Involvement of Ca2+-Dependent Hyperpolarization in Sleep Duration in Mammals

Abstract

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca(2+)-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca(2+)-dependent K(+) channels (Kcnn2 and Kcnn3), voltage-gated Ca(2+) channels (Cacna1g and Cacna1h), or Ca(2+)/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca(2+) ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca(2+)-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.

Next-generation mammalian genetics toward organism-level systems biology

“…In particular, researchers should carefully consider to what extent potential mosaicism (e.g., mutational variations in the triple-CRISPR method, or undetectable contamination of wild-type cells in the ES mouse method) would affect the final results of a scientific study. In our above experiments, the phenotypic variations of F0 mice were comparable with those in wild-type or suitable control animals, 62, 163, 164 suggesting that mutational variations (triple-CRISPR) or undetectable contamination of wild-type cells (ES mouse) do not seem problematic at lease in these cases. To further exclude the possibility of artifact phenotypes due to mutational variations or undetectable contamination of wild-type cells, we recommend that researchers independently generate a whole-body bi-allelic KO mice by using a second set of triple-CRISPR for the same gene, or to independently generate a whole-body bi-allelic KI mice using an independent clone of ES cells.…”

“…To further assess the roles of these genes in vivo, we next produced KO mice for 33 genes with the triple-CRISPR methods and eventually identified 8 genes important for regulating sleep duration. 62, 163 …”

“…This includes (1) preparation of appropriate control animals without any obvious defects in the focused phenotype (e.g., ES mice from wild-type ESCs or ESCs without genome editing for a clock gene rescue study, 164 and tyrosinase triple-CRISPR KO mice for a sleep study 62 ), (2) evaluation of genetic composition of the F0 animals with strict criteria (e.g., detection of a genomic deletion as a proof of efficient triple-CRISPR method 62, 163 or a highly sensitive detection of contaminated host embryo-derived cells 164 ). In addition, it is also useful, if necessary, to evaluate undesirable mutations in the coding sequences by exon sequencing 163 of triple-CRISPR KO mice. It is also useful to avoid cumulative mutations 165 by using ESCs with minimal passage numbers for the production of ES mice.…”

“…In fact, these non-invasive methods have already enabled sleep phenotyping of dozens of triple-CRISPR KO mice. 62, 163 Organism-level systems biology is thus coming to fruition with next-generation mammalian genetics, whole-body clearing and imaging with single-cell resolution, and non-invasive and quantitative phenotyping methods. Organism-level systems biology based on such new technologies will accelerate our understanding of complex and dynamic molecular and cellular circuits in the near future.…”

The biological function of the cellular prion protein: an update

“…Tantalizingly, recent work indicates that calcium-dependent hyperpolarization is critical to sleep duration, and that sleep deterioration is associated with impairment of calcium-dependent potassium channels, voltage-gated calcium channels (VGCC), and N-methyl-D-aspartate (NMDA) glutamate receptors [76]. Since both hyperpolarization linked to calcium dyshomeostasis and NMDA receptor-related hyperexcitability were documented in Prnp ablated mice (discussed in the next section), loss of PrP C -dependent control of these ion channels may underlie the sleep disruption in PrP C -deficient mice and perhaps also in prion diseases.…”