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Circadian rhythms in neuronal activity propagate through output circuits

Nature Neuroscience volume 19, pages 587–595 (2016)Cite this article

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Abstract

Twenty-four hour rhythms in behavior are organized by a network of circadian pacemaker neurons. Rhythmic activity in this network is generated by intrinsic rhythms in clock neuron physiology and communication between clock neurons. However, it is poorly understood how the activity of a small number of pacemaker neurons is translated into rhythmic behavior of the whole animal. To understand this, we screened for signals that could identify circadian output circuits in Drosophila melanogaster. We found that leucokinin neuropeptide (LK) and its receptor (LK-R) were required for normal behavioral rhythms. This LK/LK-R circuit connects pacemaker neurons to brain areas that regulate locomotor activity and sleep. Our experiments revealed that pacemaker neurons impose rhythmic activity and excitability on LK- and LK-R-expressing neurons. We also found pacemaker neuron–dependent activity rhythms in a second circadian output pathway controlled by DH44 neuropeptide–expressing neurons. We conclude that rhythmic clock neuron activity propagates to multiple downstream circuits to orchestrate behavioral rhythms.

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Figure 1: Lk and Lkr mutant circadian phenotypes and location of LK- and LK-R-expressing neurons.
Figure 2: The anatomy of LHLK LK-R neurons suggests they lie downstream of clock neurons.
Figure 3: LHLK neurons are downstream of LNvs.
Figure 4: LHLK neuronal activity is rhythmically regulated by clock neurons.
Figure 5: LHLK activity rhythms propagate to LK-R neurons.
Figure 6: LK and LK-R neuron signaling controls locomotor activity levels.
Figure 7: Clock electrical rhythms propagate through multiple output circuits.

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Acknowledgements

We thank B. Al-Anzi (California Institute of Technology), D. Clark (Yale University), P. Hardin (Texas A & M University), P. Herrero (Universidad Autónoma de Madrid), M. Rosbash (Brandeis University), F. Rouyer (Institut des Neurosciences Paris-Saclay), A. Sehgal (University of Pennsylvania School of Medicine), O. Shafer (University of Michigan), S. Sweeney (University of York), the Developmental Studies Hybridoma Bank and the Bloomington stock center for flies and antibodies. We thank the TRiP stock center at Harvard Medical School (NIH/NIGMS R01-GM084947) for transgenic RNAi fly stocks. We thank C. Desplan for sharing the two-photon microscope and perfusion chamber and O. Shafer for advice on calcium imaging. We thank R. Behnia, C. Desplan, C. Hackley, E. Meekhof, A. Petsakou, H. Piggins and Z. Zhu for discussions and comments on the manuscript. This investigation was conducted in facilities constructed with support from Research Facilities Improvement Grant Number C06 RR-15518-01 from the National Center for Research Resources, US National Institutes of Health (NIH). Imaging was performed at the NYU Center for Genomics & Systems Biology. This work was supported by EMBO ALTF 249-2009 (M.C.), the Charles H. Revson foundation (M.C.), EMBO ALTF 680-2009 (C.B.), HSFPO LT000077/2010-l (C.B.), NIH grant R01 EY017916 (to C. Desplan), NIH grant GM063911 (J.B.) and the NYU Abu Dhabi Research Institute (G1205).

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Author notes

  1. Matthieu Cavey & Claire Bertet

    Present address: Present address: Aix-Marseille Université, CNRS, IBDM UMR7288, Marseille, France.,

  2. Ben Collins

    Present address: Present address: Institute of Pharmacology and Toxicology, University of Zürich, Zürich, Switzerland.,

Authors and Affiliations

  1. Department of Biology, New York University, New York, New York, USA

    Matthieu Cavey, Ben Collins, Claire Bertet & Justin Blau

  2. Center for Genomics & Systems Biology, New York University Abu Dhabi Institute, Abu Dhabi, United Arab Emirates

    Matthieu Cavey & Justin Blau

  3. Program in Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates

    Justin Blau

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Contributions

M.C. and B.C. performed the RNAi screen. M.C. performed all other experiments and analyses except the immunostaining in Supplementary Figure 3, which was done by C.B. M.C. and J.B. wrote the manuscript, with comments from B.C. and C.B.

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Integrated supplementary information

Supplementary Figure 1 Characterization of Lk and Lkr mutants.

(a) Representative actograms in DD for three Lkr mutant alleles (Lkrc003, LkrMimic1 and LkrMimic2) as heterozygotes over a wild type chromosome (top row) or as hemizygotes over a deficiency (Df(3L)BSC371, bottom row) that removes Lkr. All flies are in a w1118 background. See Supplementary Table 2 for details and sample sizes. (b-c) Quantification of LK peptide levels in Lkc275 and elav > Dcr-2 + LkRNAi mutants by immunostaining. (b) Images are z-projections, color-coded to reflect pixel intensity (scale on right). LHLK cell bodies are indicated by arrowheads and shown in insets acquired at higher laser power to reveal faint LK staining in flies expressing LkRNAi. Scale bars: 20μm (c) Quantification of immunostaining. LK levels are expressed as a fraction of control (y w) levels and are indicated above each bar. Error bars indicate SEM. n=16 cell bodies from 8 brains for each sample from 1 experiment. (d) Quantification of Lkr mRNA reduction in Lkrc003 and elav > Dcr-2 + LkrRNAi mutants by quantitative real-time RT-PCR. Lkr mRNA levels are expressed as a fraction of control (y w) levels and indicated above each bar. Calmodulin was used to normalize input RNA levels. Error bars indicate SEM. n=6 replicates for each sample (see Methods).

Source data

Supplementary Figure 2 LK and LK-R are not clock neurons and the molecular clock is intact in LkRNAi mutants.

(a) LHLK neurons have no detectable GFP when UAS-nlsGFP was expressed via per-Gal4. This is despite broad GFP expression in the brain, including clock neurons identified by immunostaining (left panel, LNvs labeled by PDF) or anatomically (middle panel, DN1s and DN3s). per-Gal4 is absent from LHLKs (middle panel) and other LK neurons (SELKs, right panel) labeled by LK immunostaining. The middle and right panels are single confocal sections. Scale bar 100μm (left panel), 20μm in middle and right panels. n=8 brains examined. (b) Functional test for the presence of a molecular clock in LK and LK-R neurons. Locomotor activity rhythm strength (power) of flies over 10 days in DD after entrainment to LD cycles. Asterisks indicate significant difference (p<0.05) with the control by KS test, ns: non-significant. Expressing a cycle dominant negative construct (UAS-cycΔ) in clock neurons with tim-Gal4 (2nd bar) causes severe arrhythmicity compared to controls (UAS-cycΔ / +, 1st bar, p<0.0001, D=0.878). In contrast, UAS-cycΔ has no effect when expressed with Lk-Gal4 (3rd bar, p=0.449, D=0.238) or LkrR65C07-Gal4 (4th bar, p=0.735, D=0.186). See Supplementary Table 2 for details and sample sizes. (c-e) The molecular clock is intact in Lk mutants. (c) PDF, TIMELESS (TIM) and VRILLE (VRI) immunostaining in control (left, y w) and Lk mutant (right, elav > Dcr-2 + LkRNAi) brains dissected at 4 time points on the 2nd and 3rd days in DD. PDF staining marks s-LNvs. The brightness and contrast have been adjusted for TIM and VRI to reveal faint staining in all images. Scale bars 20μm. (d-e) Quantification of non-adjusted images reveal that TIM (d) and VRI (e) oscillations are similar in control and LkRNAi mutants. Statistics by Kruskal-Wallis ANOVA (3d.f each): y w TIM, p<0.0001, H=149.8; y w VRI, p<0.0001, H=89.6; LkRNAi TIM, p<0.0001, H=175.2; LkRNAi VRI, p<0.0001, H=175.1. Error bars indicate SEM. Sample sizes (indicated as n=x;y where x is the number of neurons and y the number of brains): y w CT12 n=60;10, y w CT18 n=73;10, y w CT24 n=64;10, y w CT6 n=60;9, LkRNAi CT12 n=61;10, LkRNAi CT18 n=68;10, LkRNAi CT24 n=65;10, LkRNAi CT6 n=75;10.

Source data

Supplementary Figure 3 Characterization of the LkrR65C07-Gal4 line.

(a-e) LkrR65C07-Gal4 labels neurons with a very similar anatomy to Lkr > FLEXAMP clones that also likely connect to LHLKs. (a) LkrR65C07-Gal4 driving CD8::GFP co-stained for LK labels neurons with their cell bodies in the lateral horn (LH) region and PI. Lateral horn LK-RR65C07 neurons project towards the FSB as observed in Fig. 2g with Lkr > FLEXAMP. (b) Single confocal section of the dashed square in a show overlap of LK-RR65C07 projections with LHLK arborizations and numerous potential sites of contact. (c) LK-RR65C07 neurons innervate several layers of the FSB. (d) LkrR65C07-Gal4 is also expressed in EB neurons. (e) LkrR65C07-Gal4 driving Syt::GFP co-stained for LK shows that LK-RR65C07 neuron synaptic terminals are found predominantly in the FSB. (f) Single confocal sections showing LK-RR65C07 projections in the FSB intermingle with neurons with previously characterized functions in locomotion and sleep. LkrR65C07-LexA driving CD8::GFP crossed to Gal4 lines driving CD8::RFP (121Y-Gal4, c5-Gal4 and c584-Gal4). Scale bars: 20μm, except in a and e: 100μm. n≥8 brains examined for each experiment.

Supplementary Figure 4 Calcium-imaging control experiments

All statistics by Kolmogorov-Smirnov (KS) test as in Fig. 3. Each experiment is from ≥2 independent replicates. (a, left) Perfusing 2.5 mM ATP significantly increases intracellular calcium in s-LNvs (n=19;4) and l-LNvs (n=26;4) of Pdf > P2X2 + GCaMP6S brains compared to 0.1% DMSO vehicle perfusion (the vehicle control is shown for s-LNvs, veh, n=22;3). p<0.0001, D=0.909 for ATP perfusion vs. veh in s-LNvs; p<0.0001, D=0.969 for ATP vs veh (n=32;3) in l-LNvs. (a, middle) No increase in intracellular calcium in LHLK neurons from Lk > GCaMP6S; Pdf-LexA > P2X2 brains with ATP perfusion (n=12;3) or vehicle (n=16;4). No statistical difference for ATP vs. veh (p=0.948, D=0.187). (a, right) Dot plots show distribution of Maximum ΔF/F0 in left and middle line graphs. (b, left) Dose response of LHLK neurons to decreasing concentrations of CCh. From top to bottom: 100 μM CCh, n=23;11; 50 μM CCh, n=23;7; 10 μM CCh, n=61;17; vehicle, n=12;4. (b, middle) LHLK response to 10 μM CCh appears to be direct since it was unaffected by 20 min pre-incubation with 2 μM TTX (n=28;7) compared to controls (veh, n=22;6, p=0.326, D=0.259). (b, right) Quantification of line graph data. For CCh dose responses, asterisks indicate a significant difference from the vehicle control by KS test (100 μM: p<0.0001, D=0.937; 50 μM: p<0.0001, D=0.937; 10 μM: p<0.0001, D=0.711). (c, left) Perfusing 10 μM PDF increases calcium gradually in s-LNvs (n=18;6) but not in l-LNvs (n=17;6). The vehicle control shown is for l-LNvs, veh, n=15;6). (c, middle) Perfusing 10 μM PDF has no effect on LHLK neurons (n=10;9, veh: n=14;7). (c, right) Quantification of line graph data. Statistics: For s-LNvs veh vs PDF: p=0.0057, D=0.567 (veh, n=15;6). For l-LNvs veh vs PDF: p=0.571, D=0.263. For LHLKs veh vs PDF: p=0.060, D=0.5. (d, left) PDF pre-incubation decreases LHLK response to 10 μM CCh as in Fig. 3c (20min 20μM PDF pre-incubation, n=38;19, veh: n=28;14, p=0.016, D=0.372). (d, middle) PDF's inhibitory effect is transient since it disappears after 15 min washout. (n=38;19, veh: n=28;14, p=0.312, D=0.231). (d, right) Quantification of line graph data. (e) Perfusing 100 μM LK (n=15;5) does not increase calcium levels in LK-R neurons in the lateral horn region in Lkr > GCaMP6S brains compared to vehicle control (n=4;2, p=0.985, D=0.233), quantified in dot plot. (f) 10 min pre-incubation with 100 μM LK does not change l-LNv responses to 100 μM CCh (n=21;7; veh: n=15;6, p=0.916, D=0.190), quantified in dot plot. (g) LK-R neurons labeled by the LkrR65C07-Gal4 line with cell bodies in the lateral horn and projecting to the FSB are inhibited by 100 μM LK pre-incubation as LK-R neurons in Fig. 3e (response to 100 μM CCh, p<0.0001, D=0.562; n=80;4 each). (h) LK-RR65C07 neuron baseline GCaMP6S levels are rhythmic in DD (p<0.0001, H=41.41, 2d.f. by Kruskal-Wallis one-way ANOVA; CT0 n=300;12, CT11 n=318;12, CT23 n=272;12).

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Supplementary Figure 5 LK and LK-RR65C07 neuron manipulations affect sleep and locomotor activity levels.

(a-d) Sleep and locomotor activity for the data shown in Fig. 6b. (e-f) Sleep for the data shown in Fig. 6c. Sample sizes and replicates are as in Fig. 6b-c, asterisks: experimental flies significantly different from both parental controls (p<0.05) by KS test as in Fig. 6, error bars in c-d: SEM. (a-b) Sleep profiles (average minutes sleeping per 30 min bin) over 3 days in DD (days indicated at bottom) with a 24hr TrpA1 activation on day 2 (red shaded area, 28°C) as in Fig. 6b-c. Control flies decreased sleep in response to heat. (a) In contrast, Lk > TrpA1 flies increased sleep while (b) LkrR65C07 > TrpA1 flies decreased sleep only slightly further than control flies, probably due to the already low sleep levels at this time of day at 28°C. The first 12hr of heat pulse are magnified in the inset. (c-d) Waking activity (average beam crossing per awake minute) calculated for 3 consecutive days over 6hr time windows. (c) Activating LK neurons caused flies to be significantly less active than control flies while awake. (d) Activating LK-RR65C07 neurons caused flies to be significantly more active than controls while awake during the first 6hr time window. Waking activity recovered to normal levels on subsequent days in both experiments although it was more variable in d than c. (e-f) Sleep profiles of flies with (e) LK or (f) LK-RR65C07 neurons inhibited with shits. (e) LK inhibition had almost no effect on sleep, whereas (f) LK-RR65C07 inhibition significantly increased sleep throughout subjective day and night.

Supplementary Figure 6 Lk>TrpA1 and LkrR65C07>TrpA1 activation phenotypes are modulated by light and require neuronal TrpA1 expression.

(a) TrpA1 activation / shits inhibition experiments performed in DD and LD. The first 12hr of heat pulse are shown for TrpA1 activation of (left) LK neurons, (middle) LK-RR65C07 neurons and (right) shits inhibition of LK-RR65C07 neurons. Upper panels show experiments performed in DD (same data as in Fig. 6b-c), lower panels show the same experiments performed in LD (from 2 independent replicates). Heat pulses start at CT0 or ZT0. Asterisks: p<0.05 by KS test as in Fig. 6b-c, error bars: SEM. (left) Light suppresses/delays the decreased locomotor activity phenotype induced by LK neuron activation for most of the day. However the phenotype is apparent during the last ~4hr of the day (~ZT8-12). (middle) Similarly, light delays the increase in locomotor activity induced by LK-RR65C07 neuronal activation seen during the first 6hr in DD. The phenotype becomes apparent during the second half of the day (~ZT7-10). (right) Light delays the phenotype caused by LK-RR65C07 neuron inhibition with Shits as with activation experiments. Decreased locomotor activity becomes apparent during the last ~5hr of the day (~ZT7-12). Sample sizes for DD experiments are the same as in Fig 6b-c. Sample sizes for LD experiments: Lk > + n=61; TrpA1 / + n=62; Lk > TrpA1 n=60; LkrR65C07 > + n=63; LkrR65C07 > TrpA1 n=62; Shits / + n=57; LkrR65C07 > Shits n=52. (b) Restricting UAS-TrpA1 expression to non-neuronal cells using elav-Gal80 overrides the locomotor activity phenotypes caused by acutely activating LK and LK-RR65C07 neurons. Population average locomotor activity for 3 days in DD (days indicated at bottom, grey and black bars indicate subjective day and night) with 24hr of TrpA1 activation on day 2 (red shaded area, 28°C). The changes in locomotor activity caused by activating LK (left) and LK-RR65C07 (right) neurons are rescued by including an elav-Gal80 transgene. Thus the locomotor activity phenotype is not due to stimulating diuresis via non-neuronal LK-R-expressing stellate cells in the Malphigian tubules. Sample sizes: (left) UAS-TrpA1 / + n=62, elav-Gal80 /+; UAS-TrpA1 / + n=51, Lk > TrpA1 n=63, Lk; elav-Gal80 > TrpA1 n=51, (right) UAS-TrpA1 / + n=94, elav-Gal80 /+; UAS-TrpA1 / + n=75, LkrR65C07 > TrpA1 n=93, LkrR65C07 / elav-Gal80 > TrpA1 n=89. Data are from 2 (left) or 3 (right) independent experiments.

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Supplementary Figure 7 Model for propagation of clock electrical rhythms through output circuits to drive circadian behavior.

Electrical rhythms originate in the clock network by interplay of the intracellular molecular clocks and communication between different clock neuron groups. For simplicity, only Morning clock neurons (s-LNvs and DN1ps; M cells, blue) are shown here. M cell activity peaks around dawn and is low around dusk (blue curve). Clock neuron activity rhythms produce 24hr oscillations in the strength of clock outputs that propagate into output circuits. LHLK neuronal activity (orange) is in antiphase with M cells since M cells inhibit LHLKs. Following the same principle, neuronal activity rhythms propagate to the next layer of the LK circuit, the LK-R neurons (brown), and the phase is reversed again since LK inhibits LK-R neurons. DH44 (green) activity is also in antiphase with M cell and the relevant subset of DH44-Receptor neurons (DH44-R) have not yet been identified. Additional output circuits include DH31-Receptor neurons (DH31-R) and PDFR-expressing neurons in the EB. Ultimately, the rhythmic activity of output circuits transfers to brain areas controlling locomotor activity and sleep, generating behavioral rhythms.

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Cavey, M., Collins, B., Bertet, C. et al. Circadian rhythms in neuronal activity propagate through output circuits. Nat Neurosci 19, 587–595 (2016). https://doi.org/10.1038/nn.4263

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