Altered SW under BL conditions in Nlgn2 KO mice

SW density and properties were compared between Nlgn2 KO mice and littermates over the course of a 24-h undisturbed recording. A significant interaction between Genotype and Interval was found for SW density (F34,561 = 2.9, p < 0.001; Fig. 1A), positive phase duration (F34,561 = 1.8, p < 0.05; Fig. 1E) and frequency (F34,561 = 1.7, p < 0.05; Fig. 1F). Nlgn2 KO mice have a higher SW density than WT and HET mice, a difference which was more prominent during the light period. Nlgn2 KO mice also show a shorter positive phase duration than both WT and HET littermates, which was mainly observed during the dark period, as well as for the first and last 3 intervals of the light period. This observation was paralleled by a higher SW frequency in KO mice in comparison to littermates at similar time intervals. A significant effect of Genotype was found for SW amplitude (F2,33 = 10.2, p < 0.001; Fig. 1B), slope (F2,33 = 12.7, p < 0.001; Fig. 1C), and negative phase duration (F2,33 = 3.8, p < 0.05; Fig. 1D). Nlgn2 KO mice showed a higher amplitude, steeper slope, and shorter negative phase duration of SW than their littermates, independent of time-of-day. These results indicate a wide range of effects of the KO of Nlgn2 in male mice on SW density and properties during undisturbed/BL SWS.

Fig. 1

SW density and properties during SWS under BL conditions in Nlgn2 KO mice and littermates. Twenty-four-hour dynamics of SW (A) density, (B) amplitude, (C) slope, (D) negative phase duration, (E) positive phase duration, and (F) frequency. Significant interactions between Genotype and Intervals were decomposed by planned comparisons and represented with different color datapoints. Significant main Genotype effects were decomposed by planned comparisons and illustrated with vertical lines accompanied by symbols. Nlgn2 KO mice datapoints identified in orange with a red contour, as well as stars (*), indicate significant differences in comparison to HET and WT mice (p < 0.05). Gray backgrounds represent the dark period

Modified sleep rebound after SD in Nlgn2 KO mice

The effect of SD on vigilance state duration and distribution was next investigated in Nlgn2 WT, HET, and KO mice. The percentage of time spent in wakefulness, SWS, and PS was first compared between genotypes for the full 24-h period including the 6-h SD and 18 h of recovery (Fig. 2A, left). A significant Genotype effect was found for the percent time spent awake (F2,36 = 4.0, p < 0.05) and in PS (F2,36 = 4.6, p < 0.05), while a tendency was found for SWS (F2,36 = 3.2, p = 0.05). Nlgn2 KO mice spent a higher percentage of time awake, a lower percentage of time in PS, and tended to spend less time in SWS in comparison to both WT and HET littermates. When specifically considering the light period (Fig. 2A, middle), a significant Genotype effect was found for wake (F2,36 = 15.6, p < 0.001) and SWS (F2,36 = 17.4, p < 0.001), but not for PS (F2,36 = 1.4, p = 0.3), showing that Nlgn2 KO mice spent a lower percentage of time awake and a higher percentage of time in SWS than both WT and HET mice. When considering the subsequent dark period (Fig. 2A, right), a significant Genotype effect was found for all states (wake: F2,36 = 12.6, p < 0.001; SWS: F2,36 = 12.4, p < 0.001; PS: F2,36 = 10.6, p < 0.001), showing that KO mice spent a higher percentage of time awake, and a lower percentage of time in both SWS and PS in comparison to WT and HET mice. The 24-h time courses of time spent in each state further support these observations, with significant interactions between Genotype and Hour for wake (F34,612 = 2.8, p < 0.001; Fig. 2B, left), SWS (F34,612 = 2.8, p < 0.001; Fig. 2B, middle), and PS (F34,612 = 2.4, p < 0.001; Fig. 2B, right). Nlgn2 KO mice spent less time awake and more time asleep across the light period following SD, while the subsequent higher amount of wake (and lower amount of both SWS and PS) observed in these mice is mainly found in the first half of the dark period following SD.

Fig. 2

Sleep architecture of Nlgn2 KO mice and littermates during a 6-h SD and 18-h recovery. (A) Percentage of time spent in wake, SWS, and PS for the total 24 h (left), the 12-h light period (middle), and the 12-h dark period (right). (B) Twenty-four-hour distribution of time spent in wake, SWS, and PS. (C) Mean duration of individual bouts (left) and number of individual bouts (right) of wake, SWS, and PS for the light and dark periods. (D) Time spent in SWS (top) and PS (bottom) during the 6-h SD. (E) Latency to initiate SWS (top) and PS (bottom) from the end of SD. (F) Accumulated difference in SWS (left) and PS (right) during SD/recovery from BL values. (G) SWS (top) and PS (bottom) recovery slopes evaluated from the 6th to the 12th intervals of corresponding data in panel F. Significant Genotype by Interval interactions were decomposed by planned comparisons and represented with different color datapoints. Significant Genotype by Light/dark period interactions and main Genotype effects were decomposed by planned comparisons and illustrated with lines accompanied by symbols. Nlgn2 KO mice red datapoints and # symbols indicate significant differences (p < 0.05) in comparison to WT mice. Nlgn2 KO mice orange datapoints and + signs indicate significant differences (p < 0.05) in comparison to HET mice. Nlgn2 KO mice datapoints in orange with a red contour and stars (*) indicate significant differences (p < 0.05) in comparison to both HET and WT mice. Dashed backgrounds represent the 6-h SD. Gray backgrounds represent the dark period. REC: 18-h recovery after SD

To verify whether effects on time spent in vigilance states were associated with the duration or number of individual bouts of wake, SWS and/or PS, these two variables were investigated separately for the light and dark periods of the SD/recovery 24-h recording. Regarding mean bout duration, a significant interaction between Genotype and Period (light/dark) was found for wake (F2,36 = 20.3, p < 0.001) and SWS (F2,36 = 3.6, p < 0.05), but not PS (F2,36 = 0.2, p = 0.8), showing that Nlgn2 KO mice had longer wake bouts than WT and HET littermates, and longer SWS bouts than WT mice, but only during the dark period (Fig. 2C, left). For the number of bouts, a significant interaction between Genotype and Period (light/dark) was found for wake (F2,36 = 13.5, p < 0.001), SWS (F2,36 = 13.4, p < 0.001), and PS (F2,36 = 7.0, p < 0.05), showing that Nlgn2 KO mice had less bouts than both littermate groups, again only in the dark period (Fig. 2C, right). These results suggest a consolidation of both wake and SWS in KO mice during the dark period. They also indicate that longer wake bout duration and lower number of sleep bouts are the respective main contributors to the increased time spent in wake and decreased time spent in sleep observed in KO mice during the dark period.

The time spent in SWS and PS during the SD was then compared between genotypes to assess potential differences in SD efficacy (Fig. 2D). A significant Genotype effect was found for time spent in SWS during SD (F2,36 = 10.4, p < 0.001), showing that Nlgn2 KO mice spent more time in SWS than WT and HET mice. Nevertheless, mice (all genotypes) were deprived of > 94% of their BL sleep amounts during SD, supporting an efficient SD procedure. No Genotype effect was found for PS (F2,36 = 0.3, p = 0.8). An elevated SWS amount during SD in Nlgn2 KO mice may result from a shorter sleep latency. The latency to enter SWS or PS after SD was thus compared between genotypes. For SWS, a Genotype effect was found (F2,36 = 3.9, p = 0.03), showing that Nlgn2 KO mice initiated SWS faster than HET littermates (trend of p = 0.09 for the comparison with WT; Fig. 2E, top). For PS, a similar significant Genotype effect was found (F2,36 = 4.6, p = 0.02), showing that Nlgn2 KO mice initiated PS faster than HET littermates (trend of p = 0.06 in comparison to WT; Fig. 2E, bottom). Overall, these results show that Nlgn2 KO mice tend to fall asleep faster, and this might explain the greater challenge when enforcing wakefulness in these animals.

Sleep loss and recovery were then compared between genotypes using accumulated differences from previously published BL values [24], and this separately for SWS and PS (Fig. 2F). A significant Genotype by Hour interaction was found for the accumulated PS differences (F34,612 = 2.3, p < 0.05), but not the accumulated SWS differences (F34,612 = 1.4, p = 0.2). Nlgn2 KO mice showed smaller accumulated PS differences than WT and HET littermates for approximately 10 h following the end of SD. These findings suggest normal SWS and altered PS recovery dynamics. However, the time spent in a given sleep state during BL affects the accumulated differences during and after SD. To obtain a refined readout of recovery dynamics, we computed the speed of SWS and PS recovery for each genotype. The slope from the 6th to the 12th intervals (recovery specifically occurring during the light period) of accumulated PS difference showed a significant Genotype effect (F2,36 = 3.6, p < 0.05; Fig. 2G, bottom), with Nlgn2 KO mice recovering PS faster than WT littermates (i.e., steeper slope). No significant Genotype effect was found for the slope of the accumulated SWS difference (F2,36 = 1, p = 0.4; Fig. 2G, top). Altogether, these results indicate that the KO of Nlgn2 modifies vigilance state duration and distribution following SD in male mice, impacting sleep recovery.

Altered ECoG response to SD in Nlgn2 KO mice

The impact of SD on vigilance state quality (i.e., ECoG spectral content) was explored in Nlgn2 WT, HET, and KO mice. Wake, SWS, and PS relative spectral activity was compared between genotypes for the 24-h recording including the 6-h SD and 18 h of recovery (Fig. 3A). For wakefulness, significant Genotype effects were found for theta (6.5–10 Hz; F2,33 = 5.8–11.1, p < 0.05), sigma (12.5–15 Hz; F2,33 = 3.4–6.1, p < 0.05), beta (15–30 Hz; F2,33 = 5.1–20.5, p < 0.05), and low gamma (30–50 Hz; F2,33 = 10.2–26, p < 0.05) frequencies: Nlgn2 KO mice had less theta, beta, and gamma activity during wake than both WT and HET mice, and less sigma activity than WT mice only (Fig. 3A, top). For SWS, significant Genotype effects were found for delta (1.5-3 Hz; F2,33 = 3.7–5.8, p < 0.05) and low gamma (32–50 Hz; F2,33 = 3.3–12.4, p < 0.05) frequencies; with Nlgn2 KO mice having more delta activity than WT, and less low gamma activity in comparison to both WT and HET mice (Fig. 3A, middle). Finally, for PS, significant Genotype effects were found for high delta/theta (3.5–8.5 Hz; F2,33 = 4.6–32.9, p < 0.05), alpha (10-13.5 Hz; F2,33 = 6.2–14.3, p < 0.05), high beta (25–30 Hz; F2,33 = 3.7–14.2, p < 0.05), and low gamma (30–50 Hz; F2,33 = 13.1–28.9, p < 0.05) frequencies: Nlgn2 KO mice had more low theta and alpha activity, and less high theta, high beta, and low gamma activity than both littermates (Fig. 3A, bottom). Interestingly, the PS theta activity alterations in KO mice could result from a shift in the theta-peak frequency, which is at 7.2 \(\pm\) 0.1 Hz in WT, 7.3 \(\pm\) 0.1 Hz in HET, while at 5.8 \(\pm\) 0.1 Hz in KO (F2,33 = 45.6, p < 0.001; Fig. 3A, bottom-left panel inset).

Fig. 3

ECoG spectral activity of Nlgn2 KO mice and littermates during SD and/or recovery. (A) Relative spectral activity between 0.5 and 50 Hz during wake (top), SWS (middle), and PS (bottom) for the total 24-h. An enlargement of the SWS activity between 0.5 and 4 Hz is presented in the inset of the middle panel. The peak frequency of PS activity is also presented in the inset of the lower panel. (B) Relative spectral activity between 0.5 and 50 Hz during wake (top), SWS (middle), and PS (bottom) for the total 24-h and expressed as percentage of BL (C) Twenty-four-hour dynamics of relative activity presented for wake theta, alpha, and gamma frequency bands, and for SWS delta frequencies. Significant Genotype by Interval interactions were decomposed by planned comparisons and represented with different color datapoints. Significant main Genotype effects were decomposed by Tuckey’s post hoc tests in panels A and B, and illustrated with lines at the top of each graphs, or decomposed by planned comparisons in panel C and illustrated with a vertical line accompanied with a star. Red lines at the top of graphs and Nlgn2 KO red datapoints indicate significant differences (p < 0.05) between KO and WT mice. Nlgn2 KO mice orange datapoints indicate significant differences (p < 0.05) in comparison to HET mice. Black lines at the top of graphs, Nlgn2 KO datapoints in orange with a red contour, and stars (*) indicate significant differences (p < 0.05) between KO mice and all littermates. Dashed backgrounds represent the 6-h SD. Gray backgrounds represent the dark period. REC: 18-h recovery after SD

We previously showed a wide range of ECoG spectral activity changes in Nlgn2 KO mice under BL conditions [24], which could contribute to the effects reported here during and/or after SD. To examine Genotype differences in SD-induced changes from BL, the power of each Hz-bin was expressed as a percentage of the BL corresponding value for each vigilance state (Fig. 3B). For wakefulness, significant Genotype effects were found for high theta/low alpha (7.5–11 Hz; F2,33 = 5.5–13.5, p < 0.05) and high beta/low gamma activity (25.5–50 Hz; F2,33 = 4.1–18.3, p < 0.05; Fig. 3B, top). Nlgn2 KO mice showed a lower SD-induced increase in activity for these frequencies in comparison to littermates. No Genotype effect was found during SWS (F2,33 = 0.01–0.6, p > 0.05; Fig. 3B, middle). For PS, significant Genotype effects were found for delta (1.5–2.5 Hz; F2,33 = 3.4–12.1, p < 0.05), high delta/low theta (3.5-7 Hz; F2,33 = 7.7–17, p < 0.05), alpha/sigma (9.5–15 Hz; F2,33 = 5.2–13, p < 0.05), and beta (15–30 Hz; F2,33 = 3.6–11.9, p < 0.05) activity, with Nlgn2 KO mice having a higher SD-induced increase in activity than WT and HET mice (Fig. 3B, bottom).

To verify whether the Nlgn2 mutation impacts the daily dynamics of relative activity in specific ECoG frequency bands during and after SD, the activity of selected frequency bands was computed across a 24-h time course (Fig. 3C). A significant interaction was found between Genotype and Interval for wake theta (6–9 Hz; F44,594 = 2.0, p < 0.05) and wake alpha (9–12 Hz; F44,594 = 2.6, p < 0.001), with Nlgn2 KO having, in general, less relative activity during the SD and at the beginning of the dark period in comparison to littermates. A significant Genotype effect was rather found for wake gamma (30–50 Hz; F10,27 = 10.6, p < 0.001) relative activity, with Nlgn2 KO mice having overall lower activity than both their littermates. A significant interaction was also found between Genotype and Interval for SWS delta (1–4 Hz; F26,429 = 7.1, p < 0.001) relative activity, with Nlgn2 KO having mainly less activity than both their littermates for the first two or three intervals following SD. These results indicate that the KO of Nlgn2 in male mice alters different ECoG responses to SD, with a particularly prominent impact on wake and PS.

Modified SW properties after SD in Nlgn2 KO mice

SW properties such as density, amplitude, and slope have been reported to be higher under elevated homeostatic sleep pressure in both humans and rodents [45, 56,57,58,59,60]. SW density and properties were thus compared between Nlgn2 KO mice and littermates for the 18 h of recovery following the 6-h SD. A significant interaction was found between Genotype and Interval for SW density (F26,429 = 5.8, p < 0.001; Fig. 4A, left), showing that Nlgn2 KO mice have a higher SW density than their littermates, with the magnitude of the genotype difference varying across the day. A significant Genotype effect was found for SW amplitude (F2,33 = 9.0, p < 0.001; Fig. 4B, left), slope (F2,33 = 10.8, p < 0.001; Fig. 4C, left), and frequency (F2,33 = 4.0, p = 0.03; Fig. 4D, left). Nlgn2 KO mice have higher SW amplitude, slope, and frequency than both littermates, independently of time-of-day.

Fig. 4

SW density and properties during SWS under recovery conditions in Nlgn2 KO mice and littermates. Twenty-four-hour dynamics of SW (A) density, (B) amplitude, (C) slope, and (D) frequency for the 18-h recovery following a 6-h SD. Left panels show absolute data, and right panels data as a percentage of the 24-h BL mean. A discontinuous line was placed at 100% on the Y axis of right panels to help data visualization. Significant Genotype by Interval interactions were decomposed by planned comparisons and represented with different color datapoints. Significant main Genotype effects were decomposed by planned comparisons and illustrated with vertical lines accompanied by symbols. Nlgn2 KO red datapoints indicate significant differences (p < 0.05) in comparison to WT mice. Nlgn2 KO orange datapoints indicate significant differences (p < 0.05) in comparison to HET mice. Nlgn2 KO mice datapoints identified in orange with a red contour, as well as stars (*), indicate significant differences in comparison to HET and WT mice (p < 0.05). Dashed backgrounds represent the 6-h SD. Gray backgrounds represent the dark period

Considering the large between-genotype differences in SW under BL conditions (Fig. 1), SW density and properties were expressed as a percentage of the BL mean values to unmask potential differences in the response to SD. Significant interactions between Genotype and Interval were found for SW density (F26,429 = 6.1, p < 0.001; Fig. 4A, right), amplitude (F26,429 = 3.3, p < 0.001; Fig. 4B, right), and slope (F26,429 = 2.0, p = 0.01; Fig. 4C, right). Nlgn2 KO mice had a blunted increase in SW density in comparison to littermates for the first three/four intervals following SD. KO mice also had a lower increase in SW amplitude than WT mice for the first interval after SD, and an increase in SW amplitude at the end of the dark period that is absent in both littermates. Finally, Nlgn2 KO had an increase in SW slope at the end of the dark period which was significantly different in comparison to HET littermates. No significant effect was found for changes relative to BL in SW frequency (Genotype x Interval: F26,429 = 1.2, p = 0.2; Genotype: F2,33 = 1.2, p = 0.3; Fig. 4D, right). These results indicate that the Nlgn2 mutation increases SW density, amplitude and slope not only in BL, but also under sleep deprived conditions, and that it generally dampens the 24-h dynamics of SW density and properties.

SD does not affect hypersynchronized ECoG event occurrence

Nlgn2 KO mice have hypersynchronized (potentially epileptic-like) ECoG activity [24, 25], and it is known that sleep loss can worsen epileptic manifestations [39]. We thus assessed the effect of SD on hypersynchronized event occurrence. Events were manually identified on the 24-h recordings including 6 h of SD and 18 h of recovery (Fig. 5A). Events were found only during wake and PS, not during SWS, as previously reported [24]. As explained in the Methods, no direct comparisons were made between BL and SD/recovery data to avoid potential scorer bias. It is however possible to assess the effect of homeostatic sleep pressure looking at event progression during and following SD. The number of events was compiled for the light and dark periods for all three genotypes and a significant interaction was found between Genotype and Period (light/dark) for both wake (F2,36 = 7.8, p = 0.001; Fig. 5B, top left) and PS (F2,36 = 30.4, p < 0.001; Fig. 5B, bottom left). Not surprisingly, Nlgn2 KO mice had more wake and PS events than their littermates, but this difference was higher in the dark period for wake events, and greater in the light period for PS events. Plotting the number of events observed in KO animals in a 24-h time course showed that the number of wake and PS hypersynchronized events follows the same 24-h dynamics as the time spent in each state (Fig. 5B, right top and bottom). To better assess an effect of SD on event occurrence, event density was calculated by dividing the number of events in a given state by the time spent in that state. A significant interaction was found between Genotype and Period (light/dark) only for PS (F2,36 = 10.7, p < 0.001; Fig. 5C, bottom left), with Nlgn2 KO animals having a higher event density than their littermates, but this difference being more prominent in the light than the dark period. Interestingly, only a significant Genotype effect was found for wake hypersynchronized event density (F2,36 = 37.1, p < 0.001; Fig. 5C, top left), with KOs having a greater event density than their WT and HET littermates, independently of the light-dark period. These phenotypes can be further visualized with a 24-h time course, in which wake event density indeed appeared relatively constant throughout the day, while the PS event density seems to peak at the end of both the light and dark periods (Fig. 5C, top and bottom right). These results suggest that homeostatic sleep pressure does not increase either wake or PS hypersynchronized event occurrence (e.g., absence of peak at the end of the SD/beginning of recovery sleep).

Fig. 5

Hypersynchronized ECoG events in Nlgn2 KO mice and littermates for BL and SD/recovery recordings. (A) Examples of wake and PS hypersynchronized events with identification criteria. (B) Number of wake (top) and PS (bottom) hypersynchronized events in all genotypes for the light and dark periods (left) of the SD/recovery 24-h recordings. The time courses of event numbers are also shown (right) for KO mice, with time spent in each vigilance state plotted on the right y axes. (C) Density of wake (top) and PS (bottom) hypersynchronized events in all genotypes for the light and dark periods (left) of the SD/recovery 24-h recordings. The time courses of event densities are also shown (right) for KO mice, with time spent in each vigilance state plotted on the right y axes. (D) Time course of wake (top) and PS (bottom) hypersynchronized events in KO mice for the 24 h of BL with time spent in each state plotted on the right y axes. Significant Genotype by Light/dark period interactions were decomposed by planned comparisons and illustrated with lines accompanied by symbols. Significant Genotype effects are represented by symbols alone. Stars (*) indicate significant differences in comparison to HET and WT mice (p < 0.05), and triangles of dots indicate significant light-dark differences (p < 0.05). Dashed backgrounds represent the 6-h SD. Gray backgrounds represent the dark period

Still, SD could have modified the 24-h variations of wake and PS hypersynchronized event occurrence compared to BL conditions. To investigate this possibility, data from BL recordings [24] were reanalysed as 24-h time courses of hypersynchronized event densities for wake and PS in Nlgn2 KO mice (Fig. 5D, top and bottom). The 24-h variations in event density during BL appeared relatively similar to those of the SD/recovery recording, with wake event density showing only small changes and PS event density peaking at the light/dark transition. These results suggest no major effect of SD on the overall dynamics of hypersynchronized event occurrence.

Nlgn2 is part of the cortical gene network decreased by SD

To explore mechanisms underlying the role of NLGN2 in the homeostatic response to sleep loss, the gene expression response to SD was interrogated in the cerebral cortex of WT mice using RNAseq. Of the 22,485 transcripts for which read number allowed comparison between SD mice and time-matched controls, the expression of 4,613 was significantly changed by SD (FDR < 0.05), which is similar in magnitude to what was reported by previous studies (3,988 transcripts in [61]; 3,201 transcripts in [62]). Hierarchical clustering of DEGs revealed two main clusters: 2,097 transcripts decreased by SD, and 2,516 increased (Fig. 6A). The lists of genes included in each of the two clusters were then separately fed to the DAVID analysis platform for GO analysis, which identifies enriched biological processes, cellular compartments, and molecular functions in each dataset. A total of 39 GO terms were significantly (FDR < 0.05) associated with the cluster of genes with expression decreased by SD, and 172 GO terms were significantly (FDR < 0.05) associated with the cluster of genes with increased expression (Fig. 6B). The transcripts with decreased expression were related to numerous synaptic GO terms including “synaptic transmission”, “presynaptic active zone”, and “synaptic vesicle” (Fig. 6C). Interestingly, “cell adhesion” and “GABAergic synapse”, which are of particular interest regarding NLGN2 functions, were amongst the decreased GO terms with the highest fold enrichment in this dataset (Fig. 6C). Concerning the GO terms enriched in the cluster of transcripts with increased expression, they were related to cellular functions such as transcription/translation (e.g., “RNApol II down/upregulation”, “RNA splicing”, “protein folding”, “chromatin-DNA binding”), stress responses (e.g., “glucocorticoid receptors”, “unfolded protein response”, “ER stress response”, “apoptotic process”), and phosphorylation, as well as to nervous system relevant functions such as “excitatory synapse” and “synaptic plasticity” (Fig. 6D).

Fig. 6

Genome-wide gene expression response to SD quantified for the cerebral cortex of WT mice. (A) Hierarchical cluster analysis of DEGs between control and sleep deprived WT mice using Ward’s method. (B) Summary of gene expression outputs, number of DEGs, as well as associated GO enrichment. GO term fold enrichment, percentage of genes from the analyzed cluster related to the specific GO terms, and selected GO term FDR for (C) decreased and (D) increased DEGs. Normalized read counts of DEGs targeted for their association to (E) GABAergic and (F) cholinergic synapses. DEGs include genes with FDR < 0.05

Considering the reported involvement of NLGN2 at GABAergic, cholinergic, and dopaminergic synapses [15,16,17,18], the effect of SD on the cortical expression of genes involved in these types of neurotransmission was examined in greater detail. As expected from GO enrichment analyses, numerous transcripts related to GABAergic synapses were significantly affected by SD (Fig. 6E). Indeed, SD decreased the cortical RNA levels of Nlgn2, Arhgef9, Slitrk3, Gabra1, Gabra4, Gabrb2, Gabrd, and Gabrg2, while increasing the levels of Igsf9b, Igsf21, Mdga1, and Gabbr2 (Fig. 6E). Amongst the decreased transcripts, Arhgef9 and Slitrk3 are of particular interest as they encode GABAergic synapse structural proteins that have been shown to interact with NLGN2 [63, 64]. The other decreased GABAergic transcripts (Gabra1, Gabra4, Gabrb2, Gabrd, and Gabrg2) code for ionotropic GABAergic receptor subunits, while the increased transcript Gabbr2 encodes a metabotropic GABA receptor. The increased Mdga1 transcript is also of relevance as it codes for a protein known to prevent NLGN2 binding to its presynaptic ligands the neurexins [65,66,67]. Finally, the increased Igsf9b and Igsf21 transcripts encode other adhesion proteins involved at GABAergic synapses [68, 69]. In contrast with the large effect of SD on GABAergic transmission-relevant gene expression, only four transcripts related to cholinergic synapse function (Chrna7, Chrnb3, Chrm3, and Ric3) were significantly affected by SD, all being decreased (Fig. 6F). As for dopamine neurotransmission-related genes, only the expression of Comt was significantly affected by SD, being increased (Control: 2,732 \(\pm\) 33 normalized read count; SD: 2,935 \(\pm\) 42 normalized read count; FDR = 0.02). Altogether, these results indicate that the expression of a substantial number of genes related to the function of NLGN2 is affected by sleep loss in the mouse cerebral cortex, especially GABAergic neurotransmission-related transcripts.