Low-voltage-activated Ca²+ channels. from burst generators to Ca²+ sources in thalamic oscillatory activity

Ca2+ ions play key signalling roles in all fundamental neurophysiological processes. The diversity of these roles is accomplished with astounding specificity, velocity and flexibility. Voltage-gated Ca2+ channels represent an important Ca2+ source in neurons, as they allow for Ca2+ influx upon excitatory electrical stimulation. My thesis has addressed the signalling roles of the third of the three major classes of VGCCs, the CaV3 Ca2+ channel family. These channels give rise to the low-voltage activated Ca2+ currents, also called T-type Ca2+ currents, and are predominantly found in neurons capable of generating rhythmic burst discharges. For example, in thalamic neurons, Ca2+ influx through these channels is well known to be critical for the generation of rhythmic burst discharges during neuronal oscillations typical during sleep. However, whether IT-mediated Ca2+ entry adopts signalling roles in thalamic neurons has not been addressed. I hypothesized that identifying Ca2+-dependent targets of CaV3 channels in the thalamus would help to elucidate Ca2+-dependent signalling processes related to sleep, and, thus, ultimately help to assess the roles of sleep in neuronal function. I have identified a dual signalling role for Ca2+ entry through CaV3 channels using electrophysiological, imaging, genetic, and computational techniques. I also demonstrate that this Ca2+ signalling process occurs in a compartmentalized structure and is highly organized. In the nRt, which is well-known for its vigorous bursting activity during sleep-related largescale oscillations, we found that IT-mediated Ca2+ signalling is the dominant Ca2+ source in nRt dendrites, raising Ca2+ to a high level of ~ 0. 7 μM/burst. In these dendrites, the Ca2+- dependent SK2-type K+ channels are selective targets of T-type Ca2+. These SK2 channels are located exclusively in nRt dendrites and are gated rapidly by IT. However, this rapid coupling between T-type Ca2+ and SK channels is not static. Instead, we identified SERCA pumps as modulators of the functional complex of CaV3 and SK2 channels. This modulation occurs in a competitive manner, since SERCA pumps sequestrate Ca2+ from the same pool of Ca2+ ions that gates SK2 channels, while Ca2+ entering through other sources is not taken up. The interplay of CaV3 channels, SK2 channels and SERCA pumps suggests that nRt dendrites are specialized in handling T-type Ca2+ to regulate oscillatory dynamics. Moreover, my work suggests that these cells have developed unique strategies to handle large, repetitive Ca2+ oscillations, and to sequester them specifically, thereby potentially using them to control endoplasmic reticulum-dependent neuronal functions, such as Ca2+-induced Ca2+ release or even protein synthesis. In collaborative efforts, we addressed the physiological relevance of our findings in vivo by studying the sleep behaviour and physiology of SK2 KO mice. Knocking out SK2, the selective target for T-type Ca2+, revealed a strong attenuation of low-frequency rhythmic activity in the EEG during NREMS and a destabilization of sleep behaviour that is manifested by an enhancement of NREMS fragmentation. These data indicate that SK2 channels strongly control sleep, and the selective weakening of frequency bands related to nRt function indicates that the signalling complex identified in my work could act as an amplifier of the synaptic and cellular events leading to large-scale thalamocortical oscillations typical for normal sleep. Altogether, my thesis presents a fully reconstructed link, from a single gene encoding a Ca2+-gated K+ channel, to the mechanism of its gating via T-type Ca2+, to its role in cellular activity, to one of the most dramatic disturbances of a healthy sleep EEG. I demonstrate that sleep is accompanied by large, unique Ca2+ signalling events in thalamic neurons that might contribute to steer the electroencephalographic manifestation and behavioural stabilization of sleep. I propose that the identification of the molecular basis of these processes will help identify targets to improve sleep quality in disease.