Astrocytic Ca 2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca 2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca 2+ activity, brain rhythms and behavior. We show that astrocytic Ca 2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca 2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca 2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca 2+ signaling in regulating slow wave sleep.

No abstract

Cortical spreading depression is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity. Accumulating evidence indicates that cortical spreading depression underlies the migraine aura and that similar waves promote tissue damage in stroke, trauma, and hemorrhage. Cortical spreading depression is characterized by neuronal swelling, profound elevation of extracellular potassium and glutamate, multiphasic blood flow changes, and drop in tissue oxygen tension. The slow speed of the cortical spreading depression wave implies that it is mediated by diffusion of a chemical substance, yet the identity of this substance and the pathway it follows are unknown. Intercellular spread between gap junction-coupled neurons or glial cells and interstitial diffusion of K+ or glutamate have been proposed. Here we use extracellular direct current potential recordings, K+-sensitive microelectrodes, and 2-photon imaging with ultrasensitive Ca2+ and glutamate fluorescent probes to elucidate the spatiotemporal dynamics of ionic shifts associated with the propagation of cortical spreading depression in the visual cortex of adult living mice. Our data argue against intercellular spread of Ca2+ carrying the cortical spreading depression wavefront and are in favor of interstitial K+ diffusion, rather than glutamate diffusion, as the leading event in cortical spreading depression.

Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 – in combination with various optical techniques – thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD.

The identity of cortical circuits mediating nociception and pain is largely unclear. The cingulate cortex is consistently activated during pain, but the functional specificity of cingulate divisions, the roles at distinct temporal phases of central plasticity and the underlying circuitry are unknown. Here we show in mice that the midcingulate division of the cingulate cortex (MCC) does not mediate acute pain sensation and pain affect, but gates sensory hypersensitivity by acting in a wide cortical and subcortical network. Within this complex network, we identified an afferent MCC-posterior insula pathway that can induce and maintain nociceptive hypersensitivity in the absence of conditioned peripheral noxious drive. This facilitation of nociception is brought about by recruitment of descending serotonergic facilitatory projections to the spinal cord. These results have implications for our understanding of neuronal mechanisms facilitating the transition from acute to long-lasting pain.

To date, it has been difficult to reveal physiological Ca 2ϩ events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca 2ϩ signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca