Drosophila neuroligin 4 regulates sleep through modulating GABA transmission

Abstract

Sleep is an essential and evolutionarily conserved behavior that is closely related to synaptic function. However, whether neuroligins (Nlgs), which are cell adhesion molecules involved in synapse formation and synaptic transmission, are involved in sleep is not clear. Here, we show that Drosophila Nlg4 (DNlg4) is highly expressed in large ventral lateral clock neurons (l-LNvs) and that l-LNv-derived DNlg4 is essential for sleep regulation. GABA transmission is impaired in mutant l-LNv, and sleep defects in dnlg4 mutant flies can be rescued by genetic manipulation of GABA transmission. Furthermore, dnlg4 mutant flies exhibit a severe reduction in GABAA receptor RDL clustering, and DNlg4 associates with RDLs in vivo. These results demonstrate that DNlg4 regulates sleep through modulating GABA transmission in l-LNvs, which provides the first known link between a synaptic adhesion molecule and sleep in Drosophila.

Figure 1.

dnlg4 mutant flies show abnormal sleep behavior. A, Western blot analysis showing the protein level of DNlg4 in wild-type (WT), dnlg4 mutant, and dnlg4/BSC516 heterozygous mutant flies. Tubulin was used as a loading control. Quantification of relative DNlg4 protein level for each genotype is presented on the bottom. B, Representative 7-d locomotor traces of WT (left) and dnlg4 mutant (right) flies. White areas indicate day, and black areas indicate night. C, Average total sleep per night in WT and dnlg4 mutant flies, plotted as a 30 min moving average. n = 200. D, E, Quantification of total sleep time per night and sleep onset latency after lights off for each genotype. n = 200. F, G, Quantification of number of sleep episodes per night and average sleep episode length for each genotype; n = 200, *p < 0.001.

Figure 2.

DNlg4 is highly expressed in l-LNvs. A, Immunostaining images indicating the expression pattern of DNlg4. PDF neurons are indicated by arrows. Scale bar, 100 μm. B, Distribution of DNlg4 in LNv terminals. Scale bar, 50 μm. A, B, Dissected whole brains were stained with anti-DNlg4 (green) and anti-PDF (red) antibodies. C, Quantification of relative DNlg4 protein amount in LNvs for each genotype. D, DNlg4 was highly expressed in l-LNvs. LNvs were labeled with mCD8-GFP under the control of the pdf-Gal4 driver. l-LNvs are indicated by the closed arrow, and s-LNvs are indicated by the open arrow. Scale bar, 10 μm. E, l-LNvs were labeled with mCD8-GFP under control of the c929-Gal4 driver. Scale bar, 10 μm. F, DNlg4 was located in the intracellular region of l-LNv somata. l-LNv somata membranes were labeled with mCD8-GFP under the control of the pdf-Gal4 driver. Scale bar, 5 μm. G, In pdf-Gal4/p[UASHgalactosyltransferase-GFP] flies, DNlg4 colocalized with the GFP signal. Dissected LNvs were costained with DNlg4 antibody (red) and GFP antibody (green, showing Golgi). Scale bar, 5 μm. H, In pdf-Gal4/p[UASHGFP-KDEL] flies, DNlg4 did not colocalize with GFP-KEDL. Dissected LNvs were costained with DNlg4 antibody (red) and GFP antibody. Scale bar, 5 mm. WT, wild type.

Figure 3.

Depletion of DNlg4 in l-LNvs leads to abnormal sleep. A, B, Total night sleep and sleep onset latency in flies with depletion of DNlg4 using UAS-dnlg4-RNAi driven by anatomically restricted Gal4 drivers. For each Gal4 line, a single copy of the Gal4 driver was used for the test; n = 32, *p < 0.001 for RNAi versus control flies. C, Average sleep traces for UAS-nlg4-RNAi;pdf-Gal4 and control flies, plotted as a 30 min moving average; n = 32. D, E, Quantification of average sleep episode length and number per night in UAS-nlg4-RNAi;pdf-Gal4 and control flies; n = 32, *p < 0.001 for UAS-nlg4-RNAi;pdf-Gal4 versus control flies. F, Expression of DNlg4 in UAS-nlg4-RNAi;pdf-Gal4 (bottom) and control (top) flies. Dissected whole brains were stained with anti-DNlg4 (green) and anti-PDF (red) antibodies. Scale bar, 50 μm. Note that DNlg4 distribution in UAS-nlg4-RNAi;pdf-Gal4 flies is comparable to control flies except in PDF neurons. Enlarged images showing expression of DNlg4 in PDF neurons are presented on the right. Scale bar, 10 μm. G, Quantification of relative DNlg4 protein amount in LNvs for each genotype.

Figure 4.

Specific expression of DNlg4 in l-LNvs rescues defective sleep in dnlg4 mutant flies. A, B, Total night sleep and sleep onset latency in flies with rescued DNlg4 expression using anatomically restricted Gal4 drivers. Flies bear one copy of the indicated drivers; n = 32, *p < 0.001 for rescued versus dnlg4 mutant flies. C, Average sleep traces for rescued and control flies, plotted as a 30 min moving average; n = 32. D, E, Quantification of average sleep episode length and number per night in rescued and control flies. n = 32. * indicates p < 0.001 for rescued vs control flies.

Figure 5.

Sleep behavior of UAS-dnlg4-RNAi;tubulin-Gal80^ts/pdf-Gal4 flies. A, Immunostaining images showing DNlg4 protein levels in UAS-dnlg4-RNAi;tubulin-Gal80^ts /pdf-Gal4 (bottom) and control (top) flies. Dissected whole brains were stained with anti-DNlg4 (green) and anti-PDF (red) antibodies. Scale bar, 10 μm. B, Quantification of relative DNlg4 protein amount in LNvs for each genotype is presented on the right. C, Continuous sleep measurements of flies expressing the dnlg4-RNAi and temperature-sensitive Gal80^ts in PDF neurons. Entrainment temperatures are shown at the top; n = 32. D, Quantification of total sleep time for individual nights. E, Average total sleep time per night in each condition. F, Quantification of sleep onset latency after lights off for each condition. G, H, Quantification of sleep episode number and average length. D–G, n = 32, and values for day 7 and 8 are quantified and presented. *p < 0.001 for UAS-dnlg4-RNAi;tubulin-Gal80^ts /pdf-Gal4 versus UAS-dnlg4-RNAi /pdf-Gal4 flies.

Figure 6.

GABA transmission is impaired in dnlg4 mutant l-LNvs. A, Representative whole-cell recording traces from wild-type (WT) and dnlg4 mutant l-LNvs at day and night. B, Percentage of spikes fired in bursts in WT and dnlg4 mutant l-LNvs. C, Average frequency of spontaneous AP firing. D, Average resting membrane potentials for each genotype and condition. E, Average spike amplitude for each genotype and condition. B–E, n = 9 for WT daytime, n = 10 for WT nighttime, n = 9 for dnlg4 daytime, n = 8 for dnlg4 nighttime, *p < 0.05 for WT versus dnlg4 mutant l-LNvs. F, GABA puffing on l-LNvs hyperpolarizes resting membrane potential and blocks spontaneous AP firing. PDF neurons were held in I-clamp (I = 0) and puffs of 10 mm GABA lasting 100 ms were applied. Arrow indicates the time point of GABA puffing. n = 6. G, GABA-gated currents with puffing of 1 and 10 mm GABA on five individual WT l-LNvs. Averaged GABA currents with puffing of 1 and 10 mm are also presented. H, Representative responses of WT (blue) and mutant (red) l-LNvs to applications of 10 mm GABA puffs in the presence (black) and absence of 200 μm PTX. The time point of GABA puffing is indicated by an arrow. I, The averaged GABA currents in WT and dnlg4 mutant l-LNvs. The number of l-LNvs for each genotype and condition are indicated. *p < 0.001 for WT versus dnlg4 mutant l-LNvs.

Figure 7.

Impaired GABA transmission leads to defective sleep in dnlg4 mutant flies. A, Average sleep traces for wild-type (WT), dnlg4, Rdl^A302S, and Rdl^A302S,dnlg4 flies plotted as a 30 min moving average, n = 32. B, C, Quantification of total sleep time and sleep onset latency after lights off for each genotype; n = 32, *p < 0.001 for dnlg4 mutant versus Rdl^A302S,dnlg4 double mutant flies. D, Effect of different CBZ concentrations on the sleep pattern of WT (top) and dnlg4 mutant (bottom) flies during the first day of drug treatment: n = 32; blue, 0.5 mg/ml; green, 1 mg/ml. Black arrow at top of graph indicates CBZ application. E, F, Quantification of total sleep time and sleep onset latency after treatment with different CBZ concentrations, n = 32.

Figure 8.

dnlg4 mutant flies exhibit reduced RDL clustering in l-LNvs, and DNlg4 associates with RDLs. A, Representative images showing the distribution of dendrite branches and number of varicosities in l-LNvs for wild-type (WT) and dnlg4 mutant flies. l-LNvs membranes were labeled with mCD8-GFP under the control of the pdf-Gal4 driver. Dissected whole brains were stained with anti-GFP (green) and anti-PDF (red) antibodies. Scale bar, 50 μm. B, Quantification of the number of l-LNv varicosities. Eight images were used for quantification for WT and dnlg4 mutant flies. Each image was taken from a different fly. C, Distribution of RDL-HA in l-LNvs neurons. RDL-HA was expressed in PDF neurons under the control of the pdf-Gal4 driver. Dissected whole brains were stained with anti-HA (green) and anti-PDF (red) antibodies. Scale bar, 20 μm. D, Quantification of the number of RDL-HA-positive varicosities in l-LNv neurons. E, Quantification of average RDL-HA protein amount in RDL-HA-positive varicosities in l-LNvs. F, Quantification of RDL-HA protein amount in l-LNv somata. C–F, Six images were used for quantification for WT and dnlg4 mutant flies. Each image was taken from a different fly. G, Co-immunoprecipitation of DNlg4 and Rdl in vivo. The precipitates and a portion (1% of the input) of the head extracts were subjected to Western blotting with anti-HA or anti-TRP (negative control) antibodies.