Sleep need-dependent responses of AMPA/NMDA ratio and silent synapses in motor cortex. A) Examples of AMPA currents at -90 mV holding potential and NMDA currents at +50 mV holding potential are shown for: control sleep (CS), 6 h of sleep deprivation (SD) and 4 h of sleep deprivation followed by 2 h of recovery sleep (RS). Traces (100msec duration) are scaled to the NMDA current for comparison (NMDA current measured @ 40msec after AMPA peak current). B) Examples of successes (black) and failures (red) at -90 mV (left, AMPA) and +50 mV (right, NMDA) after minimal stimulation of excitatory inputs to motor cortex pyramidal neurons are shown; Top row: CS sleep, middle row: SD and bottom row: RS (Cal. 10pAX20msec). Rate of failures (% of all stimuli delivered) for AMPA EPSCs (blue) and NMDA EPSCs (red) in the three conditions (+/- sem). C), D) Average (+/- sem) AMPAR and NMDAR EPSC responses (unmatched) and failure rates, respectively, for each sleep condition. E), F) Matched AMPA/NMDA EPSC response and AMPA/NMDA failure rate ratios, respectively, are shown for the three conditions (N=1 cell/slice, 2-3 slice/animal, 3 animals/condition).

snRNAseq data shows cell type and sub-type based on gene expression patterns are unaffected by sleep need. A) UMAP projection of cell-type gene expression pattern following 6 hours ad lib sleep (CS) at ZT=6hour. B) As in “A” except after 6hours sleep deprivation (SD), ZT=6hour. C) The distribution of cell numbers across subtypes is unaffected by sleep need. D) The median number of UMIs/cell is significantly increased by sleep need across all cell subtypes (see_Table S2 and text for statistics).

Cell type specific differential gene expression in response to 6 hours of SD. A) An XY bar plot of cell type specific DEGs (both up and down regulated) organized by cell type specific number of expressed genes (X axis) shows the greatest number of DEGs are found in excitatory pyramidal neurons that project within the telencephalon (ExIT). B) A pie chart of the distribution of DEGs amongst different cell types illustrates that the majority of sleep DEGs are expressed by ExIT neurons. C) An analysis of the cell type specific DEG occurrence shows the greatest probability of significant sleep loss gene response is found in ExIT cells by more than 3-fold compared to all other cell types.

DEG enrichment of cell types in response to SD by autism risk genes, synaptic shaping component genes and DEGs from MEF2c loss of function or constitutive HD4 repression of MEF2c. A) Heat map for cell type DEG enrichment by ASD risk genes, Synaptic Shaping Component genes, MEF2c-cKO DEGs and HD4cn DEGs. B) Model for the control of sleep DEGs by HD4 repression of MEF2c and by pMEF2c during low sleep need and, during high sleep need, de-repression of MEF2c by sequestration of pHD4 to the cytoplasm and dephosphorylation of MEF2c, facilitating expression of sleep genes. C) Model of downstream control by MEF2c and HD4 to facilitate SSC gene expression as sleep need increases. The SSC gene expression after waking, can then decrease AMPA/NMDA ratio and increase silent synapses during sleep to bias glutamate synapses towards decreased strength but increased potential for LTP at the start of the active phase when sleep need is low. D) An illustration of the SWS-SWA response to chronic MEF2c repression or activation. Chronic activation of MEF2c facilitated transcription leads to decreased AMPAR-mediated synaptic strength mimicking increased Ado tone that inhibits cortical-thalamic, glutamate synaptic activity, decreasing activation of cholinergic arousal centers and increasing SWS-SWA. Chronic repression of MEF2c does the opposite, mimicking loss of ADORA1 function and decreasing SWS-SWA.