Neurexin regulates nighttime sleep by modulating synaptic transmission

Abstract

Neurexins are cell adhesion molecules involved in synaptic formation and synaptic transmission. Mutations in neurexin genes are linked to autism spectrum disorders (ASDs), which are frequently associated with sleep problems. However, the role of neurexin-mediated synaptic transmission in sleep regulation is unclear. Here, we show that lack of the Drosophila α-neurexin homolog significantly reduces the quantity and quality of nighttime sleep and impairs sleep homeostasis. We report that neurexin expression in Drosophila mushroom body (MB) αβ neurons is essential for nighttime sleep. We demonstrate that reduced nighttime sleep in neurexin mutants is due to impaired αβ neuronal output, and show that neurexin functionally couples calcium channels (Cac) to regulate synaptic transmission. Finally, we determine that αβ surface (αβ_s) neurons release both acetylcholine and short neuropeptide F (sNPF), whereas αβ core (αβ_c) neurons release sNPF to promote nighttime sleep. Our findings reveal that neurexin regulates nighttime sleep by mediating the synaptic transmission of αβ neurons. This study elucidates the role of synaptic transmission in sleep regulation, and might offer insights into the mechanism of sleep disturbances in patients with autism disorders.

Figure 1. neurexin mutant flies exhibit reduced nighttime sleep.

(A) Annotated transcription of the neurexin gene. The P element XP^d08766 insertion site is marked with a diamond; the RNAi recognition site is labeled at the top. The deleted regions of neurexin mutant alleles (nrx^Δ83 and nrx²⁷³) are indicated by dashed lines. (B) Western blot analysis showing neurexin protein levels in w¹¹¹⁸ control and neurexin mutant flies. Tubulin was used as a loading control. Quantification of the relative protein level for each genotype is presented in the lower panel. (C) Sleep profiles of control w¹¹¹⁸ (black, n = 308), nrx^d08766 (light grey, n = 53), nrx²⁷³/+ (green, n = 32), nrx^273/d08766 (red, n = 63), and nrx^Δ83/273 (grey, n = 192) flies, which is plotted as a 30 min moving average. (D–G) Quantification of sleep onset latency after lights off, total nighttime sleep, average sleep episode length, and number of sleep episodes per night for each genotype. (H) After sleep deprivation overnight for 12 h, nrx^Δ83/273 mutants regain a significantly lower percentage of their lost sleep than w¹¹¹⁸ control flies (mean ± SEM, n = 32 flies per group). Quantification of total sleep time during the first 6 h recovery period is presented in the right panel.

Figure 2. Neurexin expression in mushroom body neurons is essential for nighttime sleep.

(A) Total nighttime sleep in flies with rescued neurexin expression using anatomically restricted GAL4 drivers. All rescue experiments were performed in the nrx^273/Δ83 background; flies carry one copy of the indicated drivers (n = 32). (B,C) Average sleep profiles for rescued and control flies, with curves plotted as 30 min moving averages (B); quantification of total nighttime sleep in rescued and control flies (C); n = 61. (D,E) Average sleep profiles for MB247-GAL4/UAS-nrx^RNAi and control flies, plotted as a 30 min moving average (D), and quantification of total nighttime sleep for each genotype (E); n = 32. (F) Cumulative sleep lost during 12 h of sleep deprivation and regained during subsequent recovery for 24 h in c739-GAL4/+;UAS-nrx^RNAi/+ flies and control flies (n = 16). (G) Model of the fly MB illustrating the different subsets of intrinsic Kenyon cells (KCs) within the lobes. Purple, αβ lobes; orange, α′β′ lobes; blue, γ lobes. (H) Total nighttime sleep in flies with depleted neurexin. Each GAL4 expression pattern is summarized at the top. A single copy of the driver was used for each GAL4 line (n = 32).

Figure 3. Neurexin functions in αβ_sc neurons to promote nighttime sleep.

(A–C) Projection views of confocal stacks at the level of the right MB lobes from NP5286-αβ_s (A), NP7175-αβ_c (B), and NP3208-αβ_p (C) flies driving mCD8::GFP (green). In all panels, the αβ lobes are labeled with anti-FasII (magenta). The inset shows a horizontal cross-section through the vertical collateral at the level of the dashed line in each panel. Scale bar = 20 μm. (D,E) Average sleep profiles for UAS-Dicer2/NP5286-GAL4;UAS-nrx^RNAi/+ (n = 53) and control (n = 32) flies, plotted as a 30 min moving average (D), and quantification of total nighttime sleep for each genotype (E). Note that Dicer-2 was used to enhance the transgenic RNAi effect in panels D, F, and H. (F,G) Average sleep profiles for NP7175-GAL4/Y;UAS-Dicer2/+;UAS-nrx^RNAi/+ (n = 37) and control (n = 32) flies, plotted as a 30 min moving average (F), and quantification of total nighttime sleep for each genotype (G). (H,I) Average sleep profiles for NP3208-GAL4/Y;UAS-Dicer2/+;UAS-nrx^RNAi/+ (n = 42) and control (n = 32) flies, plotted as a 30 min moving average (H), and quantification of total nighttime sleep for each genotype (I). (J) Model of the MB αβ neurons illustrating three subsets of neurons within the αβ lobes. Purple, αβ_c neurons; blue, αβ_s neurons; yellow, αβ_p neurons. (K) Total nighttime sleep in flies with rescued neurexin expression using specific GAL4 drivers for each αβ subset. Rescue experiments were conducted in the nrx^273/Δ83 background; flies bear one copy of the indicated drivers (n = 16).

Figure 4. Knockdown of neurexin expression in adult flies reduces nighttime sleep.

(A–C) Sleep profiles for c739-GAL4/tub-GAL80^ts;UAS-nrx^RNAi/+ (black) and c739-GAL4/tub-GAL80^ts control (red) flies at 21 °C (A), 30 °C (B), and 25 °C (C). Quantification of total nighttime sleep at each temperature is presented in the right panel (n = 16).

Figure 5. Blocking synaptic output from αβ_sc neurons reduces nighttime sleep.

(A,B) Average sleep profiles for NP5286/tub-GAL80^ts;UAS-Ork1^ΔC/+ and control flies, plotted as a 30 min moving average (A), and quantification of total nighttime sleep in NP5286/tub-GAL80^ts;UAS-Ork1^ΔC/+ and control flies (B) (n = 16). For (A) and (C), all flies were reared at 18 °C throughout development, and adults were entrained at 30 °C for 6 hours every day for 2 days. Then, the flies’ sleep was measured. (C,D) Average sleep profiles for NP7175/Y;tub-GAL80^ts/+;UAS-Ork1^ΔC/+ and control flies, plotted as a 30 min moving average (C), and quantification of total nighttime sleep in NP7175/Y;tub-GAL80^ts/+;UAS-Ork1^ΔC/+ and control flies (D) (n = 24). (E) Representative images of GCaMP6 fluorescence in both w¹¹¹⁸ control and nrx^273/Δ83 mutant αβ lobes. Images show the sum of fluorescence intensity from multiple layers primarily consisting of αβ lobes at ZT22. (F) Quantification of GCaMP6/RFP fluorescence ratios in w¹¹¹⁸ control and nrx^273/Δ83 mutant αβ neurons at ZT22. RFP fluorescence was used as control (n = 8). (G) Averaged eYFP/eCFP fluorescence ratio in both w¹¹¹⁸ control and nrx^273/Δ83 mutant αβ lobes at ZT22. (H) Quantification of eYFP/eCFP ratios in w¹¹¹⁸ control and nrx^273/Δ83 mutant αβ neurons at ZT22 (n = 11).

Figure 6. Neurexin functionally couples with calcium channels to mediate synaptic transmission.

(A,B) Averaged total sleep per night in cac^H18 mutants and Canton-S (CS) flies (A), and in Ca-α1T^del mutants and w¹¹¹⁸ control flies (B), plotted as a 30-min moving average (n = 64). Quantification of total nighttime sleep is presented in the right panel of each figure. Note that CS flies were used as the control flies for cac^H18 mutants, which were generated in the CS background. (C) Averaged total sleep per night in c309-GAL4/+;UAS-cac^RNAi/+ and control flies, plotted as a 30 min moving average (n = 32). Quantification of total nighttime sleep in each genotype is presented in the right panel. (D) Averaged total sleep per night in MB185B-GAL4/+;UAS-cac^RNAi/+ and control flies, plotted as a 30 min moving average (n = 20). Quantification of total nighttime sleep in each genotype is presented in the right panel.

Figure 7. The αβ_s neurons release both acetylcholine and short neuropeptide F to promote nighttime sleep, whereas α β c neurons release sNPF to maintain nighttime sleep.

(A) Expression of c739 in αβ_sp neurons is blocked by Cha-GAL80. The inset shows a horizontal cross-section through the vertical collateral at the level of the dashed line in each panel. Scale bar = 20 μm. (B,C) Averaged total sleep per night in c739-GAL4/+;UAS-nrx^RNAi/+ and c739-GAL4ⁱ/+;Cha-GAL80/UAS-nrx^RNAi flies, plotted as a 30 min moving average (B), and quantification of total nighttime sleep in each genotype (C) (n = 64). (D,E) Averaged total sleep per night in MB185B-GAL4/UAS-vAChT^RNAi and control flies (D) (n = 64), and in MB594B-GAL4/UAS-vAChT^RNAi and control flies (E) (n = 24), plotted as a 30 min moving average. (F) Quantification of total nighttime sleep in vAChT-RNAi flies and control flies. (G,H) Averaged total sleep per night in MB185B-GAL4/UAS-sNPF^RNAi and control flies (G) (n = 32), and in MB594B-GAL4/UAS-sNPF^RNAi and control flies (H) (n = 32), plotted as a 30 min moving average. (I) Quantification of total nighttime sleep in sNPF^RNAi flies and control flies.