A Neural Network Underlying Circadian Entrainment and Photoperiodic Adjustment of Sleep and Activity in Drosophila

Abstract

A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. Here we show that the Hofbauer-Buchner eyelets differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, we show that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. Our work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms.

Significance statement: In animals, circadian clocks have evolved to orchestrate the timing of behavior and metabolism. Consistent timing requires the entrainment these clocks to the solar day, a process that is critical for an organism's health. Light cycles are the most important external cue for the entrainment of circadian clocks, and the circadian system uses multiple photoreceptors to link timekeeping to the light/dark cycle. How light information from these photorecptors is integrated into the circadian clock neuron network to support entrainment is not understood. Our results establish that input from the HB eyelets differentially impacts the physiology of neuronal subgroups. This input pathway, together with input from the compound eyes, precisely times the activity of flies under long summer days. Our results provide a mechanistic model of light transduction and integration into the circadian system, identifying new and unexpected network motifs within the circadian clock neuron network.

Keywords: circadian; entrainment; photoreception; pigment dispersing factor.

Figure 1.

The HB eyelet likely forms synapses directly on the LNv clock neurons within the accessory medulla and along its ventral elongation. A–F, The Rh6-GAL4 expression pattern (green) colabeled with anti-PDF (magenta). A, One hemisphere of a UAS-GFP/+;Rh6-GAL4/+ brain coimmunolabeled for GFP and PDF. The axons of the Rh6-positive R8 photoreceptors of the compound eye cross the lamina (La) and terminate in the medulla (Me), whereas the axons of the HB eyelet directly innervate the AMe. Scale bar, 100 μm. B, Axonal terminals of the R8 photoreceptors (green) in the Me reside in close vicinity to the PDF-positive puncta of the l-LNvs (magenta). Scale bar, 15 μm. C–F, Axons of the HB eyelet terminate in the AMe and overlap with PDF arborizations (D, F, magenta). C–F, Micrographs represent 20 μm projections of the AMe consisting of 10 optical sections separated by 2 μm steps. Rh6-GAL4 is not expressed in the PDF neurons as no colocalization is visible in the cell bodies of the PDF neurons. Scale bars, C–F, 15 μm. C, D, and E, F display two different brains. G–I, Reconstitution of GFP between LNv neurons and the Rh6-positive photoreceptors. G, GFP fluorescence in a Pdf-lexA/+;Rh6-GAL4/+ parental control. H, GFP fluorescence in a Pdf-lexA/lexAop-CD4::spGFP11;Rh6-GAL4/UAS-CD4::spGFP1-10 brain reveals GFP reconstitution within the AMe and along its ventral elongation. I, GFP fluorescence in a LexAop-CD4::spGFP11/+;UAS-CD4::spGFP1-10/+ parental control. J–L, Reconstituted GFP (J, K, green) in a Pdf-lexA/lexAop-CD4::spGFP11;Rh6-GAL4/UAS-CD4::spGFP1-10 brain colabeled for PDF (K, L, magenta). Scale bars: G–L, 25 μm. C, E, K, L, Arrows indicate the central part of the AMe, which is strongly innervated by HB eyelet terminals in all brains. A, *HB eyelet. C, E, K, *Ventral elongation of the AMe.

Figure 2.

The s-LNvs, but not the l-LNvs, respond to HB eyelet excitation with increases in calcium and cAMP. A, P2X2-mediated excitation of HB eyelets. Top two traces, Average GCaMP3.0 fluorescence plots (±SEM) for HB eyelet nerves coexpressing P2X2 and GCAMP in Rh6-GAL4/UAS-GCaMP3.0;UAS-P2X2/+ brains in response to 30 s perfusion of 1 mm ATP (top, N = 24) or vehicle (bottom, N = 24). Histogram represents comparison of average maximum GCaMP responses (±SEM) for the same data. ATP caused significant GCaMP fluorescence increases compared with vehicle control. B, Effect of eyelet excitation on l-LNv Ca²⁺ in Pdf-lexA,LexA-GCaMP3.0/Rh6-GAL4; UAS-P2X2/+ brains in which P2X2 is expressed in the HB eyelet and GCaMP in the LNvs. Data arranged as in A. N = 14 for ATP and N = 15 for vehicle. The l-LNvs did not display significant GCaMP fluorescence increases in response to eyelet excitation. C, Effect of eyelet excitation on s-LNv Ca²⁺ in Pdf-lexA,LexAop-GCaMP3.0/Rh6-GAL4;UAS-P2X2/+ brains. Data arranged as in A. N = 20 for ATP, N = 13 for vehicle. Excitation of the eyelet resulted in significant GCaMP fluorescence increases relative to vehicle controls. D, In brains lacking a driver for UAS-P2X2 expression in the eyelets (Pdf-lexA,LexAop-GCaMP3.0/+;UAS-P2X2/+). ATP failed to result in an increase in s-LNv GCaMP fluorescence relative to vehicle controls. Data arranged as for A. N = 13 for ATP, N = 13 for vehicle. Calibration: A–D, 1 min (x-axis) and a 50% change in GCaMP3.0 fluorescence over baseline (y-axis). E, Effect of eyelet excitation on l-LNv cAMP in Pdf-lexA,LexAop-Epac1-camps/Rh6-GAL4;UAS-P2X2 brains. The two traces represent average inverse Epac1-camps FRET (CFP/YFP) plots for l-LNvs in response to 1 mm ATP (top, N = 17) and vehicle (bottom, N = 17). Histogram represents comparison of average maximum Epac1camps responses (±SEM) for the same data. ATP perfusion failed to produce significant inverse FRET increases in l-LNvs relative to vehicle controls. F, Excitation of the eyelet causes cAMP increases in the s-LNvs of Pdf-lexA,LexAop-Epac1-camps/Rh6-GAL4;UAS-P2X2. Data arranged as for E. ATP produced significant inverse FRET increases in the s-LNvs relative to vehicle controls. N = 15 for ATP, N = 14 for vehicle. G, Average inverse Epac1-camps FRET (CFP/YFP) plots for l-LNvs in Pdf-lexA,LexAop-Epac1-camps/+;UAS-P2X2/+ brains that express Epac1-camps in the LNvs but fail to drive P2X2 in the eyelet. The l-LNvs did not display inverse FRET increases in response to ATP (middle, N = 14) relative to vehicle controls (bottom, N = 14). The l-LNvs did display inverse FRET increases to 10⁻⁴ m nicotine (top plot, N = 12). Histogram represents comparison of maximum Epac1-camps responses for the same data. ATP perfusion failed to produce significant increases in CYP/YPF ratio in the l-LNvs relative to vehicle controls, whereas nicotine (10⁻⁴ m) produced significant cAMP increases. H, Average inverse Epac1-camps FRET (CFP/YFP) plots for s-LNvs in Pdf-lexA,LexAop-Epac1-camps/+;UAS-P2X2/+ brains. Data organized as for G. ATP perfusion (N = 19) caused small but significant increases in CYP/YPF ratio relative to vehicle controls (N = 19) in the s-LNvs, whereas nicotine (10⁻⁴ m, N = 15) produced large and significant inverse FRET increases. Calibration: E–H, 1 min (x-axis) and a 25% changes in CFP/YFP ratio (y-axis). Bars under averaged plots represent 30 s of ATP perfusion switched from a constant saline flow. n.s., Not significant (p ≥ 0.05). *p < 0.05. **p < 0.01. ***p < 0.001. I, Response of a representative l-LNv to local pressure injection of 100 mm histamine (HA) into the ipsilateral Me. Black bars represent stimulus length (0.5 s, 1 s, 10 s). Duration-dependent inhibition of ipsilateral spiking activity (large spikes) was apparent, although contralateral activity (small spikes) was not affected. J, Enlargements of the boxed regions in I showing the inhibition after drug application. Spiking stops abruptly but comes back gradually before returning to previous levels. mp, Membrane potential.

Figure 3.

PDF is required specifically in the l-LNvs for normally phased evening peaks under long day conditions. A, The averaged activity profile of a UAS-PdfRNAi control flies under LDR 16:8. The morning and evening peaks are aligned with dawn and dusk (light gray regions of the LD cycle above the plot). B, Activity of flies in which PDF has been knocked down in all LNvs through the coexpression of UAS-PdfRNAi and UAS-Dicer2 (Dcr2) under the control of Pdf-GAL4. C, Activity of flies in which PDF has been knocked down only in the l-LNvs using the c929-GAL4 driver (D). Activity of flies in which PDF has been knocked down only in the s-LNvs using the r6-GAL4 driver. E, The average evening peak phase of flies in which Pdf expression has been knocked down in different subsets of neurons. Knockdown of Pdf either in all LNvs or specifically in the l-LNvs, using Pdf-GAL4 and c929-GAL4, respectively, resulted in a significantly advanced evening peak of activity compared with Dicer2 overexpression and UAS-PdfRNAi controls. Knockdown of Pdf specifically in the s-LNvs using R6-GAL4 failed to produce an advanced evening peak relative to controls. ANOVA followed by a post hoc test revealed no significant differences in phase between Dcr2;PdfG4, PdfRNAi, Dcr2/c929G4, Dcr2/r6G4, and Dcr2/r6G4/PdfRNAi flies (p > 0.1199). However, Dcr2/PdfG4/PdfRNAi and Dcr2/c929G4/PdfRNAi flies had a significant earlier evening peak than all others (p < 0.001). In addition, the evening peak phase between these two lines was significantly different (p < 0.001). F, The average evening peak phase under LDR 16:8 of flies in which the LNvs have been electrically silenced (bottom row: Pdf-GAL4/UAS-Kir2.1). As expected, these flies display an abnormally early evening peak of activity compared with Pdf-GAL4 (top row) and UAS-Kir2.1 (middle row) controls (Kruskal–Wallis test, p < 0.001). A–F, Numbers on the right side of the panels indicate sample size. G, PDF expression in a representative UAS-Dicer2/c929-GAL4/UAS-PdfRNAi brain. Only s-LNv PDF is visible. H, PDF expression in a UAS-Dicer2/R6-GAL4/UAS-PdfRNAi brain. Only l-LNv PDF is visible. Scale bars: G, H, 20 μm. In these experiments, the GAL4 and UAS elements were always present as single copies. The GAL4 elements and UAS-RNAi elements were autosomal while the UAS-Dcr2 element was inserted into the X chromosome. Thus, flies were heterozygous for GAL4 and UAS-RNAi and hemizygous for the UAS-Dicer2 element. ***p < 0.001.

Figure 4.

The l-LNvs modulate cAMP levels in the s-LNvs. A, Averaged Epac1-camps inverse FRET plot (±SEM) of l-LNvs imaged in c929-Gal4/Pdf-LexA,LexAop-Epac1-camps;UAS-P2X2/+ brains before, during, and after 30 s perfusion of 1 mm ATP (indicated on the bottom plot in each column). ATP/P2X2-mediated excitation caused clear inverse FRET increases. B, Averaged Epac1-camps inverse FRET plot (±SEM) of s-LNvs from the same brains as in A. Excitation of the c929 network produced inverse FRET increases in the s-LNvs. C, Averaged Epac1-camps inverse FRET plot (±SEM) of large LNvs imaged in a Pdfr mutant background using han⁵³⁰⁴;c929-GAL4/Pdf-LexA,LexAop-Epac1-camps;UAS-P2X2/+ brains. c929 network excitation caused clear inverse FRET increases in these neurons. D, Averaged Epac1-camps plot (±SEM) of s-LNvs from the same brains as in C. Excitation of the c929 network failed to produce inverse FRET increases in the s-LNvs in the absence of PdfR function. E, Averaged Epac1-camps inverse FRET plot (±SEM) of large LNvs imaged in Pdf-LexA,LexAop-Epac1-camps/+;UAS-P2X2/+ brains. ATP failed to produce inverse FRET increases in the absence of the GAL4 driver. Calibration: 60 s (x-axis) and a 20% change in inverse FRET (y-axis) (also apply to A, C). F, Averaged Epac1-camps inverse FRET plot (±SEM) of s-LNvs from the same brains as in E. ATP caused no obvious inverse FRET increases. Calibration: F, 60 s (x-axis) and a 10% change in inverse FRET (y-axis) (also apply to B, D). G, Comparison of maximum Epac1-camps responses for the l-LNv data shown in A, C, E. ATP (1 mm) perfusion caused significant inverse FRET increases in both the experimental (“exp” c929-GAL4/Pdf-LexA,LexAop-Epac1-camps;UAS-P2X2/+) and PdfR mutant (“-pdfr” han⁵³⁰⁴;c929-GAL4/Pdf-LexA,LexAop-Epac1-camps;UAS-P2X2/+) conditions, relative to the negative control lacking the GAL4 driver for P2X2 expression (“-p2x2” Pdf-LexA,LexAop-Epac1-camps/+;UAS-P2X2/+). H, Comparison of maximum Epac1-camps responses for the s-LNv data shown in B, D, F. ATP (1 mm) perfusion caused significant inverse FRET increases in experimental (exp) flies relative to both PdfR mutants (-pdfr) and −p2x2 controls. Genotypes were identical to those in G. G, H, ***p < 0.001. n.s., No significant difference (p ≥ 0.05).

Figure 5.

PdfR expression in a small group of evening cells is sufficient for normal evening peak phase under long days. A, The averaged activity profile of han⁵³⁰⁴;UAS-Pdfr control flies under LDR 16:8. The evening peak is advanced compared with wild-type controls (compare withFig. 4C). B, The averaged activity profile of han⁵³⁰⁴;Pdf-GAL4/UAS-Pdfr flies. Pdfr expression in the l-LNvs and s-LNvs is not sufficient to rescue normal evening peak phase. C, The averaged activity profile of han⁵³⁰⁴;Cry-GAL4/UAS-Pdfr flies. The rescue of Pdfr expression in the cry- expressing clock neurons is sufficient to rescue normal evening peak phase. D, The averaged activity profile of han⁵³⁰⁴;UAS-Pdfr/+;R78G02-GAL4/+ flies. Pdfr expression in the R78G02-GAL4-expressing neurons is sufficient to rescue normal evening peak phase. E, The averaged activity profile of han⁵³⁰⁴;npf-GAL4/UAS-Pdfr flies. Pdfr expression in the NPF expressing clock neurons is sufficient to rescue normal evening peak phase. F, A comparison of evening peak phases between wild-type (WT) flies, flies lacking functional Pdfr (PdfR-/UAS-PdfR), and flies in which Pdfr expression has been rescued in various subsets of clock neurons using various GAL4 (G4) drivers. Only rescues that included the fifth s-LNv- and CRY-positive LNds (bottom five genotypes) resulted in rescue of evening peak under LDR 16:8. ANOVA followed by a post hoc test revealed highly significant differences in evening peak phase between the fly lines marked by asterisks and the ones without asterisks (p < 0.001). The lines with asterisks were not significantly different from each other (p = 1.000). Neither were the unmarked lines significantly different from each other (p > 0.838). G, PDF (cyan) and CRY (magenta) expression in a single hemisphere of a UAS-StingerGFP/+; R78G02-GAL4/+ brain. Only the region bordering the central brain and Me is shown. H, The same brain region as in G coimmunolabeled for GFP (green) and CRY (magenta). R78G02-GAL4 drives GFP expression in the CRY-positive LNds and the fifth s-LNv. Another, nonclock neuron-expressing GFP (asterisk) is also present in this brain region. I, A GRASP brain preparation in which GFP (green) has been reconstituted between LNvs (magenta) and R78G02-GAL4 expressing neurons. Reconstituted GFP is visible in the AMe near the s-LNvs. J, A GRASP brain preparation as in I. Reconstituted GFP is visible along dorsal projection (dp) of the s-LNvs. pot, Posterior optic tract of the l-LNvs. Scale bars, G–I, 20 μm. ***p < 0.001.

Figure 6.

Network model of light input in the circadian clock neuron network that governs bimodal locomotor rhythms. The l-LNvs receive excitatory cholinergic from the compound eyes (ACh) and inhibitory histaminergic (His) input from the HB eyelets, whereas the s-LNvs receive excitatory ACh input from the HB eyelets. Thus, HB eyelet activity antagonizes compound eye- and CRY-mediated light excitation in the l-LNvs while synergizing CRY-mediated excitation in the s-LNvs. Although we separate ACh and His terminals for the eyelet in the model, we do not mean to suggest that these two transmitters are differentially trafficked to different presynaptic termini. Rather, the differential effects of eyelet excitation are most likely due to the differential expression of receptors on the l-LNvs and s-LNvs. The l-LNvs modulate (red arrows) the s-LNvs and the E1 and E2 evening neurons via PDF. Such modulatory signaling likely sets the phase of the molecular clocks in target neurons, although it results in a modest excitation of targets as well (see Discussion). We hypothesize that the effects of the HB eyelets wane over the course of the day. Consequently, the excitatory influence of the compound eyes on the l-LNvs prevails under long days. The neuropeptides expressed by the various classes of clock neurons are indicated by color as is the pattern of CRY expression (dark blue outline). For the exception of the l-LNvs, PdfR is expressed wherever CRY is expressed. M, Morning neurons (s-LNv); E3, evening neurons that are not receptive to PDF.