level, SDs entail complete breakdown of ionic gradients, persisting for seconds to minutes. It is unclear whether these transient events have a more lasting influence on neuronal function. Here, we describe electrophysiological changes in cortical neurons after recovery from hypoxia-induced SD. When examined with standard measures of neuronal excitability several hours after recovery from SD, layer 5 pyramidal neurons in brain slices from mice of either sex appear surprisingly normal. However, we here introduce an additional parameter, dynamic gain, which characterizes the bandwidth of action potential encoding by a neuron, and thereby reflects its potential efficiency in a multineuronal circuit. We find that the ability of neurons that recover from SD to track high-frequency inputs is markedly curtailed; exposure to hypoxia did not have this effect when SD was prevented pharmacologically. Staining for Ankyrin G revealed at least a fourfold decrease in the number of intact axon initial segments in post-SD slices. Since this effect, along with the effect on encoding, was blocked by an inhibitor of the Ca2+-dependent enzyme, calpain, we conclude that both effects were mediated by the SD-induced rise in intracellular Ca2+ Although effects of calpain activation were detected in the axon initial segment, changes in soma-dendritic compartments may also be involved. Whatever the precise molecular mechanism, our findings indicate that in the context of cortical circuit function, effectiveness of neurons that survive SD may be limited.SIGNIFICANCE STATEMENT Spreading depolarization, which commonly accompanies cortical injury, entails transient massive breakdown of neuronal ionic gradients. The function of cortical neurons that recover from hypoxia-induced spreading depolarization is not obviously abnormal when tested for usual measures of neuronal excitability. However, we now demonstrate that they have a reduced bandwidth, reflecting a significant impairment of their ability to precisely encode high-frequency components of their synaptic input in output spike trains. Thus, neurons that recover from spreading depolarizations are less able to function normally as elements in the multineuronal cortical circuitry. These changes are correlated with activation of the calcium-dependent enzyme, calpain.

Role of sodium channel subtype in action potential generation by neocortical pyramidal neurons.

Neocortical pyramidal neurons express several distinct subtypes of voltage-gated Na+ channels. In mature cells, Nav1.6 is the dominant channel subtype in the axon initial segment (AIS) as well as in the nodes of Ranvier. Action potentials (APs) are initiated in the AIS, and it has been proposed that the high excitability of this region is related to the unique characteristics of the Nav1.6 channel. Knockout or loss-of-function mutation of the Scn8a gene is generally lethal early in life because of the importance of this subtype in noncortical regions of the nervous system. Using the Cre/loxP system, we selectively deleted Nav1.6 in excitatory neurons of the forebrain and characterized the excitability of Nav1.6-deficient layer 5 pyramidal neurons by patch-clamp and Na+ and Ca2+ imaging recordings. We now report that, in the absence of Nav1.6 expression, the AIS is occupied by Nav1.2 channels. However, APs are generated in the AIS, and differences in AP propagation to soma and dendrites are minimal. Moreover, the channels that are expressed in the AIS still show a clear hyperpolarizing shift in voltage dependence of activation, compared with somatic channels. The only major difference between Nav1.6-null and wild-type neurons was a strong reduction in persistent sodium current. We propose that the molecular environment of the AIS confers properties on whatever Na channel subtype is present and that some other benefit must be conferred by the selective axonal presence of the Nav1.6 channel.

Functional implications of axon initial segment cytoskeletal disruption in stroke.

Axon initial segment (AIS) is the proximal part of the axon, which is not covered with a myelin sheath and possesses a distinctive, specialized assembly of voltage-gated ion channels and associated proteins. AIS plays critical roles in synaptic integration and action potential generation in central neurons. Recent evidence shows that stroke causes rapid, irreversible calpain-mediated proteolysis of the AIS cytoskeleton of neurons surrounding the ischemic necrotic core. A better understanding of the molecular mechanisms underlying this "non-lethal" neuronal damage might provide new therapeutic strategies for improving stroke outcome. Here, we present a brief overview of the structure and function of the AIS. We then discuss possible mechanisms underlying stroke-induced AIS damage, including the roles of calpains and possible sources of Ca(2+) ions, which are necessary for the activation of calpains. Finally, we discuss the potential functional implications of the loss of the AIS cytoskeleton and ion channel clusters for neuronal excitability.