Gap Junctions and NCA Cation Channels Are Critical for Developmentally Timed Sleep and Arousal in Caenorhabditis elegans

Abstract

An essential characteristic of sleep is heightened arousal threshold, with decreased behavioral response to external stimuli. The molecular and cellular mechanisms underlying arousal threshold changes during sleep are not fully understood. We report that loss of UNC-7 or UNC-9 innexin function dramatically reduced sleep and decreased arousal threshold during developmentally timed sleep in Caenorhabditiselegans UNC-7 function was required in premotor interneurons and UNC-9 function was required in motor neurons in this paradigm. Simultaneous transient overexpression of UNC-7 and UNC-9 was sufficient to induce anachronistic sleep in adult animals. Moreover, loss of UNC-7 or UNC-9 suppressed the increased sleep of EGL-4 gain-of-function animals, which have increased cyclic-GMP-dependent protein kinase activity. These results suggest C. elegans gap junctions may act downstream of previously identified sleep regulators. In other paradigms, the NCA cation channels act upstream of gap junctions. Consistent with this, diminished NCA channel activity in C. elegans robustly increased arousal thresholds during sleep bouts in L4-to-adult developmentally timed sleep. Total time in sleep bouts was only modestly increased in animals lacking NCA channel auxiliary subunit UNC-79, whereas increased channel activity dramatically decreased sleep. Loss of EGL-4 or innexin proteins suppressed UNC-79 loss-of-function sleep and arousal defects. In Drosophila, the ion channel narrow abdomen, an ortholog of the C. elegans NCA channels, drive the pigment dispersing factor (PDF) neuropeptide release, regulating circadian behavior. However, in C. elegans, we found that loss of the PDF receptor PDFR-1 did not suppress gain-of-function sleep defects, suggesting an alternative downstream pathway. This study emphasizes the conservation and importance of neuronal activity modulation during sleep, and unequivocally demonstrates that gap junction function is critical for normal sleep.

Keywords: Caenorhabditis elegans sleep; NCA channel; cGMP-dependent kinase; gap junction.

Figure 1

UNC-7 and UNC-9 gap junction proteins are required for normal L4/A developmentally timed sleep and arousal and are downstream of EGL-4 PKG. (A) Total time in sleep bouts for wild-type, unc-7(e5lf), unc-9(e101lf), and unc-9(e101lf) unc-7(e5lf) animals during L4 to adult lethargus. (B) Percent of wild-type, unc-7(e5lf), unc-9(e101lf), and unc-9(e101lf) unc-7(e5lf) animals not responding to gentle touch during L4 to adult sleep bouts. Because of the response defect of unc-7(e5lf) and unc-9(e101lf) animals to blue light stimuli, the arousal threshold was measured by gentle touch instead. Three independent trials were tested. n = 10 per genotype for each trial. (C) Total time in sleep bouts for wild-type, unc-1(e719lf), unc-24(e138lf), and unc-24(e138lf) animals during L4 to adult lethargus. (D) Total time in sleep bouts for egl-4(ad450gf), unc-7(e5lf), egl-4(ad450gf); unc-7(e5lf), unc-9(e101lf), and egl-4(ad450gf); unc-9(e101lf) animals during L4 to adult lethargus. Error bars show the SEM. * P < 0.05, *** P < 0.001. All error bars represent standard error of the mean.

Figure 2

UNC-7 and UNC-9 is partially required in premotor interneurons and motor neurons, respectively, during sleep. (A) Total time in sleep bouts for unc-7(e5lf) animals in L4 to adult lethargus rescued with myo-3 promoter, aex-3 promoter, glr-1 promoter, and rig-3 promoter driven unc-7 and their corresponding promoter driven GFP as controls. Colors represent three independent extrachromosomal arrays tested for each condition (strain information in Table S2). (B) Total time in sleep bouts for unc-9(e101lf) animals in L4 to adult lethargus rescued with myo-3 promoter, aex-3 promoter, glr-1 promoter, and unc-4 promoter driven unc-9 and their corresponding promoter driven GFP as controls. Colors represent two or three independent extrachromosomal arrays tested for each condition (strain information in Table S2). (C) Percent of animals with anachronistic sleep in wild-type animals, animals overexpressing hsp::gfp ([control]), hsp::unc-7, hsp::unc-9, and both hsp::unc-7 and hsp::unc-9. Data from three independent extrachromosomal arrays were pooled for each genotype. At least three independent trials were tested. n = 10 per genotype for each trial. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars represent standard error of the mean.

Figure 3

Normal NCA cation channel activity is required for proper response latency during sleep bouts. (A) Total time in sleep bouts for wild-type, nca-1(gk5lf); nca-1(gk9lf) [simplified as nca(lf)], unc-79(ec1lf), unc-80(e1272lf), and nca-1(e625gf) animals during L4 to adult lethargus. (B) Response latency of wild-type, nca(lf), unc-79(ec1lf), and unc-80(e1272lf) animals to blue light stimulation during sleep bouts (left panel) and motion bouts (right panel) in L4 to adult lethargus. (C) Response latency of nca-1(e625gf) animals to blue light stimulation during sleep bouts (left panel) and motion bouts (right panel) in L4 to adult lethargus. (D) Total time in sleep bouts for wild-type, unc-79(ec1lf), unc-9(e101lf) unc-7(e5lf), and unc-79(ec1lf); unc-9(e101lf) unc-7(e5lf) animals during L4 to adult lethargus. All sleep bouts and motions bouts were assessed during L4 to adult lethargus. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars represent standard error of the mean.

Figure 4

EGL-4 PKG is downstream of NCA channels during L4/A lethargus. (A) Total time in sleep bouts for wild-type, unc-79(ec1lf), egl-4(n479lf), unc-79(ec1lf); egl-4(n479lf), egl-4(ad450gf), and unc-79(ec1lf); egl-4(ad450gf). (B) Response latency of wild-type, unc-79(ec1lf), egl-4(n479lf), and unc-79(ec1lf); egl-4(n479lf) animals to blue light stimulation during sleep bouts (left panel) and motion bouts (right panel). (C) Response latency of wild-type, unc-79(ec1lf), egl-4(ad450gf), and unc-79(ec1lf); egl-4(ad450gf) animals to blue light stimulation during sleep bouts (left panel) and motion bouts (right panel). All sleep bouts and motions bouts were assessed during L4 to adult lethargus. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars represent standard error of the mean.

Figure 5

PDF signaling acts upstream or in parallel to NCA channels. (A) Total time in sleep bouts for wild-type, pdfr-1(ok3425lf), pdf-1(tm1996lf), pdf-2(tm4393lf), and pdf-1tm1996(lf); pdf-2(tm4393lf) animals. (B) Response latency of wild-type, pdfr-1(ok3425lf), pdf-1(tm1996lf), pdf-2(tm4393lf), and pdf-1(tm1996lf); pdf-2(tm4393lf) animals to blue light stimulation during sleep bouts (left panel) and motion bouts (right panel). (C) Total time in sleep bouts for wild-type, pdfr-1(ok3425lf), nca-1(e625gf), and pdfr-1(ok3425lf); nca-1(e625gf) animals. (D) Model for genetic pathways of the conserved cation channel and gap junction proteins regulating sleep across species. Dashed arrows represent possible parallel pathway. All sleep bouts and motions bouts were assessed during L4 to adult lethargus. * P < 0.05, ** P < 0.01. All error bars represent standard error of the mean.