Modulation of the Critical Small Ventrolateral Pacemaker Neurons in Drosophila.

Almost every organism on earth carries with it a timepiece that bestows a critical sense of time. In many organisms, including humans, that timepiece is a network of neurons that each expresses an ancient molecular circadian clock. Like setting a clock, modulation of clock neurons adjusts the timing of an organism’s daily rhythms. However, it is not well understood how neuronal modulation of clock neuron activity translates into adjustments of molecular clocks and synchronization of daily rhythms to environment cycles. To investigate this question, I looked to the fruit fly’s small ventral lateral clock neurons (s-LNv), which are not only located in a rich medulla of environmental and interneuronal inputs, but are critical components of the fly’s timepiece. To survey these small clock neurons, I employed, validated and expanded a burgeoning method of genetically encoded circuit interrogation to determine s-LNv receptivity and connectivity. Acetylcholine and GABA are neuromodulators from the visual system and sleep/arousal circuits, respectively. I found that cholinergic agonists and GABA inversely modulate Ca2+ and cAMP levels in the s-LNv. Cholinergic modulation likely comes from the fly’s eyelets, as I further showed that activation of the larval and adult eyelets lead to increases in Ca2+ and cAMP from the s-LNv. The results of my studies identify concrete neuronal connections between the s-LNv and the visual system. My work also suggests that the mechanism by which environmental and interneuronal modulation adjusts the clock is through modulation of critical signaling molecules like cAMP. Overall, this work contributes to a growing body of evidence that shows cAMP to be a conserved signaling molecule involved in clock resetting in mammals and insects. Furthermore, the results of this study support continued investigation into the simple fruit fly to understand the more complex circuitry that underlies the rhythms of our life.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108826/1/ktszu_1.pd

Functional conservation of clock output signaling between flies and intertidal crabs

Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (β-pdh-I and β-pdh-II). Phylogenetically, β-pdh-I is more closely related to insect pdf than to β-pdh-II, and we hypothesized that β-PDH-I may represent a canonical clock output signal. Accordingly, β-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf(01)) as a heterologous system, we show that β-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but β-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and β-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but β-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.Fil: Beckwith, Esteban Javier. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Lelito, Katherine R.. University Of Michigan; Estados UnidosFil: Hsu, Yun Wei A.. University Of Washington; Estados UnidosFil: Medina, Billie M.. University Of Washington; Estados UnidosFil: Shafer, Orie. University Of Michigan; Estados UnidosFil: Ceriani, Maria Fernanda. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: de la Iglesia, Horacio O. University Of Washington; Estados Unido

Differentially Timed Extracellular Signals Synchronize Pacemaker Neuron Clocks

<div><p>Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in <i>Drosophila</i> larvae, manipulating either the four master pacemaker neurons (LN_vs) or two dorsal clock neurons (DN₁s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LN_vs and DN₁s to maintain synchronized LN_v clocks. We also found that glutamate is a second synchronizing signal that is released from DN₁s and perceived in LN_vs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing <i>Pdfr</i> and <i>mGluRA</i> expression in LN_vs severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LN_vs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LN_vs. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LN_vs. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.</p></div

PDF and glutamate signal at different times of day to regulate LN_v cAMP levels.

<p>Statistical comparisons are by ANOVA with Tukey's post hoc test, unless otherwise stated. Desynchrony data were calculated from three independent experiments, each consisting of at least three brains. Total number of LN_v clusters analyzed are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959.s010" target="_blank">Table S1</a>. Error bars show SEM. Whiskers represent 95% confidence. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.005. (A–C) Larvae were subjected to a heat pulse (6 hours at 31°C) from either CT9 to CT15 on day 2 (CT12 shift) or from CT21 on day 2 to CT3 on day 3 of DD (CT24 shift). Larvae were then dissected at CT3 on day 3 of DD and immunostained with αTIM (red), αPDP1 (blue), and αPDF (green). (A) Representative images of control (+/<i>UAS-Shi^ts</i>) LN_vs or LN_vs of larvae expressing the temperature-sensitive allele of <i>Shibire</i> in DN₁s (<i>DN₁>Shi^ts</i>). At 31°C, <i>Shi^ts</i> is inactive, blocking synaptic transmission. Left: Effect of heat pulse at CT12. Right: Effect of heat pulse at CT24/0. (B) Histograms showing the percentage of LN_v clusters where TIM was detected in either none or all four of the four LN_vs (“synchronized,” green bars) or in one, two, or three LN_vs (desynchronized, red bars). (C) Desynchrony was quantified as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio-1001959-g001" target="_blank">Figure 1</a> by measuring ST DEV in TIM expression. A CT12 heat pulse significantly increased ST DEV of TIM expression in <i>DN₁>Shi^ts</i> brains compared to controls and to <i>DN₁>Shi^ts</i> larval brains with a CT24 heat pulse (F_3,60 = 6.423, <i>p</i> = 0.0008). (D) Larval LN_vs were immunostained for TIM at ZT3 and CT3 on days 1 and 2 of DD in Control (<i>+/UAS-Dti</i>), <i>DN₁>Dti</i>, and <i>Pdf⁰¹</i> mutants. DN₁ ablation and <i>Pdf⁰¹</i> mutants do not affect LN_v TIM levels at ZT3 (F_3,41 = 1.53, <i>p</i> = 0.22). On the first day of DD, only <i>Pdf⁰¹</i> increases TIM expression in LN_vs (F_3,51 = 11.43, <i>p</i><0.0001). <i>DN₁>Dti</i> increases TIM levels in LN_vs on day 2 in DD (Student's <i>t</i> test, <i>p</i> = 0.0004). (E) Desynchrony of LN_vs in LD and on days 1 and 2 of DD was quantified by measuring ST DEV of TIM expression in Control (<i>+/UAS-Dti</i>), <i>DN₁>Dti</i>, and <i>Pdf⁰¹</i> mutants. The STDEV in TIM is significantly higher in <i>Pdf⁰¹</i> LN_vs compared to control or <i>DN₁>Dti</i> LN_vs on the first day of DD, reflecting increased desynchrony (F_2,38 = 16.48, <i>p</i><0.0001). <i>DN₁>Dti</i> increases desynchrony as measured by TIM ST DEV only on day 2 in DD (Student's <i>t</i> test, <i>p</i> = 0.019).</p

Model for regulation of cAMP levels and the molecular clock in clock neurons.

<p>Black arrows and text show established pathways; grey arrows and text reflect pathways inferred but not yet demonstrated. Left panel: In LN_vs, PDF signals via PDFR and Gα/AC3 to boost intracellular cAMP <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959-Mertens1" target="_blank">[18]</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959-Duvall1" target="_blank">[21]</a>. In this study, we show that glutamate (glu) signals received via mGluRA reduce cAMP levels, likely by inhibiting AC3. Differentially timed release of PDF and glutamate signals results in cAMP rhythms. PKA responds to cAMP to increase stability of the PER/TIM dimer via PER <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959-Li1" target="_blank">[46]</a> and likely also via TIM (data here and inferred from non-LN_vs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959-Seluzicki1" target="_blank">[45]</a>). Right panel: In non-LN_v clock neurons, PDF signals via PDFR through Gα and unknown Adenyl cyclase(s) (AC) to boost intracellular cAMP. By analogy with what we show here for LN_vs, we propose that an inhibitory signal released at a different time of day from PDF inhibits AC activity to generate a cAMP rhythm in non-LN_vs. PKA responds to cAMP to increase stability of the PER/TIM dimer through TIM <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959-Seluzicki1" target="_blank">[45]</a> and likely also PER (by analogy with LN_vs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959-Li1" target="_blank">[46]</a>).</p

LN_v and non-LN_v clock neurons maintain LN_v synchrony.

<p>All experimental lines and <i>Pdf></i>+control larvae in RNAi experiments include <i>UAS-Dcr-2</i>, but this is omitted from written genotypes for simplicity. Desynchrony data were calculated from 3–4 independent experiments, each consisting of at least three but usually five or more brains. Total number of LN_v clusters analyzed are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio.1001959.s010" target="_blank">Table S1</a>. ** <i>p</i><0.01; *** <i>p</i><0.001. (A) Representative images of LN_vs in control larvae (<i>+</i>/<i>UAS-Pdfr^RNAi</i>) or in larvae with reduced <i>Pdfr</i> levels in LN_vs (<i>Pdf>Pdfr^RNAi</i>) or all clock neurons except LN_vs (<i>tim-Gal4; Pdf-Gal80</i>><i>Pdfr^RNAi</i>) immunostained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD. The lower panels for each genotype are the same images with the green channel (PDF) removed and replaced by a dashed white line outlining LN_vs. (B) Box plots showing the ST DEV in TIM expression as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001959#pbio-1001959-g001" target="_blank">Figure 1</a>. Statistical comparisons by ANOVA with Tukey's post hoc test show both <i>Pdf>Pdfr^RNAi</i> (F_2,49 = 12.33, <i>p</i><0.0001) and <i>tim-Gal4; Pdf-Gal80</i>><i>Pdfr^RNAi</i> (F_2,51 = 8.158, <i>p</i> = 0.0008) significantly increase the ST DEV of TIM levels compared to parental controls, reflecting increased desynchrony. (C) Representative images of larval LN_vs stained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD. From left to right, Control <i>DN₁>+</i>, and +/<i>UAS-Dti</i> LN_v clusters, and a representative desynchronized <i>DN₁>Dti</i> LN_v cluster. The green channel (PDF) has been removed from the lower panel and replaced by a dashed white outline of LN_vs. (D) Box plots showing quantification of desynchrony through measurement of ST DEV in TIM expression in larval LN_vs in control or DN₁ ablated larvae at ZT3, CT3, and CT9. <i>DN₁>Dti</i> increases ST DEV at CT 3 compared to both parental controls (ANOVA with Tukey's post hoc test, F_2,49 = 10.5, <i>p</i><0.0001). There was no significant difference between <i>DN₁>Dti</i> and controls at ZT3 (Student's <i>t</i> test, <i>p</i> = 0.35) or CT9 (Student's <i>t</i> test, <i>p</i> = 0.31).</p

PdfR and mGluRA are required in LN_vs for normal evening activity and timing of sleep onset.

<p>All experimental lines and <i>Pdf>+</i>control larvae also include <i>UAS-Dcr-2</i> for RNAi experiments, but this is omitted from written genotypes for simplicity. Error bars show SEM. *** <i>p</i><0.001. (A) Locomotor activity was recorded for 3–4 days in LD cycles, followed by 10 days in DD (shaded area of actograms). Representative actograms are shown for <i>Pdf>+</i> control flies and for <i>Pdf>GluCl^RNAi</i> and <i>Pdf>Pdfr^RNAi+mGluRA^RNAi</i> experimental flies. (B) Graphs show average locomotor activity over the first 5 days in DD. Each panel shows two control genotypes: <i>Pdf>+</i> (blue, <i>n</i> = 19) and <i>+/UAS-mGluRA^RNAI</i>; +/<i>UAS-Pdfr^RNAi</i> (green, <i>n</i> = 26). Experimental genotypes are shown in red. Top left: <i>Pdf>GluCl^RNAi</i> (<i>n</i> = 37). Top right: <i>Pdf>mGluRA^RNAi</i> (<i>n</i> = 54). Bottom left: <i>Pdf>Pdfr^RNAi</i> (<i>n</i> = 33). Bottom right: <i>Pdf>Pdfr^RNAi+mGluRA^RNAi</i> (<i>n</i> = 37). Activity between ∼CT6 and 18 is elevated in <i>Pdf>mGluRA^RNAi</i>, <i>Pdf>Pdfr^RNAi</i>, and <i>Pdf>Pdfr^RNAi+mGluRA^RNAi</i> flies compared to controls or <i>Pdf>GluCl^RNAi</i>. (C) Histogram shows the average sleep latency on the first day in DD. <i>Pdf>Pdfr^RNAi+mGluRA^RNAi</i> flies show significantly increased sleep latency compared to <i>Pdf>+</i>, <i>+/UAS-mGluRA^RNAI</i>; +/<i>UAS-Pdfr^RNAi</i>, and <i>Pdf>GluCl^RNAi</i> controls (ANOVA F = 6.83, <i>p</i> = 0.0003).</p

mGluRA and PdfR regulate intracellular cAMP.

<p>Statistical comparisons are by ANOVA with Tukey's post hoc test. Error bars show SEM. Whiskers represent 95% confidence. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; **** <i>p</i><0.0001. (A) Larvae were dissected and analyzed on day 2 in DD. CFP and YFP levels were measured in the projections of <i>Pdf>Epac1-camps</i> LN_vs. The ratio of CFP/YFP reflects the basal level of cAMP. The CFP/YFP ratio oscillates in control (<i>Pdf>Epac1-camps</i>) LN_v projections, peaking at CT24 (ANOVA F_3,62 = 2.933, <i>p</i> = 0.04). There is no significant oscillation in <i>Pdf>Epac1-camps</i>+<i>mGluRA^RNAi</i> (F_3,59 = 0.815, <i>p</i> = 0.49) or <i>Pdf>Epac1-camps</i>+<i>Pdfr^RNAi</i> (F_3,47 = 1.068, <i>p</i> = 0.37). The CFP/YFP ratio is significantly increased at CT12 in <i>Pdf>Epac1-camps</i>+<i>mGluRA^RNAi</i> compared to control LN_vs (F_2,38 = 5.021, <i>p</i> = 0.0017) but not in <i>Pdf>Epac1-camps</i>+<i>Pdfr^RNAi</i>, consistent with glutamate signals inhibiting cAMP at CT12. (B) Averaged Epac-1-camps CFP/YFP ratio responses to bath application of 100 nM PDF or vehicle (arrow). The wild-type (<i>Pdf>Epac1-camps</i>) response to 100 nM PDF is shown in blue, and the wild-type response to vehicle is shown in black. Knockdown of <i>GluCl</i> (<i>Pdf>Epac1-camps</i>+<i>GluCl^RNAi</i>, green) had no significant effect on the response to PDF, but knockdown of <i>mGluRA</i> (<i>Epac1-camps</i>+<i>mGluRA^RNAi</i>, magenta) significantly increased the cAMP response of LN_vs to PDF. Vehicle traces represent 10 LN_v cell bodies from five brains (10, 5), wild-type PDF (10, 5), <i>Pdf>GluCl^RNAi</i> PDF (20, 9), and <i>Pdf>mGluRA^RNAi</i> PDF (27, 12). (C) Comparison of mean maximum Epac-1-camps CFP/YFP ratio changes between 0 and 240 s [dashed line in (B)] [genotypes and sample sizes as in (B)]. Application of 100 nM PDF significantly increased cAMP in LN_vs of <i>Pdf>Epac1-camps</i> flies compared to vehicle (<i>p</i><0.0001 by unpaired <i>t</i> tests). PDF responses of <i>Pdf>Epac1-camps</i>+<i>GluCl^RNAi</i> LN_vs were not significantly different from wild-type LN_vs (<i>p</i> = 0.6217). PDF responses of <i>Pdf>Epac1-camps</i>+<i>mGluRA^RNAi</i> LN_vs were significantly higher than wild-type (<i>p</i> = 0.024) and <i>Pdf>Epac1-camps</i>+<i>GluCl^RNAi</i> (<i>p</i> = 0.0193) LN_vs. (D) Model: We propose that LN_vs signal to each other via PDF around dawn. This signal is received by PdfR, which acts via Gαs/AC3 to increase intracellular cAMP. DN₁s release glutamate around dusk. This signal is received by mGluRA in LN_vs, which acts via Gαi to inhibit AC3 and reduce intracellular cAMP. Daily regulation of cAMP by external signals promotes robust TIM oscillations and LN_v synchrony.</p