The HisCl1 histamine receptor acts in photoreceptors to synchronize Drosophila behavioral rhythms with light-dark cycles

Abstract

In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. Here we show that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. Our results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina.

Fig. 1

HisCl1 and Ort histamine receptors can each transmit light input from Rh1- and Rh6-expressing PRs. a, b Average double plots of locomotor activity (actograms) of flies exposed to an 8 h advance of the LD/RD cycle, with corresponding phase plots (see Methods). Flies were initially entrained with both LD and temperature cycles (TC: 25–20 °C). a T°C was kept constant (25 °C) from the beginning of the day 3 light phase until the end of the experiment and the day 3 light phase was shortened by 8 h. Thus, flies were exposed to a novel advanced LD regime for 8 cycles followed by constant darkness (6 DD cycles). (Top) Wild-type flies (left) as well as flies with no Cry (center) or no histamine receptors (right) synchronize with phase advanced LD cycles. (Bottom) Flies with no Cry and no histamine receptors do not synchronize (left), whereas flies with no cry and only Ort (center) or only HisCl1 (right) do synchronize. b T°C was kept constant (25 °C) from the beginning of the day 2 light phase until the end of the experiment. Between days 2 and 3, the dark phase was shortened by 8 h and red light was used for the advanced light phase. Thus, flies were exposed to a novel advanced RD (red light) regime for 8 cycles followed by 6 DD cycles. Each of the four genotypes with only one histamine receptor and either Rh1 or Rh6 rhodopsin synchronizes with advanced RD cycles. Bars above actograms indicate the initial LD/TC cycles (black: lights-off, 20 °C; white: lights-on, 25°). White or red areas indicate the light phase of the cycle with white or red light respectively, and gray areas the dark phase of the cycle. Dots of the phase plots indicate the peak value of evening activity in LD/RD cycles and of activity in DD. n is the number of flies. Some genotypes (e.g., CHO, in (a) bottom left) have a free-running period that is slightly shorter than 24 h, which results in gradual advancement of the activity peak in DD or in LD/RD (also see other figures) when not entrained

Fig. 2

Ort and HisCl1 define different neuronal pathways for clock synchronization. Actograms and phase plots of flies with expression of the two histamine receptors in the triple mutant (HisCl1¹³⁴ ort¹ cry⁰²–CHO) genetic background using different gal4 drivers. The experimental design is as described in Fig. 1 legend. a Expression of either HisCl1 (top) or ort (bottom) in tim-expressing cells rescues entrainment. b HisCl1 cannot rescue entrainment when expressed in ort-expressing neurons (top), whereas ort can (bottom). c Neither HisCl1 (top) nor ort (bottom) rescues entrainment when expressed in most of the clock neurons. d Entrainment is rescued by HisCl1 expression in photoreceptors (top) and is not rescued by HisCl1 expression in tim-expressing cells when expression in the photoreceptors is blocked by the Gal4 inhibitor Gal80 (GMR-gal80) (bottom)

Fig. 3

HisCl1 but not Ort is expressed in photoreceptors. a–d Expression of HisCl1-gal4 UAS-mCD8-gfp, detected with anti-GFP antibody (green); photoreceptors are marked with anti-Chaoptin antibody (red). HisCl1 expression is detected in the lamina epithelial cells and very faintly in the eyelet (a, b). Weak labeling is observed in some R7 and R8 photoreceptors axons in the medulla (c, d). e–g Expression of GFP-tagged HisCl1 protein expressed from the HisCl1 promoter detected with anti-GFP antibody (green); photoreceptors are marked with ey-rfp from the same fosmid construct (red). HisCl1::GFP is detected in the lamina (e), H-B eyelet (e, g), and both in the R7 (f) and R8 (g) photoreceptors, identified by their respective position in the retina (arrows), upper layer for R7 and lower layer for R8. h–m HisCl1 (h-k) and ort (l, m) mRNA expression (blue) visualized by RNAscope^® in situ hybridization (ISH) (see Methods). Anti-Rh6 antibody (red) labels yR8 and eyelet photoreceptors in (j, k, m). HisCl1 mRNA in the retina appears as puncta primarily in two layers: proximally, near the R8 photoreceptor nuclei (h pink bracket) and distally, near the R1-7 photoreceptor and other retinal cell nuclei (h yellow bracket). No puncta were detected in the HisCl1¹³⁴ mutant (i). In the R8 photoreceptor layer HisCl1 mRNA is expressed in the Rh6-positive (red) yR8 retinal photoreceptors, as well as in the surrounding cells that likely include Rh5-expressing pR8 photoreceptors (j). HisCl1 mRNA is also present in the Rh6-expressing H-B eyelet (k). ort mRNA is expressed in the lamina but not in the retina (l, m). In particular, ort is undetectable in the Rh6-expressing cells (red in m) both in the retina (outlined in l, m) and in the eyelet. In (h, i), the retinas are surrounded by the autofluorescent cuticle. Images in (h–m) are maximal projections of short confocal stacks (thickness: 10 (h, i), 12 (j, k), 13.5 (l-m) µm). La lamina, Me medulla, Re retina. White arrows point to the H-B eyelet. Scale bars represent 20 (a–i), 10 (j, k), and 50 (l, m) µm

Fig. 4

HisCl1 acts in Rh6-expressing photoreceptors to support circadian entrainment. Actograms and phase plots. The experimental design is as described in Fig. 1 legend for advanced LD/RD cycle (a, b, c, top). For delayed LD cycles (c, bottom), the dark phase was lengthened by 8 h between days 2 and 3. a Expression of HisCl1 specifically in Rh6 photoreceptors with a Rh6-gal4 driver rescues entrainment in CHO triple mutants (left). This rescue is blocked by simultaneous expression of Gal80 in all photoreceptors (right). b In the absence of Ort and Cry, RNAi knock-down of HisCl1 in Rh6 photoreceptors slows down the synchronization with advanced RD cycles (right), in comparison to controls (left). c In contrast to control flies with HisCl1+ photoreceptors (left), flies with HisCl1¹³⁴ mutant photoreceptors (right) very poorly synchronize with an advanced LD cycle (top) and fail to synchronize with a delayed LD cycle (bottom)

Fig. 5

HisCl1 in Rh6-expressing photoreceptors receive inputs both from outer and inner photoreceptors. a–c Actograms and phase plots. The experimental design is as described in Fig. 1 legend. a The RD phase advance rescue of CHO flies by HisCl1 expression in Rh6 photoreceptors (left) is slowed down by the absence of Rh1 (ninaE¹⁷ mutation, center) and is abolished by the absence of both Rh1 and Rh6 (ninaE¹⁷ Rh6¹ genetic background, right). b Flies lacking Cry, compound eyes, and ocelli but retaining the H-B eyelet synchronize with advanced LD cycles (so¹; cry⁰² double mutants, left), but do not if HisCl1 expression is restricted to Rh6 photoreceptors (center). CHO mutants with HisCl1 rescue in Rh6 cells and genetic ablation of the eyelet (in addition to Rh5-expressing photoreceptors) synchronize with advanced RD cycles (right). c The LD phase advance rescue of CHO flies by HisCl1 expression in Rh6 photoreceptors is retained in the absence of either Rh1 and Rh6 (top) or Rh1, Rh5, and Rh6 (bottom)

Fig. 6

Model for the retinal input pathways to the brain clock. All retinal photoreceptors receive light inputs and release histamine. Ort-expressing interneurons of the optic lobe receive inhibitory histamine signals from most or all rhodopsin-expressing photoreceptors and transmit light information to the clock neurons through direct or indirect pathways. At least some of the Rh6-expressing photoreceptors express HisCl1 and receive histamine signals from other photoreceptors, in addition to receiving direct light inputs. These Rh6-expressing photoreceptors use a histamine-independent neurotransmission (possibly cholinergic) to transmit light information to the clock neurons. This connection could be either direct or via interneurons of the optic lobe