The GABA(A) receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila

Abstract

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. To analyze the role of the circadian clock, we have used the fruit fly Drosophila. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. We assessed sleep-wake modulation by a core set of circadian pacemaker neurons that express the neuropeptide PDF. We find that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. We find that RNA interference-mediated knockdown of the GABA(A) receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo.

Figure 1. Loss of PDF results in increased sleep and reduced responsiveness to external stimuli

Hourly sleep amount plots for (A) pdf-GAL4/UAS-hid (black lines with square data points; N=35) and pdf-GAL4/+ (gray lines with circle data points; N=34), (B) pdf⁰¹ (black lines with square data points; N=72) and WT sibling controls (gray lines with circle data points; N=64), and (C) pdfr^han5304 (black lines with square data points; N=75) and WT sibling controls (gray lines with circle data points; N=70). Horizontal white and black boxes along the X-axis indicate light and dark periods of the last day of LD (LD5), respectively. Horizontal gray boxes indicate the subjective light period during the first day of DD (DD1). Data points represent mean ± SEM of three independent experiments. *p<0.05 as determined by Student’s t-test. (D) Bar graph indicating the corrected % of flies that responded to a mechanical stimulus administered at CT3 on DD1 (see methods for calculation). Data points represent mean ± SEM of four independent experiments. *p<0.05 as determined by Student’s t-test.

Figure 2. Knockdown of Rdl transcript in PDF cells results in decreased sleep

Averaged total sleep amount for LD days 2–5 (LD2–5) and DD days 1–5 (DD1–5). “L” indicates the light period of LD2–5, “D” indicates the dark period of LD2–5, “Subj L” indicates the subjective light period of DD1–5, and “Subj D” indicates the subjective dark period of DD1–5. All data bars represent mean ± SEM of three independent experiments. * at least p<0.05 to all controls, ** at least p<0.01 to all controls, and *** at least p<0.001 to all controls as determined by one-way ANOVA, Tukey HSD post hoc. Sample sizes for each genotype and for each condition can be found within Supplemental Table 5.

Figure 3. Inhibitory inputs to the lateral neurons

(A) GABA_A receptor subunit RDL is expressed in lLNv and at much lower levels in the sLNv. Left panel, maximum projection; upper and lower halves comprise different regions of the same brain. Right panels show individual optical sections through the center of each cell soma, with large arrows pointing to each lLNv and small arrows to each sLNv. Blue = PDF, red = RDL. Note the RDL-stained somata below the sLNv in the final panel. (B) Individual PDF-containing fibers in the accessory medulla colocalize with RDL puncta (small arrows) in single sections. (C) PDF varicosities formed by lLNv neurites in the optic lobe also express RDL (asterisks). (D) The response of lateral neurons to puffer applications of 1 mM GABA in the presence and absence of 100 µM picrotoxin. Holding potential = −90mV. 100 ms puffs of 1 mM GABA were applied to the neurons as indicated. See methods for ionic conditions. Dark lines represent the average current of six cells in each condition; light lines indicate the standard error. Green (gray) indicates control with high chloride (high Cl) concentration. Blue (dark) indicates picrotoxin(ptx) treated cells with high chloride (high Cl). Red indicates reduced chloride concentration (low Cl).