Phase Coupling of a Circadian Neuropeptide With Rest/Activity Rhythms Detected Using a Membrane-Tethered Spider Toxin

It has recently been demonstrated that peptide ion channel toxins from venomous predators can be heterologously expressed as fusion proteins covalently tethered to GPI anchors inserted in the extracellular leaflet of the plasma membrane [34]. Membrane-tethered toxins are expressed as chimeric fusion protein comprising (from N to C termini) a secretory signal sequence, peptide toxin sequence, glycine-asparagine repeat hydrophilic linker, and GPI targeting sequence. Membrane-tethered toxins are targeted to the secretory pathway, where the signal sequence is cleaved, and the GPI targeting sequence is substituted by covalent bond to GPI, thus retaining the toxin on the surface of the cell in which it is expressed. The modulatory effects of these tethered toxins are limited to ion channels on the surface of the cells in which the toxin is expressed, and do not influence channels on neighboring cells not expressing membrane-tethered toxin [34].

We selected 19 different peptide ion channel toxins from the venoms of cone snails, scorpions, bees, and spiders (Table 1) for testing in vivo in membrane-tethered form in the nervous system of transgenic Drosophila melanogaster. Of the 19 membrane-tethered toxins tested, four of them cause complete embryonic/larval lethality when expressed pan-neuronally using an elav-GAL4 driver (Figure 1), indicating activity against functionally essential fly ion channel subtypes. Three of these four membrane-tethered toxins are derived from the venom of the Australian funnel web spider Hadronyche versuta. δ-ACTX-Hv1a inhibits inactivation of Na⁺ channels in isolated cockroach giant axon and dorsal unpaired median neurons and rat dorsal root ganglion neurons, and induces spontaneous repetitive action potential (AP) firing accompanied by plateau potentials [35]. κ-ACTX-Hv1c has highly conserved structural features common to multiple K⁺ channel blockers and has been shown to target Drosophila K⁺ channels [36,37]. ω-ACTX-Hv1c is a blocker of mid/low- and high-voltage–activated insect Ca²⁺ channels [38,39]. PLTXII, a toxin from Plectreurys tristis spider venom, inhibits neurotransmission at the Drosophila neuromuscular synapse and targets the presynaptic Ca²⁺ channel encoded by the cacophony gene [40,41]. As shown in Figure 1, pan-neuronal expression of any of these four toxins induces embryonic/early-larval lethality when expressed from any one of multiple independent chromosomal insertions. In contrast, μO-MrVIA, a blocker of vertebrate voltage-gated Na⁺ channels from the venom of a fish-eating cone snail [42], has no effect when expressed pan-neuronally (Figure 1). Lack of effect of μO-MrVIA when expressed pan-neuronally indicates absence of activity against fly Na⁺ channels, at least in its tethered form. We thus utilize tethered μO-MrVIA as a negative control for all experiments. The fact that the only four toxins showing signs of bioactivity when expressed in membrane-tethered form in the Drosophila nervous system are derived from spiders, and not from ocean-dwelling cone snails, makes sense in light of the natural prey species of spiders and cone snails.

To determine whether these four tethered spider toxins are active against ion channels important for circadian pacemaker neuron function, we expressed membrane-tethered toxins solely in the LN_V subset of approximately 20 clock neurons that secrete PDF and are considered key pacemakers of the clock circuit [12,16,17]. Three of the four potent membrane-tethered toxins substantially disrupt free-running circadian rhythms of locomotor activity when expressed in PDF neurons using a pdf-GAL4 driver (Figure 2, Table S1). δ-ACTX-Hv1a or κ-ACTX-Hv1c expression in PDF neurons induces arrhythmicity or complex rhythmicity in individual toxin-expressing flies. Complex rhythmicity occurs when an individual fly exhibits multiple rhythms of locomotor activity free-running simultaneously with different periods (Figure 2). In contrast, expression of tethered vertebrate Na⁺ channel blocker μO-MrVIA in PDF neurons has no behavioral effect on locomotor rhythms in comparison either with the background strain used for transgenesis (unpublished data) or with pdf-GAL4 driver flies (Figure 2, Table S1). Membrane-tethered PLTXII Ca²⁺ channel blocker has no effect on circadian behavior when expressed in PDF neurons (Figure 2). Membrane-tethered ω-ACTX-Hv1c expression in PDF neurons using the pdf-GAL4 driver, which is active in both PDF-expressing clock neurons as well as in neurosecretory cells in the thoracic ganglion starting in early larval development [8,17], causes death by approximately 1 wk posteclosion, suggesting the possibility of toxin-induced developmental defects. To eliminate such possible developmental effects of toxin expression, we exploited temperature-dependent temporal control of GAL4-driven transgene expression using a temperature-sensitive mutant form of GAL80 transcriptional repressor (GAL80^ts), which binds to GAL4 and inhibits downstream transcription that would otherwise be activated by GAL4 [43,44]. At 18 °C, GAL80^ts dominates GAL4′s activator function and is active at repressing transcription, but at 30 °C, it is inactive. Thus, GAL4-mediated transgene expression can be temporally activated by shifting flies that also express GAL80^ts from 18 °C to 30 °C. Flies expressing membrane-tethered ω-ACTX-Hv1c with both pdf-GAL4 and tub-GAL80^ts were allowed to develop to adulthood at 18 °C, and then were shifted to 30 °C to induce membrane-tethered ω-ACTX-Hv1c expression. These flies have a normal lifespan, but exhibit circadian rhythm defects of arrhythmicity or complex rhythmicity, as shown in Figure 2 and Table S1. These results indicate the presence of functionally important ion channels sensitive to κ-ACTX-Hv1c, ω-ACTX-Hv1c, and δ-ACTX-Hv1a in PDF-secreting LN_V clock neurons.

δ-ACTX-Hv1a has been shown to inhibit inactivation of insect and mammalian voltage-gated Na⁺ currents [35]. The predominant Drosophila voltage-gated Na⁺ channel is encoded by the para gene [45,46]. Functional expression of para in Xenopus oocytes is enhanced by coexpression of the tipE accessory subunit [47,48]. We coexpressed membrane-tethered δ-ACTX-Hv1a with para/tipE to examine the effect of the tethered toxin on para voltage-gated Na⁺ currents. Uninjected oocytes showed no voltage-gated inward currents (unpublished data). The inward Na⁺ current recorded from oocytes injected with para/tipE complementary RNA (cRNA) exhibits voltage-dependent activation, with rapid and complete inactivation occurring within several milliseconds (Figure 3A). Coexpression of tethered δ-ACTX-Hv1a dose-dependently inhibits the inactivation of para Na⁺ current. We used the ratio of steady-state current to peak current during 40 ms of depolarization to −30 mV to compare the extent of inhibition by membrane-tethered δ-ACTX-Hv1a of para Na⁺ current inactivation. Coinjection of para/tipE and either H₂O or tethered PLTXII Ca²⁺ channel toxin cRNAs (1:1) induces inward Na⁺ current with the current ratio of 0.023 ± 0.015 (n = 4) or 0.029 ± 0.004 (n = 16), respectively (mean ± standard error of the mean [SEM]). A low concentration of 1:1,000 tethered δ-ACTX-Hv1a cRNA significantly decreased the inactivation of Na⁺ current to a current ratio of 0.169 ± 0.018 (n = 21, p < 0.001). A high concentration of toxin (1:10) completely abolishes inactivation of Na⁺ current resulting in a current ratio of 1.005 ± 0.003 (n = 8, p < 0.001; Figure 3). In contrast, tethered δ-ACTX-Hv1a has no effect on kinetics and amplitude of Drosophila Shaker K⁺ current, which also exhibits fast activation and inactivation (Figure 3C), indicating retained specificity of membrane-tethered δ-ACTX-Hv1a for inhibiting inactivation of voltage-gated Na⁺ channels.

To confirm membrane targeting of membrane-tethered δ-ACTX-Hv1a in vivo, we examined the expression of Myc-tagged δ-ACTX-Hv1a in PDF-secreting clock neurons. The ten-amino acid Myc epitope tag is located in the middle of the glycine-asparagine repeat hydrophilic linker domain. Brains of flies with two, four, or six copies of UAS-δ-ACTX-Hv1a and pdf-GAL4 were double immunostained with anti-Myc and anti-PDF fluorescence, with parental 4×UAS-δ-ACTX-Hv1a flies as negative control for anti-Myc staining. As shown in Figure 4, control UAS-δ-ACTX-Hv1a flies show no anti-Myc immunofluorescence in the LN_V cell bodies or terminals. Anti-PDF labels the cell bodies of PDF neurons (LN_Vs) and their processes, including dorsomedial terminals of sLN_Vs (Figure 4A) and projection of large LN_Vs (lLN_Vs) to the contralateral optic lobe through posterior optic tract (unpublished data). Flies expressing δ-ACTX-Hv1a in LN_Vs exhibit red anti-Myc immunofluorescence colocalized with anti-PDF green fluorescence in the cell bodies of LN_Vs. δ-ACTX-Hv1a expression dose-dependently increases anti-Myc immunofluorescence detected in the cell bodies of both lLN_Vs and sLN_Vs, as well as sLN_V dorsomedial terminals (Figure 4). Red anti-Myc immunofluorescence is barely detectable in cell bodies of lLN_V in pdf>2×UAS-δ-ACTX-Hv1a flies, and not at all in the sLN_Vs. Introduction of four or six copies of UAS-δ-ACTX-Hv1a transgene significantly increases anti-Myc immunofluorescence detected in the cell bodies of both lLN_Vs and sLN_Vs (p < 0.001, Figure 4). Anti-Myc immunofluorescence in sLN_V dorsomedial terminals is not seen in brain hemispheres of pdf>2×UAS-δ-ACTX-Hv1a or pdf>4×UAS-δ-ACTX-Hv1a 4 flies. In pdf>6×UAS-δ-ACTX-Hv1a flies, 11 out of 16 hemispheres (68.8%) exhibit detectable anti-Myc immunofluorescence in the sLN_V dorsomedial terminals (Figure 4B). Projection of lLN_Vs to the opposite optic lobes through the posterior optic tract is also visualized with anti-Myc immunofluorescence in these brains (unpublished data). These results establish that membrane-tethered δ-ACTX-Hv1a is expressed in LN_V clock neurons and is transported throughout their neuronal arbors. Membrane-tethered ω-ACTX-Hv1c, a Ca²⁺ channel blocker, expression in LN_Vs is shown in Figure S1. High-resolution confocal images of the dorsomedial terminals of sLN_Vs expressing membrane-tethered ω-ACTX-Hv1c demonstrate green anti-Myc fluorescence signal surrounding “puncta” of red anti-PDF signal. This is consistent with targeting of ω-ACTX-Hv1c to the plasma membrane of the terminals that encloses regions of concentration of PDF-containing dense-core vesicles. These results indicate that membrane-tethered spider toxins are expressed by LN_V clock neurons and targeted to the plasma membrane where they can interact with their target ion channels.

In order to confirm functional expression of membrane-tethered δ-ACTX-Hv1a, we performed whole-cell electrophysiological recordings on lLN_V clock neurons expressing δ-ACTX-Hv1a. Previous studies indicate that lLN_V RMP and AP firing rate is regulated by the circadian clock to encode time of day [49,50]. In 12-h:12-h light:dark (LD) conditions, lLN_V RMP varies within the range of −40 mV to −70 mV. For any given cell steady state, lLN_V RMP remains relatively stable, albeit frequently with approximately 5−10-mV membrane potential oscillation. In the representative example shown in Figure 5A, this WT lLN_V RMP is relatively stable at −40 mV. Membrane-tethered δ-ACTX-Hv1a–expressing lLN_V RMP exhibits dramatically different membrane-activity from WT lLN_Vs. A representative example is shown in Figure 5B. Membrane-tethered δ-ACTX-Hv1a–expressing lLN_V membrane potential oscillates in a very wide range from approximately −140 to approximately 0 mV. Membrane-tethered δ-ACTX-Hv1a–expressing lLN_V RMP becomes gradually depolarized, and spontaneous AP firing rate increases as the membrane potential level increases. When the RMP reaches approximately −40 mV, a burst of 5−30 APs lasting approximately 0.2–0.5 s occurs. After the burst, the membrane potential remains at a depolarized plateau (−15−0 mV) for 2−7 s. Following the plateau, membrane potential repolarizes rapidly down to approximately −140 mV within 2 s. Following this huge hyperpolarization, membrane potential gradually depolarizes to eventually initiate another cycle of this unique behavior. We have recorded from 53 membrane-tethered δ-ACTX-Hv1a–expressing lLN_Vs, and 36 of them exhibit this basic cyclic temporal pattern of membrane potential. However, the exact frequency of these cycles, the duration of AP bursts and depolarized plateau, and the number of APs during the AP burst, exhibit substantial variation from cell to cell, even when expressing the same number of copies of δ-ACTX-Hv1a transgene. lLN_Vs expressing different numbers of δ-ACTX-Hv1a transgene do not show consistent differences in their membrane potential dynamics. Since membrane-tethered δ-ACTX-Hv1a inhibits inactivation of para voltage-gated Na⁺ channels (Figure 3), the AP burst and prolonged depolarized plateau in membrane-tethered δ-ACTX-Hv1a–expressing lLN_Vs most likely involve sustained opening of para channels, substantial Na⁺ influx, and increased intracellular [Na⁺]. We thus tested the hypothesis that the massive postplateau hyperpolarization well below the reversal potential for K⁺ is induced by electrogenic Na⁺/K⁺-ATPase pump currents expelling all that intracellular Na⁺, by blocking those currents with ouabain [51]. In the representative example shown in Figure 5C, the application of 0.1 mM ouabain for 1 min substantially reduces the massive postplateau hyperpolarizations in membrane-tethered δ-ACTX-Hv1a–expressing lLN_Vs, and this effect is at least partially reversible. These results suggest that Na⁺/K⁺-ATPase underlies the postplateau hyperpolarization.

To determine whether these physiological modifications of LN_Vs induced by expression of membrane-tethered δ-ACTX-Hv1a affect the function of the circadian control network, we examined free-running locomotor activity patterns of flies expressing either membrane-tethered δ-ACTX-Hv1a or μO-MrVIA specifically in the PDF-expressing LN_Vs using pdf-GAL4 driver. Control pdf>μO-MrVIA flies expressing membrane-tethered μO-MrVIA exhibit rhythmic free-running circadian behavior of locomotor activity with a single statistically significant period of approximately 24.4 h (Figure 6A), of 298 μO-MrVIA–expressing flies tested, 93% of them exhibited a single statistically significant periodogram peak (Figure 6, Table S1). In contrast, substantial numbers of experimental flies expressing membrane-tethered δ-ACTX-Hv1a in the LN_Vs exhibit arrhythmicity or complex rhythmicity (i.e., multiple superimposed free-running circadian rhythms of locomotor activity with different periods: one of approximately 25 h and one of approximately 22 h). Free-running behavioral effects induced by membrane-tethered δ-ACTX-Hv1a expression in the LN_Vs either with single independent chromosomal insertion, or with multiple chromosomal insertions are relatively similar. The remaining rhythmic δ-ACTX-Hv1a–expressing flies exhibit single-period free-running circadian rhythms with period corresponding to either the long or short period of complex rhythmicity, suggesting a similar effect on the circadian control network in these flies, albeit with a different behavioral manifestation (unpublished data). Arrhythmicity or complex rhythmicity is only rarely observed in control pdf>μO-MrVIA flies, which almost always exhibit single free-running rhythms (Figure 6, Table S1). The differences in proportion of behavioral phenotypes between membrane-tethered δ-ACTX-Hv1a–expressing flies and control (either membrane-tethered μO-MrVIA–expressing flies or parental UAS flies) were all statistically significant (χ²; p < 0.001).

To exclude a role for developmental effects of membrane-tethered δ-ACTX-Hv1a expression in inducing these behavioral phenotypes, we exploited the GAL80^ts system for temporal control of GAL4-driven transgene expression. Flies possessing UAS-δ-ACTX-Hv1a, pdf-GAL4, and tub-GAL80^ts transgenes were allowed to develop at 18 °C from egg to adult in the absence of δ-ACTX-Hv1a expression, and then shifted to 30 °C to induce membrane-tethered δ-ACTX-Hv1a expression. Experimental pdf,tub>δ-ACTX-Hv1a flies exhibit arrhythmicity or complex rhythmicity at 30 °C similar to pdf>δ-ACTX-Hv1a flies (Figure S2), indicating that the behavioral phenotype caused by membrane-tethered δ-ACTX-Hv1a expression in the LN_Vs is not due to developmental effects. The percentage of arrhythmic or complex rhythmic flies expressing δ-ACTX-Hv1a from two or four copies of UAS-δ-ACTX-Hv1a transgene is significantly greater than control pdf>μO-MrVIA or UAS-toxin flies (χ²; p < 0.0001). The exact manifestation of complex rhythmicity in pdf,tub>δ-ACTX-Hv1a flies is somewhat different than in pdf>δ-ACTX-Hv1a flies. The latter tend to simultaneously manifest strong short- and long-period rhythms immediately after transfer from 12-h:12-h LD into DD, while the former tend to initially manifest mainly a long-period rhythm and then, after about a week in DD, transition to predominately just a short-period rhythm, as seen in published studies of complex rhythmic flies [20]. The most likely interpretation of this particular form of complex rhythmicity at the cellular level is—as in the case in which strong short- and long-period rhythms manifest themselves simultaneously—that there are independently free-running subsets of clock neurons running with short and long periods, with the difference that their respective ability to gate locomotor activity waxes and wanes reciprocally over time in DD. These results indicate that the physiological changes induced by LN_V expression of membrane-tethered δ-ACTX-Hv1a interfere with the ability of the circadian control network to drive coherent free-running behavioral rhythms in constant darkness.

PDF-secreting LN_Vs are important not only for driving free-running locomotor rhythms in DD, but also for generating the “morning” peak of locomotor activity that anticipates lights-on in 12-h:12-h LD conditions [12,16,17,30,31]. To determine whether LN_V expression of membrane-tethered δ-ACTX-Hv1a interferes with the LN_V-dependent morning activity peak, we performed a refined analysis of the average relative activity in LD of flies expressing membrane-tethered μO-MrVIA or δ-ACTX-Hv1a using pdf-GAL4 driver. As shown in Figure 7A, control μO-MrVIA–expressing flies exhibit robust morning and evening circadian anticipatory peaks of locomotor activity. Membrane-tethered δ-ACTX-Hv1a–expressing pdf>δ-ACTX-Hv1a flies also exhibit robust morning and evening circadian anticipatory peaks of locomotor activity, albeit with phase-advanced morning activity (Figure 7A) compared to control pdf>μO-MrVIA flies. We quantified phase advance of morning activity peaks by computing an anticipation phase score for each fly, defined as the ratio of total relative activity in the 3-h period before lights-on to that after. Increase in anticipation phase scores reflects greater anticipatory activity before lights-on compared to after, thus quantifying phase advances in anticipation. As seen in Figure 7B, membrane-tethered δ-ACTX-Hv1a expression induces dose-dependent increase in morning anticipation phase score (p < 0.001), indicating dose-dependent phase advance of morning anticipation caused by inhibition of para Na⁺ channel inactivation in LN_V morning cells. The dose-dependent phase advance is also apparent in the shape of envelope of relative activity occurring before lights-on, being concave for pdf>μO-MrVIA flies and then becoming progressively more convex with increasing dose of membrane-tethered δ-ACTX-Hv1a. These effects are observed in multiple independent replicate experiments (unpublished data).

PDF has been hypothesized to be a daily phase signal transmitted from LN_Vs to other clock neurons (and potentially non-clock targets) that entrains both clock neuron cellular oscillation and locomotor rhythms [27]. To determine whether advanced phase of morning activity in flies expressing membrane-tethered δ-ACTX-Hv1a is due to altered phase of PDF secretion rhythms, we compared the daily rhythm of accumulation of PDF in the sLN_V dorsomedial terminals (thought to reflect daily rhythm of secretion; [18,22]) of flies expressing either δ-ACTX-Hv1a or μO-MrVIA in the LN_Vs in LD. There is a recent study demonstrating absence of detectable PDF cycling in the sLN_V terminals of a particular transgenic strain of flies that nonetheless still exhibit normal circadian locomotor activity [52]. However, this result is still consistent with a key role for cyclic secretion in circadian rhythms, and with cyclic PDF accumulation as a readout of cyclic secretion, as steady-state accumulation will only reflect secretion rhythms when synthesis rate and secretion rate are similar in magnitude; under conditions in which synthesis rate outpaces secretion rate, a rhythm of secretion may not result in a detectable rhythm of steady-state accumulation. Furthermore, there is functional, albeit indirect, evidence in the literature that rhythmic-activity–dependent PDF secretion is important for circadian rhythmicity [18,20,49,50].Therefore, it is reasonable to conclude that rhythmic steady-state accumulation of PDF reflects rhythmic PDF secretion.

In LD conditions, control flies expressing membrane-tethered μO-MrVIA in the LN_Vs exhibit significantly greater anti-PDF immunofluorescence in the sLN_V dorsomedial terminals at zeitgeber time (ZT)2 and ZT6, in comparison to the night at ZT14 and ZT18 (p < 0.001; Figure 7C). In contrast, PDF accumulation in the sLN_V dorsomedial terminals of experimental flies expressing membrane-tethered δ-ACTX-Hv1a in the LN_Vs cycles with advanced phase, peaking late night, rather than early in the morning, with peak anti-PDF immunofluorescence at around ZT22 (p < 0.001). This approximately 4-h phase advance of PDF cycling induced by LN_V expression of membrane-tethered δ-ACTX-Hv1a, in combination with the induced phase advance of morning anticipation, provides experimental support for the hypothesis that the phase of rhythmic PDF secretion determines the phase of morning anticipation. Figure S4 shows an independent replicate of this experiment, confirming δ-ACTX-Hv1a–induced phase advance of PDF accumulation.

We also examined the effect of membrane-tethered δ-ACTX-Hv1a on accumulation of PDF in the sLN_V dorsomedial terminals in constant darkness. Anti-PDF immunoreactivity in the dorsomedial terminals was assayed on the 2nd, 4th, or 6th d after release from LD entraining conditions into DD. As shown in Figure 8, LN_V expression of membrane-tethered δ-ACTX-Hv1a induces phase advance of PDF accumulation in DD consistent with the effect in LD. On the 2nd d in DD (DD-D2), anti-PDF immunofluorescence in the sLN_Vs dorsomedial terminals of control membrane-tethered μO-MrVIA–expressing flies peaks at CT2 to CT6 (p < 0.001). In contrast, experimental flies expressing membrane-tethered δ-ACTX-Hv1a exhibit phase-advanced peak PDF accumulation at circadian time (CT)22 to CT2 (p < 0.001). On DD-D4, control pdf>μO-MrVIA flies exhibit peak PDF accumulation at CT6–CT10 (p < 0.001). Experimental pdf>δ-ACTX-Hv1a flies exhibit phase advance of PDF cycling with peak at CT22 (p < 0.001). On DD-D6, control pdf>μO-MrVIA flies exhibit peak PDF accumulation at CT10 (p < 0.001), while experimental pdf>δ-ACTX-Hv1a flies exhibit peak PDF accumulation around CT22 (p < 0.001). The gradual accumulation of a phase delay relative to the 24-h d of PDF oscillation in control pdf>μO-MrVIA flies over 6 d in DD is consistent with their free-running behavioral period of greater than 24 h (Figures 2 and 6). The phase advance of PDF accumulation in sLN_V terminals of membrane-tethered δ-ACTX-Hv1a–expressing flies in LD and DD relative to μO-MrVIA–expressing control and the correlated phase advance of morning anticipatory locomotor activity support the hypothesis that phase of LN_V PDF secretion rhythms determines phase of morning anticipation.

The results described above show that membrane-tethered δ-ACTX-Hv1a expression in PDF-secreting LN_Vs induces a phase-advance of both sLN_V PDF secretion rhythms and morning anticipatory locomotor behavior in LD, and disrupts free-running locomotor rhythms in DD. In order to determine the relationship between these phenotypes and cellular transcriptional feedback oscillation, par domain protein 1 (PDP1) clock protein levels were assayed in the sLN_V, dorsal lateral neuron (LN_D), dorsal neuron (DN)1, and DN2 neurons of flies expressing membrane-tethered δ-ACTX-Hv1a in the LN_Vs. PDP1 is an oscillating transcription factor that has been implicated in a second interlocking circadian transcriptional feedback loop [8]. Regardless of the validity of the assertion that PDP1 does not participate in the cellular time-keeping mechanism and is solely a clock output factor [53], PDP1 is a high-fidelity phase marker for circadian transcriptional feedback oscillation and has been used as such by multiple laboratories [18,32,54,55]. Flies expressing membrane-tethered μO-MrVIA or δ-ACTX-Hv1a are entrained in LD conditions and then released into DD. Brains are fixed at different circadian times, followed by anti-PDP1 immunofluorescence. Anti-PDP1 immunofluorescence is detected in nuclei of clock neurons, and fluorescence levels are quantified by counting the number of stained nuclei in the various anatomical cell groups. To confirm the reliability of this counting method for assaying cellular transcriptional oscillation, we compared it to the established background-subtracted pixel intensity method [13,18,54]. As shown in Figure S3, these two methods reveal the identical temporal patterns of PDP1 oscillation on DD-D4 in DN1 neurons of flies expressing either membrane-tethered μO-MrVIA or δ-ACTX-Hv1a in LN_Vs. This establishes the reliability of counting PDP1-stained nuclei as a much less labor-intensive method for assessing transcriptional feedback oscillation at the cellular level.

Consistent with previous studies [8,13,18,54], control pdf>μO-MrVIA flies expressing membrane-tethered μO-MrVIA in the LN_Vs exhibit PDP1 oscillation in sLN_Vs with peak levels late at night or subjective night and trough levels during day or subjective day (Figure 9). In LD or on DD-D2, sLN_Vs exhibit peak of PDP1 accumulation at CT18–CT22 with approximately four stained sLN_Vs detected (p < 0.001). PDP1 accumulation in sLN_Vs of control pdf>μO-MrVIA flies peaks at CT18–CT22–CT2 on DD-D4 and at CT22–CT2 on DD-D6 (p < 0.001). This gradually increasing slight phase delay of PDP1 accumulation in control flies free-running in DD is consistent with the free-running phase delays of PDF oscillation in the sLN_V dorsomedial terminals and moderately long period of free-running locomotor rhythms (Figures 2, 6, and 8, Table S1). PDP1 accumulation in the nuclei of sLN_Vs of experimental pdf>δ-ACTX-Hv1a flies expressing membrane-tethered δ-ACTX-Hv1a in the LN_Vs exhibits the same temporal pattern in LD as that of control flies. Thus, membrane-tethered δ-ACTX-Hv1a expression in the LN_Vs has no effect on PDP1 oscillation in sLN_Vs in LD, while inducing phase advance of PDF oscillation in their dorsomedial terminals. This phase shift between sLN_V PDP1 and PDF oscillations in LD induced by LN_V expression of membrane-tethered δ-ACTX-Hv1a was also observed in the independent replicate experiment depicted in Figure S4. The phase advance of morning anticipatory locomotor activity induced by LN_V expression of membrane-tethered δ-ACTX-Hv1a thus is determined by the phase of PDF rhythms and not of PDP1. In DD conditions, experimental membrane-tethered δ-ACTX-Hv1a–expressing flies exhibit similar temporal pattern of PDP1 accumulation in the nuclei of sLN_Vs as control pdf>μO-MrVIA flies on DD-D2 or DD-D4, with a peak at CT18–CT22 (p < 0.001, Figure 9). On DD-D6, PDP1 accumulation in the sLN_Vs of pdf>δ-ACTX-Hv1a flies peaks at CT18 (p < 0.001), slightly advanced compared with control pdf>μO-MrVIA flies. These results provide experimental support for the hypothesis that the phase of sLN_V PDF secretion determines the phase of morning anticipatory locomotor activity, and that PDF transmits phase information from sLN_Vs to downstream PDF-sensitive targets.

We used anti-PDP1 immunofluorescence to assess the effects of phase-advanced PDF cycling in δ-ACTX-Hv1a–expressing PDF-secreting LN_Vs on cellular oscillation of non-LN_V clock neurons that express functional PDF receptor [26]. Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LN_D cell group in both LD and DD conditions, with peak levels late at night or subjective night, and trough levels during day or subjective day (Figure 10; p < 0.001). This constant phase of PDP1 accumulation over 6 d in DD indicates that LN_D cellular oscillation free-runs with approximately 24-h period in control flies. Experimental pdf>δ-ACTX-Hv1a flies exhibit a similar temporal pattern of LN_D PDP1 oscillation to control pdf>μO-MrVIA flies in LD and on DD-D2. However, by DD-D4, PDP1 oscillation in LN_Ds of experimental pdf>δ-ACTX-Hv1a flies has begun to phase shift relative to control, with blunting of peak accumulation late in subjective night (p < 0.001). By DD-D6, the peak of LN_D PDP1 accumulation in flies expressing membrane-tethered δ-ACTX-Hv1a in LN_Vs has phase advanced to CT10. This phase advance indicates that membrane-tethered δ-ACTX-Hv1a expression in the LN_Vs induces an increase in the pace of LN_D cellular oscillation. The approximately 12-h phase advance that accumulates after 6 d in DD is consistent with the approximately 22-h free-running period of the short-period component of the complex locomotor rhythms exhibited by many pdf>δ-ACTX-Hv1a flies.

In control pdf>μO-MrVIA flies, DN1s exhibit a peak of PDP1 accumulation in LD around ZT18 (Figure 11). In DD conditions, peak of PDP1 accumulation in DN1s of control flies broadens, but does not exhibit any phase shift. Experimental pdf>δ-ACTX-Hv1a flies expressing membrane-tethered δ-ACTX-Hv1a in LN_Vs exhibit a similar pattern of PDP1 oscillation as control pdf>μO-MrVIA flies in LD, with peak around ZT18 (p < 0.001). In DD, however, PDP1 oscillation of DN1s in pdf>δ-ACTX-Hv1a flies gradually phase advances with accumulation of an approximately 12-h delay after 6 d in DD, consistent with the approximately 22-h free-running locomotor rhythm component of complex rhythmic pdf>δ-ACTX-Hv1a flies.

The DN2s of control pdf>μO-MrVIA flies exhibit peak PDP1 accumulation at ZT18–ZT22 in LD (Figure 12). The DN2s of control flies exhibit a peak of PDP1 accumulation at CT14 on DD-D2, CT10 on DD-D4, and CT6 on DD-D6. This gradual phase advance of DN2 PDP1 oscillation out of synchrony with the other cell groups after release into DD (Figures 9–12) is consistent with published observations of DN2 cellular oscillation assayed with anti-PER, anti-TIM, or anti-PDP1 immunostaining [18,54,56] and reflects inherent accelerated cellular oscillation in DN2s in DD. Experimental pdf>δ-ACTX-Hv1a flies also exhibit gradual phase advance of PDP1 oscillation after release into DD, peaking around CT0 on DD-D6, but this advance is greater than in control flies, which peak around CT6. Acceleration of non-LN_V clock neurons in free-running DD conditions has also been observed in flies in which LN_V membrane excitability is altered by ectopic ion channel subunit expression [18,54]. Taken together, all these results indicate that altered membrane activity of LN_Vs induced by expression of membrane-tethered δ-ACTX-Hv1a (1) phase advances sLN_V PDF secretion rhythms and morning anticipatory locomotor activity in LD without phase advance of sLN_V PDP1 oscillation and (2) accelerates free-running PDP1 oscillation of LN_D, DN1, and DN2 PDF receptor-expressing clock neurons in DD.

pdf-GAL4 and pdf-gal4;UAS-DsRedII fly lines are as described previously [17,54,63]. pdf-GAL4 was recombined with tubGAL80^ts (from Bloomington Drosophila Stock Center) on the second chromosome. UAS-toxin fly strains are generated via standard P element transgenesis techniques [64]. Membrane-tethered toxin transgenes were chemically synthesized with optimal Drosophila codon usage and an optimal Drosophila Kozak translation initiation sequence [AAA]. Encoded chimeric membrane-tethered toxin sequence is based on the long-linker version of Ibañez-Tallon et al. [34], except Myc epitope tag is substituted for FLAG. pdf-GAL4 or pdf-GAL4,tubGAL80^ts virgins were crossed to UAS males to generate male progeny for behavioral analysis or immunostaining. pdf-GAL4;UAS-DsRedII virgins were crossed with UAS males to generate progeny for electrophysiological recordings. Multiple independent chromosomal insertions of the UAS-toxin transgene were recombined using classical genetic methods to generate second and third chromosomes bearing two independent insertions each for dose-dependent experiments.

Locomotor activity of individual adult male flies (1–4 d posteclosion) was measured at 25 °C using the TriKinetics infrared beam-crossing system. The flies were first monitored for 5∼6 d in 12-h:12-h LD conditions, and free-running locomotor activity was then monitored in constant darkness (DD) for two further weeks. Total infrared beam crosses in 10-min bins were recorded and circadian rhythms were analyzed in 20-min blocks by Lomb-Scargle periodogram using Actimetrics Clocklab software [65]. Significant circadian rhythmicity was defined as presence of a peak in periodogram power that exceeds the p = 0.05 significance line, and the percentages of arrhythmic flies were compared using chi-square analyses. Rhythm strength is defined by the height of the relevant periodogram peak, expressed in arbitrary units. Average numbers of activity events per half-hour bin per fly were also calculated, and histograms for relative activities were generated for LD behavior of the flies among 5 d. In addition, the morning anticipation phase score in LD was computed as relative activity in 3-h period before versus after lights-on transition for individual flies, and these peak phases among different genotypes were statistically analyzed by the ANOVA with Tukey-Kramer multiple comparisons.

δ-ACTX-Hv1a and PLTXII were subcloned into pCS2+ plasmid for Xenopus oocyte expression. The gene constructs for cloning Drosophila voltage-gated Na⁺ channel α subunit (para) and its auxiliary β subunit (tipE), pGH19-13-5 para [47,48] and pGH tipE, are from M. Williamson (Rothamsted Research, Harpenden, United Kingdom). pBSc-ShH37 for Drosophila voltage-gated K⁺ channel, Shaker, is from L. Salkoff (Washington University, St. Louis, Missouri). Xenopus laevis oocytes were prepared, injected, and recorded using standard methods in ND96 solution [66]. Oocytes were injected with less than 50 nl in vitro transcribed cRNAs encoding para/tipE and either δ-ACTX-Hv1a or PLTXII. The full-strength (1:1) of para, tipE, δ-ACTX-Hv1a, and PLTXII cRNAs are 1.66 ng/nl, 1.32 ng/nl, 2.24 ng/nl, and 1.95 ng/nl, respectively. δ-ACTX-Hv1a cRNA was diluted into the various dilutions (1:10, 1:100, 1:1,000, et al.). Then para, tipE, and either δ-ACTX-Hv1a or PLTXII cRNAs were mixed at 1:1:1 ratio of volume and injected into the oocytes. Two days after para/tipE cRNA injection, inward Na⁺ current evoked by 40-ms or 100-ms depolarizing steps from −70 mV to 40 mV with 10-mV increments at the holding potential of −90 mV was recorded using two-electrode voltage clamp. The full strength of 2.89 ng/nl Shaker cRNA was diluted 10-fold and mixed with equal volume of H₂O or 1:1 δ-ACTX-Hv1a cRNA. Three days after Shaker cRNA injection, outward K⁺ current evoked by 400-ms depolarizing steps from the holding potential of −90 mV was recorded.

Whole-cell recordings on lLN_V of fly brain explants are performed as described [49,54]. Briefly, the brains of flies 3–7 d posteclosion are dissected in external recording solution, which consists of (in mM): 101 NaCl, 3 KCl, 1 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, 5 glucose, 20.7 NaHCO₃ (pH 7.2) with osmolarity of 250 mmol/kg. The brain is placed ventral side up and secured in a recording chamber with a mammalian brain-slice “harp” holder and is continuously perfused with external solution bubbled with 95% O₂/5% CO₂ at room temperature. LN_vs are visualized by DsRed fluorescence, and the immediate area surrounding the lLN_Vs is enzymatically digested with focal application of protease XIV (2 mg/ml, Sigma). Whole-cell recordings are performed using borosilicate standard-wall capillary glass pipettes (Sutter Instrument Company), and data are acquired with Axopatch 200B amplifier, Digidata 1200 A/D hardware, and pClamp 8.0 software (Axon Instruments). Recording pipettes are filled with internal solution consisting of (in millimolar): 102 potassium gluconate, 17 NaCl, 0.085 CaCl_2, 4 Mg-ATP, 0.5 Na-GTP, 0.94 EGTA, and 8.5 HEPES (pH 7.2) and osmolarity of 235 mmol/kg. Gigaohm seals are achieved before recording in cell-attached configuration in voltage-clamp mode, followed by break-in to whole-cell configuration. Then current-clamp is employed to monitor RMP and AP firing rate.

Adult fly brains were dissected and processed for anti-PDP1 (1:2,000, rabbit) and anti-PDF (1:50, mouse) immunocytochemistry as described previously [18,54]. PDP1 and PDF were visualized using a Cy3-conjugated anti-rabbit secondary antibody (1:200) and a Cy2-conjugated anti-mouse secondary antibody (1:200). Because the DN2 subgroup of clock neurons is frequently indistinguishable anatomically from the nearby DN1 clock neurons, DN2 staining was scored for each brain hemisphere only as the presence or absence of a distinct pair of neurons ventral to the DN1s, and analyzed by comparing the percentage of brain hemispheres exhibiting such a distinct pair. Anti-PDP1 and anti-PDF immunofluorescent images were collected using a charge-coupled device (CCD) camera mounted on a Zeiss Axiophot epifluorescent photomicroscope and used for analysis. In our previous studies, the average background pixel value for each image was subtracted from the threshold-selected pixels of that image to yield the final threshold-selected background-subtracted images. The integrated pixel values of the threshold-selected background-subtracted images were normalized within each cell group and genotype in LD cycle or DD to the time point with the greatest average integrated pixel value. In this study, a simpler and much less labor-intensive method was used to analyze the PDP1 staining pattern, wherein recognizable nuclei exhibiting anti-PDP1 antibody are counted. We have validated that this counting method reveals identical temporal pattern of PDP1 nucleus accumulation as the previous staining-intensity method (Figure S3). The statistical analysis of anti-PDP1 staining was performed using ANOVA with Tukey-Kramer multiple comparisons.

For dose-dependent expression of toxin δ-ACTX-Hv1a in the LN_Vs and LN_V terminals, adult fly brains were processed with anti-Myc (1:200, mouse) and followed by Cy3-conjugated anti-mouse secondary antibody (1:200). For colocalization of PDF and δ-ACTX-Hv1a expression in the LN_Vs and LN_V terminals, adult fly brains were processed with anti-Myc and anti-PDF (1:2,000, rabbit) accompanied with Cy3-conjugated anti-mouse (1:200) and Cy2-conjugated anti-rabbit (1:200) secondary antibodies. For expression of other toxins, the primary antibodies are the same, and the secondary antibodies are Cy2 (1:200) and Texas-red (1:300)-conjugated antibodies, respectively. Anti-myc (clone 9E10) is from Upstate Cell Signaling Solutions. Cy2-, Cy3-, and Texas-red–conjugated secondary antibodies are from Jackson ImmunoResearch Laboratories.