Synaptic targets of circadian clock neurons influence core clock parameters

Abstract

Neuronal connectivity in the circadian clock network is essential for robust endogenous timekeeping. In the Drosophila circadian clock network, the small ventral lateral neurons (sLN_vs) serve as critical pacemakers. Peptidergic communication mediated by the neuropeptide Pigment Dispersing Factor (PDF), released by sLN_vs, has been well characterized. In contrast, little is known about the role of the synaptic connections that sLN_vs form with downstream neurons. Connectomic analyses revealed that the sLN_vs form strong synaptic connections with previously uncharacterized neurons called superior lateral protocerebrum 316 (SLP316). Here, we show that silencing the synaptic output from the SLP316 neurons via tetanus toxin expression shortened the free-running period, whereas hyperexciting them by expressing the bacterial voltage-gated sodium channel resulted in period lengthening. Under light-dark cycles, silencing SLP316 neurons caused lower daytime activity and higher daytime sleep. Our results reveal that the main postsynaptic partners of key Drosophila pacemaker neurons are a nonclock neuronal cell type that regulates the timing of sleep and activity.

Fig. 1.. SLP316 neurons are the main postsynaptic partner of the sLN_vs.

(A and B) Connectomic mapping from the hemibrain dataset. (A) Map of top input/output neuronal groups to/from the SLP316s and from the sLN_vs. The number of neurons in each group is indicated in parentheses if greater than one and reflected by circle size. Connection strength between cell types is represented by arrow width and decreases in a clockwise direction for each connection type (input/output). SLP232 likely corresponds to DN3s. (B) Strong shared inputs to and (B′) outputs of the SLP316s (>10 synapses to/from at least two SLP316 neurons) with neuronal morphology shown above or below. (C) Input synapses to and output synapses of the three SLP316 neurons (SLP316_R_1, SLP316_R_2, and SLP316_R_3) shown in green, excluding synapses from neuron segments. Synapse color corresponds to neuron color in (A) and (B), and all other synapses are shown in gray.

Fig. 2.. The SLP316 driver labeled nonclock neurons that share functional synapses with the sLN_vs.

(A to D) Representative confocal images of a brain from a 7-day-old SLP316 > GFP male fly stained with antibodies against green fluorescent protein (GFP; green) and PDF (magenta). (A) Whole brain, (B) SLP z-stack, and anterior (C) and posterior (D) single optical slices (step size of 0.9 μm). D, dorsal; V, ventral; L, lateral; M, medial. (E) Quantification of cell body count on the left hemisphere labeled by the SLP316 driver in 7- and 1-day-old male and female flies from three replicate experiments (n > 21 for each group). (F to H) Representative confocal images of a brain hemisphere of a 6- to 7-day-old SLP316 > GFP male brain stained with antibodies against GFP (green) and PERIOD (PER) (magenta). (I to M) Representative confocal images of SLP316-Gal4 and Pdf-LexA driving expression of BAcTrace components. Red fluorescent protein (RFP) signal can only be detected in the sLN_vs. (J) sLN_v dorsal termini show PDF and RFP expression. (K) Cell bodies of sLN_vs and lLN_vs. (L) sLN_v cell bodies plus dorsal projections show RFP signal, whereas the lLN_vs do not. (M) Synaptobrevin (syb)-GFP expression in the small and large LN_vs in experimental brains. (N to P) Representative confocal images of Pdf-LexA driving expression of BAcTrace components without the SLP316-Gal4 driver. (P) syb-GFP expression in the small and large LN_vs in control brains. Scale bars, 50 μm (all). OT, Optic Tract. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.. Manipulation of the SLP316s altered free-running period length.

(A) Representative double-plotted actograms, (B) free-running period calculated using the chi-square periodogram, and (C) percentage of rhythmic flies for control (SLP316 split-GAL4 and UAS-NaChBac) and experimental (SLP316 > NaChBac) flies. n.s., not significant. (D) Activity plot on the eighth day of free running, indicated in (A) with a yellow box. (E) Phase of activity from (D), calculated in ClockLab using a sine fit of the waveform of activity. CT, circadian time. (F) Representative actograms, (G) free-running period, and (H) percentage of rhythmic flies for control (w;UAS-hid;UAS-GFP and w;pUAS-tetx-LC-TNT;) and experimental (SLP316 > hid + GFP or SLP316 > tetx-LC-TNT) flies. (I) Free-running period, and (J) percentage of rhythmic flies for control (SLP316 split-GAL4 and ;pUAS-tetx-LC-TNT;) and experimental (SLP316 > tetx-LC-TNT) flies. (K) Activity plot and (L) phase of activity on the eighth day of free running for control (SLP316 split-GAL4 and ;pUAS-tetx-LC-TNT;) and experimental (SLP316 > tetx-LC-TNT) flies. Actograms show 5 days of LD, followed by 9 days of DD. Flies were raised, and activity-rest behavior was recorded at 25°C. Rhythmic power was calculated using the chi-square periodogram in ClockLab using 30-min activity bins, and flies with a rhythmic power of 10 or greater were classified as rhythmic. Free-running period was calculated for rhythmic flies using the chi-square periodogram routine of ClockLab using 1-min activity bins. Statistical comparisons were conducted using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test for free-running period and phase of activity, and using Fisher’s exact test for the number of rhythmic and arrhythmic flies. Data from three replicate experiments are plotted for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.. Silencing or eliminating SLP316 neurons did not affect the eclosion rhythm.

(A) Records showing the time course of emergence of flies under DD conditions (left), their corresponding autocorrelation analysis (middle), and periodicities (right). (B) Period of eclosion of populations of flies of the indicated genotypes.

Fig. 5.. Pdf and SLP316 disruption have opposite effects on daytime activity and sleep quantities under cold temperature.

(A to D) Activity and sleep phenotypes of SLP316 > hid + GFP flies during 3 days of 12-hour:12-hour LD cycle at 18°C. Representative activity (A) and sleep (C) plots averaged for control (SLP316 split-GAL4 and w;UAS-hid;UAS-GFP) and experimental (SLP316 > hid + GFP) flies. Daytime (ZT0 to ZT12) (B) activity counts and total sleep amount (D). (E to H) Activity and sleep phenotypes of SLP316 > ;tetx-LC-TNT flies and parental controls. Representative activity (E) and sleep (G) plots, and their respective quantifications (F and H). (I to L) Activity and sleep phenotypes for wild-type w¹¹¹⁸ and pdf ⁰¹ flies. Representative activity (I) and sleep (K) plots, and total daytime activity counts (J) and sleep amount (L). Flies were raised at 25°C, and activity-rest behavior was recorded at 18°C. Statistical comparisons were conducted using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test for comparisons between three genotypes in (B), (D), and (F) and (H), and using the Mann-Whitney test for comparisons between two genotypes in (J) and (L). Data plotted are from two replicate experiments for (B), (D), and (F) and (H) and three replicate experiments for each genotype for (J) and (L). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6.. Silencing or eliminating SLP316 neurons increased the amount of daytime sleep.

(A) Representative sleep plot averaged from 3 days of 12-hour:12-hour LD cycle at 25°C for control (SLP316 split-GAL4 and ;UAS-hid;UAS-GFP) and experimental (SLP316 > ;hid;GFP) flies. (B) Total daytime sleep over 3 days for each genotype. (C) Representative sleep plot and (D) total daytime sleep for control (SLP316-Gal4 and ;pUAS-tetx-LC-TNT;) and experimental (SLP316 > tetx-LC-TNT) flies. (E) P(doze) from the same 3 days of LD, plotted in 3-hour bins for controls (SLP316 split-GAL4 and ;UAS-hid;UAS-GFP) and experimental (SLP316 > ;hid;GFP) flies. (F) Average P(doze) throughout the entire 3-day period. (G) P(doze) plot and (H) average P(doze) for control (SLP316-Gal4 and ;pUAS-tetx-LC-TNT;) and experimental (SLP316 > tetx-LC-TNT) flies under the same conditions. Sleep was quantified using PHASE based on periods of inactivity of 5 min or more and plotted in (A) and (C) as minutes of sleep per 30-min bin. P(doze) was quantified in R using routines from the Griffith Laboratory (70). Statistical comparisons were conducted using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. Data are plotted from four replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.