Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex

Abstract

Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25-P30, ≥ 50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments.

Keywords: adolescence; secondary motor cortex; sensitive period; sleep loss.

Figure 1.

Experimental Timeline and MOs Projections. A, Experimental timeline. Between P25 and P30, mice were allowed to sleep normally (C) or subjected to CSR. All mice were injected with AAV-GFP between P44 and P47, and each mouse was perfused exactly 3 weeks later. Middle, Right, The location of the viral injections on the skull and in a coronal brain section. A, Anterior; P, posterior; D, dorsal; V ventral; B, bregma. B, Example of projections from MOs in two representative mice (C and CSR) 3 weeks after injection of AAV-GFP. Measurements are given in millimeters from bregma. Final analysis included 14 mice in each group.

Figure 2.

Projection Fractions in Controls and CSR Mice. A, A plot of projection fractions (normalized by injection volume) across all mice (y-axis) and all regions (x-axis). B, Correlations of projection fraction patterns in mice with injection site. The mouse with the most extreme distance in injection location is taken as the basis for correlation (and is by definition equal to one). All regions were first normalized to have unit means to account for differences between injections. Without this normalization, the mean pairwise correlation of all animals is 0.903 ± 0.07. Distance in micrometers is measured to the center of the injection site from the midline (826 ± 140, left), the midline merging of the anterior commissure (1771 ± 262, middle) or the pial surface (636± 60, right). Values are given for Pearson’s rho. C, Left, Box plot of the condition influence, $k_{cj}$, of the GLM for all regions. A positive $k_{cj}$ indicates that the control group has a higher connectivity than the CSR group (see Results for details). Right, An expanded version that is aligned and color-coded to match the subdivision in major anatomical regions.