Neuroligin-1 links neuronal activity to sleep-wake regulation

Abstract

Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation.

Keywords: ChIP; EEG; gene expression; sleep homeostasis; synaptic plasticity.

Fig. 1.

Effect of SD on the expression of Nlg1 transcript variants. (A) Scheme of the Nlg1 mRNA showing position of splice sites and qPCR amplicons (A and NA or B and NB, with and without insert A or B, respectively; C, common probe). Dark gray, cholinesterase domain (94–1,878 bp); blue, transmembrane domain (2,086–2,142 bp); light blue, PSD95-binding domain (2,512–2,529 bp). (B) Relative expression of Nlg1 transcripts in the forebrain of C57BL/6J (B6) and AKR/J (AK) mice at ZT6 (6 h after light onset) under the control condition (n = 4 for B6 and AK) or after a 6-h SD (n = 3 for B6, 4 for AK). SD significantly decreased the expression of Nlg1 with insert B and common Nlg1 (condition effects: F_1,12 ≥ 5.1, *P < 0.05) and increased the expression of Nlg1 with A only in AK mice (interaction: F_1,12 = 6.8, *P < 0.05: compared with Control). AK mice also expressed more common Nlg1 and Nlg1 without A and without B than B6 mice (strain effects: F_1,12 ≥ 9.8, P < 0.01). (C) Expression of Nlg1 transcripts in the forebrain of DBA/2J (D2) mice at ZT6 under the control condition or after a 6-h SD in mice submitted to either a sham surgery (n = 7/group) or an ADX (n = 6/group). SD decreased the expression of Nlg1 without A and with B (condition effects: F_1,22 ≥ 4.2, *P ≤ 0.05), and a similar tendency was found for common Nlg1 (F_1,22 = 3.7, ^∆P < 0.07).

Fig. 2.

Effect of SD on NLG1 protein level. (A) Western blot showing NLG1 in the SN fraction of the anterior (Ant) forebrain in four control (C) and four SD C57BL/6J (B6) mice. Endogenous control Actin is also shown. (B) Quantification of Western blots for NLG1 in total protein fraction (Tot) and SN of the anterior and posterior (Post) parts of B6 mice forebrain (n = 10 or 11/group). SD significantly decreased the level of NLG1 in Ant SN (t = 2.4, *P < 0.05). (C) Western blot showing NLG1 (and Actin) in the SN of the Ant brain in five C and five SD AKR/J (AK) mice. (D) Quantification of NLG1 Western blots in Tot and SN of Ant and Post parts of AK mice forebrain (n = 5–7/group). SD significantly increases the level of NLG1 in Ant SN (t = −3.5, *P < 0.05).

Fig. 3.

Sleep structure and cortical synchrony in Nlg1 KO mice. (A) Vigilance state duration in WT (+/+, n = 10) and Nlg1 KO (−/−, n = 12) mice for 24 h, 12-h light, and 12-h dark periods. For the 24-h and 12-h dark periods, Nlg1 KO mice spent less time in wakefulness and more in NREM sleep (t ≥ 2.5 and t = −2.6, respectively; *P < 0.05). (B) State duration per hour in WT and KO mice. Genotype-by-time interactions were found for wake, NREM, and REM sleep (F_23,460 > 1.9, P < 0.01). Differences between KO and WT are indicated by red symbols (P < 0.05; same in D, E, and G). (C) Mean duration of wake bouts in WT and KO mice for 24 h, 12-h light, and 12-h dark periods. For the 24 h and the 12-h dark period, the duration of wake bouts was lower in KO mice than in WT (t ≥ 2.2, *P < 0.05). (D) Spectral power for the three states in WT and KO mice. (E) Forty-eight-hour time course of delta power in WT and KO mice. Genotype-by-time interactions were found for the 24-h baseline (F_17,340 = 2.3, P < 0.05) and for the 18-h recording after a 6-h SD (F_13,247 = 3.7, P < 0.05). Pink symbols indicate a trend for difference between WT and KO (P < 0.1). (F) NREM sleep measured during the 6-h SD did not differ significantly between WT and KO (t = 1.8, P = 0.08). The same was observed for loss of time spent in NREM sleep and latency to sleep after SD (t = 1.5, P = 0.15). (G) Time course of accumulated differences in NREM sleep between the 6-h SD followed by 18 h of recovery and baseline conditions. Significant genotype-by-time interaction was found (F_23,460 = 9.1, P < 0.001). Gray backgrounds indicate dark periods and black rectangles indicate the 6-h SD.

Fig. 4.

Rhythmic expression of Nlg1 and binding of its gene by clock transcription factors. (A) Relative expression of Nlg1 transcripts measured at ZT0, -6, -12, and -18 in the forebrain of C57BL/6J (B6) and AKR/J (AK) mice (n = 5–8/group). The expression of Nlg1 containing insert B is highest at ZT6 (time effect: F_3,49 = 11.7, P < 0.01). A significant strain effect was observed for Nlg1 with A and without B (F_3,49 ≥ 4.4, P < 0.05). Gray backgrounds indicate dark periods. *P < 0.05 between indicated values (also in C). (B) Diagram of the mouse, rat, and human Nlg1 gene showing the position of E-boxes relative to the transcription start sites (arrows). Red E-boxes represent CANNTG and orange CACGTG. Absence of E in the box indicates the presence of multiple E-boxes. (C) Binding of BMAL1 and CLOCK to the CACGTG of Nlg1 in B6 mouse cerebral cortex measured at ZT0, -6, -12, and -18 (n = 3/point, except n = 2 for ZT12). BMAL1 and CLOCK bind to Nlg1 in a time-of-day–dependent manner (F_3,7 ≥ 4.2, P = 0.05). (D) Binding measured at ZT6 after 6 h of SD and expressed relative to the binding in undisturbed control (n = 5/group). SD decreased CLOCK binding to Nlg1 (t = −15.1, **P < 0.001), whereas a tendency was observed for BMAL1 (t = −1.7, P = 0.17). (E) Model showing how elevated sleep pressure may change Nlg1 expression: prolonged wakefulness could lead to detachment of CLOCK/BMAL1 from the gene, which would decrease expression of Nlg1 with insert B and synaptic NLG1 and down-regulate NMDAR function.