Balance of activity between LN(v)s and glutamatergic dorsal clock neurons promotes robust circadian rhythms in Drosophila

Abstract

Circadian rhythms offer an excellent opportunity to dissect the neural circuits underlying innate behavior because the genes and neurons involved are relatively well understood. We first sought to understand how Drosophila clock neurons interact in the simple circuit that generates circadian rhythms in larval light avoidance. We used genetics to manipulate two groups of clock neurons, increasing or reducing excitability, stopping their molecular clocks, and blocking neurotransmitter release and reception. Our results revealed that lateral neurons (LN(v)s) promote and dorsal clock neurons (DN(1)s) inhibit light avoidance, these neurons probably signal at different times of day, and both signals are required for rhythmic behavior. We found that similar principles apply in the more complex adult circadian circuit that generates locomotor rhythms. Thus, the changing balance in activity between clock neurons with opposing behavioral effects generates robust circadian behavior and probably helps organisms transition between discrete behavioral states, such as sleep and wakefulness.

Figure 1. Pre- and post-synaptic DN₁ terminals are located close to LN_v axonal termini

(A) The nuclei of larval clock neurons were marked with the circadian transcription factor Par Domain Protein 1 (PDP1, red). LN_vs were co-labeled with PDF (blue). cry13-Gal4; Pdf-Gal80 driven expression of the Dscam17.1-GFP post-synaptic marker (green) labels DN₁ projections very close to LN_v axons. The 5^th LN_v was identified by lack of PDF and GFP staining and its location. (B) cry16-Gal4; Pdf-Gal80 driven expression of UAS-CD8-GFP (green) and the pre-synaptic marker UAS-Syt-HA (anti-HA, red) co-localize to DN₁ projections adjacent to LN_v axons (labeled with PDF, blue). (C) Same image as (B) with GFP channel removed to show Syt expression in DN₁ projections adjacent to LN_v axons. This image shows a 20μm stack but single 4μm sections also show DN₁ projections adjacent to LN_v axons.

Figure 2. LN_vs promote and DN₁s inhibit larval light avoidance

Larval light avoidance was measured by counting the number of larvae on the dark sides of a Petri dish after 15min. Transgenes were targeted to either LN_vs using Pdf-Gal4 (Pdf >), or DN₁s using cry-Gal4; Pdf-Gal80 (DN₁ >). Control lines are either the Gal4 line crossed to the non-conducting UAS-dORKΔNC transgene (Con) or the relevant UAS-transgene crossed to y w (transgene / +). Error bars show SEM. All statistical comparisons to the relevant control line were made using the students t-test. *p<0.05, **p<0.01. (A) Light avoidance was assayed between ZT 3–6 at 150lux. Hyperpolarizing LN_vs with UAS-dORKΔC (Pdf > dORK) or ablating LN_vs with UAS-Dti (Pdf > Dti) had no significant effect on light avoidance vs control larvae. Hyperexciting LN_vs via NaChBac (Pdf > NaCh, p<0.005) increased larval light avoidance. (B) Light avoidance was assayed as in (A). Hyperpolarizing DN₁s with UAS-dORKΔC or UAS-mKir2.1 (DN₁ > dORK, p<0.05 and DN₁ > Kir, p<0.005), or DN₁-ablation (DN₁ > Dti, p<0.01) significantly increased larval light avoidance. Hyperexciting DN₁s (DN₁ > NaCh) had no significant effect on light avoidance.

Figure 3. Altering CLK/CYC activity has opposite effects on LN_v and DN₁ excitability

All Statistical comparisons were made by ANOVA with Tukey's post-hoc test. *p<0.05, **p<0.01, ***p<0.001 (A) Light avoidance was assayed between ZT 3–6 in LD at 150lux. Expressing Clk^DN (p<0.05) or cyc^DN (p<0.001) in LN_vs increased larval light avoidance at 150lux compared to controls (Pdf-Gal4 or UAS-transgene crossed to y w). Hyperpolarization of LN_vs expressing cyc^DN (Pdf > cyc^DN + dORK) restored light avoidance to wild-type levels while hyperexcitation of LN_vs expressing cyc^DN (Pdf > cyc^DN + NaCh) did not. See also Fig S1. (B) Light avoidance was assayed as in (A). Expressing Clk^DN (p<0.01) and cyc^DN (p<0.01) in DN₁s increased larval light avoidance at 150lux compared to controls (DN₁ > Con, reproduced from Fig 2B, or UAS-transgenes crossed to y w, reproduced from Fig 3A). Hyperexcitation of DN₁s expressing cyc^DN (DN₁ > cyc^DN + NaCh) restored light avoidance to wild-type levels. (C) Light avoidance was assayed between ZT 3–6 in LD at 750lux. Hyperexciting LN_vs (per⁰¹; Pdf > NaCh, p<0.01) rescued the low levels of light avoidance of per⁰¹ larvae whilst hyperpolarizing LN_vs did not (per⁰¹; Pdf > dORK). Hyperpolarizing DN₁s (per⁰¹; DN₁ > dORK, p<0.01) also increased light avoidance whilst hyperexciting DN₁s (per⁰¹; DN₁ > NaCh) did not.

Figure 4. A signal from DN₁s is necessary and sufficient for light avoidance rhythms

All Statistical comparisons are as specified below. *p<0.05, **p<0.01, (A) Light avoidance was assayed on day 2 (CT12, 18, 24) or day 3 (CT6) of DD after prior LD entrainment. Control UAS-Dti / + larvae (grey) show time-dependent light avoidance at 150lux (CT12 vs CT24, t-test p<0.01). DN₁-ablated larvae (green) show no time-dependent light avoidance, (ANOVA p=0.79). 2-Way ANOVA between control and DN₁-ablated larvae for CT12 and 24 reveals a significant Genotype × Time interaction (F_1,11=8.53, p<0.05). No time-dependent differences in light avoidance were observed in control or DN₁-ablated larvae at 50lux (t-test). (B) Light avoidance was assayed as in (A) at 150lux. All statistical comparisons by students t-test. Light avoidance scores were higher at CT24 than CT12 in control (per⁺ UAS-per, p<0.05) but not in per⁰¹UAS-per larvae. Rhythms were rescued by restoring per expression to LN_vs (blue, p<0.005) or DN₁s (green, p<0.05) in per⁰¹ mutants. See also Fig S2. (C) Light avoidance was assayed as in (A). Light avoidance scores were lower at CT12 than at CT24 in control UAS-CLK^DN / + larvae at 150lux (t-test p<0.001). DN₁ > CLK^DN increased light avoidance compared to controls (2 Way ANOVA F_1,31=5.81, p<0.05), with no time-dependent differences in light avoidance observed at either 150lux (ANOVA) or 50lux (t-test). (D) Light avoidance was assayed on day 2 in DD at 150lux using larvae reared at 20°C. Light avoidance scores were lower at CT12 than at CT24 in DN₁ > TrpA1 larvae when assayed at 20°C (t-test p<0.01) but not 26°C. At 26°C, temperature-induced activation of DN₁s via TrpA1 reduces light avoidance at CT24 to CT12 levels (2 Way ANOVA, Temperature × Time interaction F_1,12=5.73, p<0.05). See also Fig S3

Figure 5. DN₁s release glutamate to inhibit light avoidance

For all RNAi experiments UAS-dcr-2 was co-expressed to improve efficacy. The Gal4 control lines shown also express UAS-dcr-2. Statistical comparisons are as stated below. *p<0.05 (A–C) Larval light avoidance was measured as in Fig 2. (A) Expression of a VGlut-RNAi transgene (GD2574) in all clock neurons (tim > VGlut^RNAi increased light avoidance at 150lux compared to control larvae. These data are significantly different (ANOVA p<0.005). Tukey's post-hoc comparison gives a significant difference only between tim > VGlut^RNAi and UAS-VGlut^RNAi / +. However, light avoidance in tim > VGlut^RNAi is higher than tim > + by t-test (p<0.05) and tim > VGlut^RNAi larvae also lose circadian rhythms in light avoidance (Fig S4A). (B) Expression of Glutamate decarboxylase (UAS-Gad1) in DN₁s (DN₁ > Gad1) significantly increased light avoidance at 150lux compared to UAS-Gad1 / + control larvae (Gad1 / +, t-test p<0.05). See also Fig S4B. (C) A GluCl-RNAi transgene expressed in LN_vs (Pdf > GluCl^RNAi) significantly increased light avoidance at 150lux compared to control larvae (ANOVA p<0.05). An mGluRA-RNAi transgene expressed in LN_vs (Pdf > mGluRA^RNAi) had no effect on light avoidance compared to controls (ANOVA). See also Fig S4C. (D) Light avoidance was assayed in DD at 150lux as in Fig 4. Light avoidance is higher at CT24 than CT12 in control larvae (UAS-GluCl^RNAi / +, which also contain a UAS-dcr-2 transgene, t-test p<0.05). No rhythms in light avoidance were detectable when GluCl-RNAi was expressed in LN_vs (Pdf > GluCl^RNAi, ANOVA). Rhythmic light avoidance was still detectable in larvae expressing mGluRA-RNAi in LN_vs (Pdf > mGluRA^RNAi, ANOVA p<0.05). By 2 Way ANOVA comparison of CT12 and 24 time points, Pdf > GluCl^RNAi is different to control (F_1,22=9.17, p<0.01) whilst Pdf > mGluRA^RNAi is not (F_1,24=0.00, p=0.9547) (E) Glutamate-mediated inhibition of ACh-stimulated Ca²⁺ transients in dissociated larval LN_vs. Representative relative fluorescence (F/Fo) recordings are shown from dissociated larval LN_vs expressing UAS-GCaMP1.6. Solution changes, including neurotransmitter applications, are indicated by black bars. Lowering extracellular Cl⁻ to 13.6mM completely relieved glutamate-dependent inhibition. Glutamate completely blocked ACh-stimulated transients when physiological Cl⁻ was restored. (F) A 2min incubation of a larval LN_v with 500nM ivermectin irreversibly blocks subsequent ACh-induced Ca²⁺ transients.

Figure 6. Adult CRY+ non-LN_vs are required for robust locomotor activity rhythms

Locomotor activity was recorded from flies entrained to 12:12 LD (white area of actogram), then transferred to DD (shaded). Comparisons to UAS-Control / + are by ANOVA with Tukey's post-hoc test. *p<0.05, **p<0.01, ***p<0.001 (A) Representative, double-plotted, normalized actograms are shown for tim-Gal4; Pdf-Gal80 > dORK and tim-Gal4; cry-Gal80 > dORK flies. (B) The power of rhythms is plotted for flies expressing UAS-dORK under the control of tim-Gal4, Pdf-Gal4, tim-Gal4; Pdf-Gal80 and tim-Gal4; cry-Gal80 drivers. Power is significantly reduced in Pdf > dORK, tim > dORK and tim-Gal4; Pdf-Gal80 > dORK flies compared to UAS-dORK / + control flies. (C) Representative, double-plotted, normalized actograms are shown for tim-Gal4; Pdf-Gal80 > NaCh and tim-Gal4; cry-Gal80 > NaCh flies. (D) The power of rhythms is plotted for flies expressing UAS-NaCh under the control of tim-Gal4, Pdf-Gal4, tim-Gal4; Pdf-Gal80 and tim-Gal4; cry-Gal80 drivers. Power is significantly reduced in tim-Gal4; cry-Gal80 > NaCh flies compared to UAS-NaCh / + control flies. (E) Average locomotor activity over the first 7 days in DD plotted for UAS-NaCh / + control (black) and tim-Gal4; Pdf-Gal80 > NaCh flies (grey). tim-Gal4; Pdf-Gal80 > NaCh fly activity is substantially reduced during the subjective morning. Each genotype shows the average of 16 flies from a single experiment.

Figure 7. Glutamate signaling from Adult CRY+ non-LN_vs is required for robust locomotor activity rhythms

Locomotor activity was recorded from flies entrained to 12:12 LD (white area of actogram), then transferred to DD (shaded). Comparisons to UAS-Control / + by ANOVA with Tukey's post-hoc test. *p<0.05, **p<0.01 (A) Representative, double-plotted, normalized actograms are shown for UAS-VGlut^RNAi/ + and tim > VGlut^RNAi flies. UAS-dcr-2 was co-expressed to improve RNAi efficacy and the Gal4 control line also expresses UAS-dcr-2. (B) The average power of rhythms is shown for tim > dcr2 + VGlut^RNAi flies and tim > dcr2 and UAS-VGlut^RNAi / + control flies. Power is significantly reduced in tim > VGlut^RNAi flies compared to tim > dcr2 and UAS-VGlut^RNAi control flies. Error bars represent SEM. (C) Data from (B) plotted as % of flies that are “arrhythmic” (power >100) “weakly rhythmic” (power from 100–250) or “rhythmic” (power > 250). (D) Representative, double-plotted, normalized actograms are shown for tim-Gal4; Pdf-Gal80 > Gad1 and tim-Gal4; cry-Gal80 > Gad1 flies. (E) The average power of rhythms is shown for flies expressing UAS-Gad1 with the tim-Gal4; Pdf-Gal80 and tim-Gal4; cry-Gal80 drivers. Power is significantly reduced in tim-Gal4; Pdf-Gal80 > Gad1 flies compared to UAS-Gad1 / + control or tim-Gal4; Pdf-Gal80 crossed to the UAS-dORKΔNC control (con) flies. Error bars represent SEM. (F) Data from (E) plotted as in (C)