Introduction
Sleep disturbances are predominant in patients suffering from neurodevelopmental disorders and often associated with worse disease outcomes. Decreased sleep duration, increased sleep fragmentation, and alterations in electroencephalographic activity have notably been reported in autism spectrum disorders (ASD) and schizophrenia [
1‐
5]. Importantly, less total sleep time was correlated with worse social and communication skills in patients with ASD [
2]. Likewise, poorer sleep hygiene and sleep spindle deficits were associated with worse positive/negative symptoms and cognitive functions in patients with schizophrenia [
5,
6]. Epileptic syndromes also present with numerous sleep disturbances, which can include less time spent in paradoxical sleep (PS) and/or longer PS latency [
7‐
10], and increased subjective sleepiness [
11]. Sleep complaints were even found to associate with a worse quality of life in epileptic patients [
12]. Accordingly, understanding the molecular mechanisms underlying sleep disturbances in neurodevelopmental disorders could help alleviate disease symptomatology.
Neuroligins (NLGNs) are adhesion proteins usually expressed at postsynaptic sites and involved in both neurodevelopment and synaptic function [
13,
14]. Neuroligin-2 (NLGN2), which mainly regulates gamma aminobutyric acid (GABA)ergic neurotransmission, but also cholinergic and dopaminergic transmission [
15‐
18], was repeatedly associated with neurodevelopmental disorders. In humans, mutations in
NLGN2 have been linked to ASD and schizophrenia [
19,
20], while rodents with NLGN2 deregulations have been found to have behavioral impairments, such as anxiety-like behaviors and impaired social interactions [
21‐
23]. Moreover, both knocking out (KO) and overexpressing
Nlgn2 in mice was shown to cause abnormal (potentially epileptic-like) hypersynchronized cerebral activity [
21,
24,
25]. Interestingly, our group previously reported important sleep disturbances in
Nlgn2 KO mice, such as decreased time spent asleep and a wide range of changes in electrocorticographic (ECoG) activity during wakefulness and sleep states [
24]. Thus, NLGN2 represents a good candidate to unravel the etiology of sleep disorders in neurodevelopmental pathologies.
During slow-wave sleep (SWS), cortical neurons alternate between a hyperpolarized/silent (down) state and a depolarized/firing (up) state, creating slow waves (SW) of low frequency (< 4 Hz) and high amplitude [
26,
27]. SW and slow-wave activity, which are implicated in cognitive functions including memory consolidation and extinction [
28,
29], were reported to be altered in autistic and epileptic patients [
30‐
32]. Our previous findings show a large increase in SWS delta (1–4 Hz) activity in
Nlgn2 KO mice [
24]. However, it remains to be defined whether this effect originates from an increased capacity to generate SW during SWS (increased density) or in more synchrony between individual neurons contributing to SW generation (higher amplitude and steeper slope). This is of relevance to help define the role of NLGN2 in SW generation during SWS as well as in sleep-related cognitive alterations observed in neurodevelopmental disorders.
Sleep deprivation (SD) was shown to impact GABAergic neurotransmission in key sleep regulatory areas of the rodent brain [
33‐
36]. Given the involvement of NLGN2 at GABAergic synapses [
15,
16], and that increased immunostaining of NLGN2 was reported after SD on wake-promoting orexin neurons [
33], the KO of
Nlgn2 in mice might affect the response to sleep loss (e.g., sleep duration and distribution, ECoG activity). Our observation that
Nlgn2 KO mice have an altered 24-h dynamics of SWS delta activity under baseline (BL) conditions [
24] also supports this hypothesis. Indeed, the dynamics of SWS delta activity is well-recognized to reflect sleep homeostasis, being increased by prolonged wakefulness and dissipating during sleep [
37,
38]. The altered 24-h dynamics of SWS delta activity in
Nlgn2 KO mice could thus indicate potential dysfunctions in homeostatic sleep regulation, which can be further investigated by assessing the response to SD. Interestingly, an impairment of SW slope dissipation across the night was observed in epileptic patients [
30], and SD was reported to increase the occurrence of epileptic discharges and to trigger seizures [
39]. Considering the above-mentioned epileptic-like ECoG activity observed under abnormal expression of
Nlgn2 in mice [
21,
24,
25], this further underscores the relevance of investigating the involvement of NLGN2 in the response to sleep loss.
We here tested the first hypothesis that different SW properties would be altered during SWS in
Nlgn2 KO mice. Given the elevated SWS delta activity in these mutant animals [
24], we specifically expected to find SW of higher amplitude and steeper slope, in addition to a higher density of SW per minute of SWS. Our second hypothesis was that KO mice would show modifications in their response to SD both at the level of vigilance state duration and ECoG activity. We further hypothesized that hypersynchronized event occurrence would be increased in the course of SD in KO mice, peaking at the end of the SD. To test these hypotheses, the ECoG of
Nlgn2 KO mice and littermates was recorded during undisturbed/BL conditions and following a 6-h SD, and the ECoG signal was submitted to state identification, spectral analysis and SW detection. Our findings support the first two hypotheses, revealing multiple alterations in SW properties during SWS and modifications in sleep duration and quality after enforced wakefulness in the absence of NLGN2. However, hypersynchronized events did not show the anticipated changes during SD. We additionally verified the effect of a 6-h SD on
Nlgn2 expression in the cerebral cortex of WT mice and found it to be decreased, along with the expression of many other GABAergic transcripts. Altogether, these results bring further insight into basic sleep-wake regulatory mechanisms involving adhesion molecules, which could help clarifying the etiology of sleep disturbances in neurodevelopmental disorders.
Discussion
We here reported striking alterations in the density and properties of individual SW during SWS, as well as in the response to sleep loss in the absence of NLGN2 protein in mice. More specifically, Nlgn2 KO mice have higher SW density, amplitude, slope, and frequency in comparison to WT and HET mice under BL conditions and following SD. In addition, KO mice spend more time in SWS during early recovery in the light period following SD, more time awake during the subsequent dark period, show an accelerated recovery of lost PS, and an altered ECoG response to sleep loss. Importantly, hypersynchronized wake and PS ECoG events do not peak at the end of the 6-h SD, and the 24-h variations in event occurrence is relatively preserved after SD. In parallel, we have reported that SD decreases the gene expression of Nlgn2 in the mouse cerebral cortex, together with the expression of several genes associated to GABAergic neurotransmission. Altogether, our findings support a role for NLGN2 in shaping SW during SWS and in modulating the electrophysiological response to sleep loss.
NLGN2 regulates SW generation and characteristics
Our prior findings of increased SWS delta activity in
Nlgn2 KO mice under BL conditions [
24], has led to the hypothesis that KO mice would show increased SW density, amplitude, and/or slope. We here report remarkable changes that support this hypothesis with differences so large that they could be used to differentiate KO animals from WT and HET littermates. A higher SW density can be interpreted as an increase in SW generation. In the current study, SW were detected between frequencies of 0.5 and 4 Hz, which includes both slow oscillations (< 1 Hz) and delta waves (1–4 Hz), or slower (delta 1: 0.75-2 Hz) and faster (delta 2: 2.5–3.75 Hz) delta frequencies as recently studied [
70]. Slow oscillations and delta waves appear to originate from different brain networks; slow oscillations being mainly generated by the cerebral cortex and delta waves originating from thalamocortical circuits [
70‐
72]. It is thus likely that the absence of NLGN2 in both intracortical and thalamocortical circuits contributes to the increased SW density observed in
Nlgn2 KO mice. Alterations in inhibitory synaptic transmission in
Nlgn2 KO mice were indeed reported for both the cortex [
16,
73,
74] and the ventrobasal thalamus [
25]. However, considering our finding of higher SW frequency in the absence of NLGN2 (i.e., mean WT between 2.35 and 2.44 Hz; mean KO between 2.42 and 2.59 Hz), it is expected that the specific generation of delta waves by thalamocortical circuits is particularly boosted in the absence of NLGN2. In parallel, a higher SW amplitude is indicative of a greater number of neurons firing (and being silent) simultaneously, while a steeper SW slope can be interpreted as faster and more synchronous switching between firing and silent states, both signs of higher cortical synchrony [
57]. The higher SW amplitude and slope here observed in
Nlgn2 KO mice can thus be interpreted as greater cortical synchronization, as was interpreted before for older mice in comparison to younger ones [
75].
One molecular mechanism by which NLGN2 could modulate SW density and properties is through the chloride exporter KCC2. The knockdown of
Nlgn2 was shown to reduce the expression of KCC2 in vitro, which causes a depolarization of GABAergic equilibrium potential [
76]. In parallel, chloride transporters (NKCC1 and KCC2) and GABAergic equilibrium potential were recently shown to regulate cortical synchronization and SWS delta activity [
77]. For instance, raising intracellular chloride concentration via KCC2 pharmacological inhibition or optogenetic manipulation increases SWS delta activity and neuronal firing during the up state of SW [
77]. Accordingly, the KO of
Nlgn2 could boost SWS delta activity and cortical synchronization by reducing KCC2-mediated chloride transport to the extracellular space (and consequently increasing intracellular chloride). This hypothesis could be tested by assessing the effect of a KCC2 activator on SW density/properties in
Nlgn2 KO mice.
NLGN2 is involved in the electrophysiological response to sleep loss
We also hypothesized that
Nlgn2 KO mice have an altered response to SD, which is supported by our findings showing alterations in sleep rebound and modifications in ECoG activity, including in SD-driven changes in SW density and properties. Regarding sleep architecture,
Nlgn2 KO mice have more SWS than their littermates during the SD, suggesting difficulties to maintain wakefulness under high sleep pressure, and a globally accelerated sleep recovery. We propose that
Nlgn2 KO mice, already spending more time awake under BL conditions [
24], present some rigidity/elevated sensitivity to additional sleep pressure such as experienced during SD. These findings could also suggest that different brain regions/neuronal circuits are involved in NLGN2-dependent modulation of normal sleep and of the response to sleep loss, which could be addressed by targeting manipulations of NLGN2 to different sleep-wake regulatory areas such as the basal forebrain, reticular tegmental nuclei or lateral hypothalamus [
78].
Nlgn2 KO mice also showed major alterations in the ECoG response to SD. During SD, WT and HET mice showed a large increase in wake theta-alpha and gamma activity compared to BL, which was almost completely absent in KO mice. One could speculate an implication of the basal forebrain, which projects to the cerebral cortex and contains cholinergic neurons shown to burst maximally during wake and PS, as their bursting correlates with cortical theta and gamma oscillations [
78‐
81]. Moreover, the firing of GABAergic wake-active basal forebrain neurons was also shown to correlate with cortical wake gamma activity [
78,
81]. Considering the involvement of NLGN2 at both cholinergic and GABAergic synapses [
15,
17], reduced inputs from the basal forebrain might thus be involved in the dampened SD-induced increase in wake theta/gamma activity in
Nlgn2 KO mice. Such altered circuits could also underlie the observed lower PS theta peak frequency, as the firing of PS-active cholinergic basal forebrain neurons specifically correlates with cortical activity around 7 Hz [
80].
Interestingly, increased wake theta activity during extended wakefulness was shown to positively correlate with delta activity during recovery sleep in humans [
82]. Wake theta and gamma activity were further proposed to be the main drivers for the build-up of homeostatic sleep pressure reflected in SWS delta activity in mice [
48]. The absence of theta/gamma-rich wake ECoG activity during SD could therefore greatly contribute to the blunted increases in SWS delta activity and SW density observed in
Nlgn2 KO mice after SD when compared to littermates. A molecular mechanism could again involve KCC2 and intracellular chloride concentration, as the above-mentioned study showed that preventing the shift in GABAergic equilibrium potential after SD reduces the SWS delta activity rebound [
77]. It is possible that a downregulation of NLGN2 is required for proper SW rebound, by way of reducing KCC2 function and increasing intracellular chloride. At the gene expression level, we did find a decreased
Nlgn2 expression after SD, which is in line with previous observations [
83], and related GABAergic transcripts, which could both contribute to changes in intracellular chloride. Interestingly, a reduced KCC2 expression (mRNA and protein) was reported after SD for the hippocampus in rats [
84], and further studies should investigate the mouse cerebral cortex.
Nlgn2 KO mouse model relevance to neurodevelopmental disorders
Considering that sleep loss is known to increase epileptic events in patients [
39], the third hypothesis of this work was that hypersynchronized ECoG events observed in
Nlgn2 KO mice would increase over the course of SD. This hypothesis was not confirmed, with wake event density being mostly constant through SD and recovery, and PS event density peaking at the light-dark transition. Still, it is possible that sleep loss increases overall 24-h event density in comparison to BL, or changes event duration and spectral composition. However, as the BL and SD/recovery hypersynchronized events were here manually identified, years apart and by different scorers, direct comparison could not be done. An automatic event detector is currently being developed to refine the analysis of this phenotype and ensure reproducible quantification across cohorts/projects.
Nonetheless, the current observations have a notable relevance for neurodevelopmental disorders involving epileptic phenotypes,
NLGN gene mutations and/or abnormal excitation/inhibition ratio. Epileptic patients were found to have reduced time spent in PS [
7,
9,
10], increased SWS delta activity [
31,
85], and impaired SW slope dissipation during the sleep episode [
30]. These observations are all reminiscent of findings in
Nlgn2 KO male mice, which indicates that understanding the mechanisms by which NLGN2 modulates sleep amounts and quality could help unraveling the cause of comorbid sleep disturbances in the context of neurodevelopmental disorders such as epilepsy. This is also of relevance considering that poor sleep quality has been associated with more frequent/severe seizures and worse quality of life in patients [
86,
87]. Future investigations should consider the therapeutic potential of manipulating
NLGN2 expression to improve sleep quality in epileptic patients, including patients with co-morbid ASD.
Limitations
The main limitation of our work is the inclusion of male mice only, a choice that was made to reduce variability considering the mixed genetic background of the mouse strain studied. Future efforts will however include both sexes as there are known sex differences in both sleep phenotypes [
88,
89] and neurodevelopmental disorders prevalence/symptomatology [
90‐
93]. In addition, it is here not possible to differentiate potential neurodevelopmental effects from adult stage-specific phenotypes considering the use of a constitutive
Nlgn2 KO mice. Manipulations of
Nlgn2 expression in young and adult animals will bring further insights into the involvement of NLGN2 in sleep-wake regulation in the context of neurodevelopment.
Conclusion
We here show that NLGN2 is a key regulator of SW density and properties during SWS, and that its absence affects the response to sleep loss at multiple levels, potentially involving multiple separate sleep regulatory circuits. We specifically uncover that although SW density is strikingly increased in the absence of NLGN2, daily variations and the typical SD-driven increase in SW density are importantly impaired; thus identifying SW density as the main driver of the dampened 24-h dynamics of SWS delta activity previously reported [
24]. In parallel, we found that hypersynchronized ECoG events observed in
Nlgn2 KO mice are not changed in the course of SD, and that SD decreases the expression of
Nlgn2 in the cerebral cortex together with a molecular network associated with GABAergic neurotransmission. Given that the current RNAseq data indicate a higher expression of RNA splicing-related transcripts in the cerebral cortex following SD, and that NLGN2 exists in two different isoforms, with one preferentially found at GABAergic synapses [
94], investigating the role of different NLGN2 isoforms in sleep regulation will be an important future step.
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