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REPORT
Department of Biology and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts
Submitted 2 February 2006; accepted in final form 15 March 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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C, which has been shown to disrupt circadian locomotor rhythms, hyperpolarizes these neurons, and blocks firing. These data imply that the firing properties of large PDF neurons are both regulated by and critical for clock function. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). These groups form a network of interconnected oscillators that drive a variety of biological rhythms (Grima et al. 2004; Lin et al. 2004; Peng et al. 2003; Stoleru et al. 2004). One of these subsets, the ventral group of lateral neurons (LNvs), has been shown to be important for both the maintenance of rhythms in constant conditions (Lin et al. 2004; Peng et al. 2003) and for generation of the morning peak of locomotor activity (Grima et al. 2004; Stoleru et al. 2004). LNvs express genes that are known to be components of the molecular clock, such as per and tim (Helfrich-Forster 1995; Kaneko and Hall 2000). The LNvs also contain, and presumably release, PDF, a neuropeptide that is homologous to crustacean pigment-dispersing hormone (Helfrich-Forster and Homberg 1993), which may function to synchronize multiple subgroups of clock neurons (Lin et al. 2004; Nitabach et al. 2006). Mutation of the pdf gene causes circadian defects that are similar to those produced by ablation of the LNvs, i.e., a loss of rhythmicity in constant light conditions (Renn et al. 1999).
The electrical activity of clock cells appears to be critical to their function. Oscillations in membrane potential underlie rhythms in the molluscan eye (McMahon and Block 1987) and in mammalian suprachiasmatic nucleus, changes in membrane properties lead to circadian alterations in firing rates (de Jeu et al. 1998; Pennartz et al. 2002). In Drosophila, the ion channel mutant slowpoke has been shown to have weak circadian rhythms (Ceriani et al. 2002) and expression of open rectifier potassium channels in the LNvs disrupts locomotor rhythms (Nitabach et al. 2002). In this study we demonstrate that spiking of large LNvs is blocked by expression of a leak potassium channel, dORK-C, and that the light-dependent depolarization of the LNv resting potential is also blocked by this channel. These results support the idea that the modulation of LNv firing is critical for its function in the clock.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The pdf-GAL4 line (Park and Hall 1998) was a gift of J. Levine and J. Hall (Brandeis University, Waltham, MA). UAS-dORK–NC and UAS-dORK-C lines (Nitabach et al. 2002) were a gift of M. Nitabach (Yale University, New Haven, CT) and T. Holmes (New York University, New York). To generate experimental animals, UAS-mCD8-GFP; Pin/CyO and pdf-GAL4 were crossed to generate a homozygous stock of UAS-mCD8-GFP; pdf-GAL4. The homozygous line was crossed to the UAS-dORK-C lines to generate heterozygous UAS-mCD8-GFP/+; pdf-GAL4/UAS-dORK–NC; and UAS-mCD8-GFP/+; pdf-GAL4/+; UAS-dORK–C/+ animals. Animals were kept in a vial in a 25°C incubator with 12 h:12 h light:dark cycle until the day of experiment. For dark–dark (DD) experiments, UAS-mCD8-GFP/+; pdf-GAL4/+ animals were raised in light–dark (LD) to synchronize their clocks then transferred to darkness. Animals for experiments were taken from vials during the first 24 h of DD. The published effects of dORK-C on circadian rhythms were verified in our GFP-expressing genotypes by assessment of locomotor activity (J. Levine, unpublished results).
Brain dissection
Female flies aged 6 to 8 days, were anesthetized with CO2 and pinned ventral side up through thorax on a Sylgard-coated recording chamber. The chamber was filled with the external recording solution (in mM: 124 NaCl, 40 KCl, 5 Trehalose, 5 HEPES, 4 MgCl2, 2 CaCl2, 4 NaHCO3, 1 NaH2PO4, 35 sucrose, pH 7.3, with osmolarity of 290 mmol/kg). The head cuticles, eyes, proboscis, and trachea were carefully removed with fine forceps, exposing the brain. The detached brain was then placed ventral side up on a coverslip coated with poly-D-lysine (0.5 mg/ml, Sigma–Aldrich, St. Louis, MO) in the recording chamber.
Dissection was done under light from a dissecting microscope, and patching required fluorescence, so there is some concern that during this short time light might activate cryptochromes, which are cell-autonomous light sensors for the clock (Emery et al. 2000). To minimize this possibility for "dark" cells, microscope and room lights were turned off after patching. In addition, we note that the effect of dissection in light on maintaining the clock has been tested, and the dark state of molecular components of the clock is stable during dissection (Kaneko et al. 1997), although the possibility of the electrical properties of the cell being more sensitive than the clock mechanism cannot be ruled out.
Patch clamping
Ventral lateral neurons (LNvs) were visualized and identified under an Olympus upright microscope with GFP fluorescence. The immediate area surrounding the LNvs was enzymatically digested (protease XIV, 2 mg/ml, Sigma) and mechanically disrupted as previously described for the larval brain (Choi et al. 2004). External recording solution bubbled with 95% O2-5% CO2 was continuously perfused over the preparation.
A whole cell giga seal was formed using a filamented thick-walled capillary pipette (WPI, Sarasota, FL) which was fire polished to a resistance of 10–20 MOhm and contained internal solution (in mM: 120 potassium gluconate, 20 KCl, 10 HEPES, 1.1 EGTA, 2 MgCl2, and 0.1 CaCl2, pH 7.2 and osmolarity of 280 mmol/kg). Resting membrane potential and action potentials were recorded under current clamp. Action potentials were evoked by injecting small amounts of current by the recording pipette.
For anatomical studies, the recording pipette was backfilled first with a fluorescent dye mixture of Alexafluora 568 (10 mg/ml) and tetramethylrhodamine dextran 3,000 MW (10 mg/ml, Molecular Probes, Eugene, OR) before filling it with the internal solution. Diffusion of the dye mixture was observed immediately after rupture of cell membrane under the patch pipette. A small hyperpolarizing current was injected for 30 min to 2 h to facilitate filling. Dye-filled brains were fixed in 4% paraformaldehyde for 30 min at room temperature. Fixed brains were washed three times with the external saline followed by a 10-min incubation each with 30, 70, and 100% glycerol. Brains were mounted on slides with Vectashield (Vector Laboratories, Burlingame, CA) and confocal images were acquired using a Leica microscope system.
Data analysis
Data acquisition was carried out using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and ITC-16 data acquisition board (National Instruments, Austin, TX) with Igor software (Wavemetrics, Lake Oswego, OR). Voltage traces were analyzed with Igor and Excel (Microsoft, Redmond, WA) software. Statistical analysis was done with JMP (SAS Institute, Cary, NC). P values were obtained with ANOVA and post hoc analysis unless otherwise indicated.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Dye filling allowed us to investigate the morphology of individual LNvs for the first time in wild-type adult Drosophila. Two distinct types of LNvs were previously described: the large LNvs and the small LNvs. Each side of the brain has four to five of each type and they are believed to have different projection patterns (Helfrich-Forster and Homberg 1993). Small LNvs are believed to be the morning oscillator that is responsible for the anticipation of lights-on (Grima et al. 2004). Large LNvs have been postulated to be important for keeping the independent oscillators in the two hemispheres in synch (Helfrich 1986; Helfrich-Forster and Homberg 1993) by direct connections between the LNv clusters. Figure 2 shows representative dye fills of a large LNv (Fig. 2A) and a small LNv (Fig. 2B). Large LNvs have both ipsilateral and contralateral optic lobe projections in addition to a branch that goes ventrally to the accessory medulla. This branch has a "smooth" profile without the varicosities associated with the distal medullary branches. Small LNvs do not project to optic lobes, but instead have a major ventral projection that reverses to run dorsally from the accessory medulla, eventually reaching the dorsal part of the brain. We have focused on the large LNv class for further analysis.
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In the medullae, the complexity of ipsilateral and contralateral branches in the medulla from an individual LNv differed. Ipsilateral branches had a higher complexity than that of contralateral branches (2.3 ± 0.2 vs. 1.8 ± 0.5 branches), indicating that the majority of LNv contacts in a given optic lobe are from the ipsilateral group of neurons. Previous immunohistochemical studies suggested the opposite (Helfrich-Forster and Homberg 1993), in analogy to the arborization pattern of similar neurons in larger flies (Strausfeld 1976).
We also analyzed the initial branching structure of the large LNvs (Fig. 2C) and found it to be highly stereotyped. Like many invertebrate neurons, large LNvs are unipolar, having a single ventrally projecting process. This process bifurcates, sending a ventral process to the accessory medulla. The other branch splits to run in the mediolateral axis to innervate the ipsilateral medulla and the contralateral medulla (by the POT). This pattern was seen in eight of eight fills where the initial branching pattern could be determined.
Electrophysiological characterization of large LNvs and the effect of dORK-C
The function of LNvs in generation and maintenance of circadian rhythms was investigated using a number of genetic manipulations, including expression of the open rectifier potassium channel dORK-C (Nitabach et al. 2002). Expression of this channel protein, which in Xenopus oocytes produces a voltage-insensitive leak current and reduces resting membrane potential, suggested that the excitability of LNvs might be key to their function in the clock. No recordings of these neurons in situ had been done to determine what effects expression of these channels actually had on cell properties. We have used whole cell patch clamp to investigate the basic electrophysiological properties of large LNvs and the effect of expression of dORK-C on cell function. To assess the effects of circadian rhythms on LNv function, animals were kept in a 12 h:12 h light/dark (LD) cycle and the time at which recordings were made was noted. Lights-on occurred at ZT 0 and lights-off at ZT 12. Data for cells recorded in the 4 h after light transitions was analyzed to determine the effect of light. The control genotypes UAS-mCD8/+; pdf-GAL4/+ and UAS-mCD8-GFP/+; pdf-GAL4/+; UAS-dORK-NC/+ (dORK-NC is a nonconducting dORK channel; Nitabach et al. 2002) were statistically indistinguishable when compared between ZT 0–4 and ZT 12–16, so were pooled and constitute the "Control" group in LD conditions in Table 1 and Fig. 3E. The DD "Control" genotype consisted entirely of UAS-mCD8/+; pdf-GAL4/+ animals. All dORK-C animals also expressed the mCD8-GFP gene to allow visualization of the LNvs.
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C/+ large LNv neurons. Injection of increasing amounts of current elicited action potentials in all control neurons, but not in most (9/14) dORK-C neurons (Table 1). In cases where current injection did induce spikes after lights on, as shown in Fig. 3C, the amount of current required for spiking was elevated, with the LD control genotypes requiring significantly less current than dORK-C flies (P < 0.05, Table 1). Neurons expressing dORK-C had hyperpolarized membrane potentials compared with control neurons in LD (P < 0.05). This was true regardless of the time in the light:dark cycle at which the recordings were made. Input resistance appeared to be decreased in dORK-C neurons, but the effect was not statistically significant. The threshold for spiking was not significantly altered by dORK-C (P > 0.05), suggesting that expression of this channel is not altering sodium channel function in any gross way. Spontaneous activity was seen in only a small number of recordings, most likely as a result of the low amount of calcium and high magnesium levels present in our saline. Effects of light on the properties of large LNv neurons
Large LNvs have been postulated to have a role in the synchronization of brain hemispheres and in the maintenance of rhythms under constant conditions. To determine whether there were light- or clock-driven changes in the properties of LNvs, we measured resting membrane potential, input resistance, spiking, and threshold in control UAS-mCD8/+; pdf-GAL4/+ neurons from animals that were maintained in constant darkness (DD). Recordings were made from animals at the beginning of the subjective day (CT 0–4) and the beginning of the subjective night (CT 12–16). The properties of LNvs in animals maintained in DD showed no significant differences from LD animals at ZT 12–16, which is right after lights-off for LD animals. Interestingly, resting membrane potential was significantly more hyperpolarized in DD animals than in LD animals right after lights-on (Fig. 3E, P < 0.05). This correlated with a significant (P < 0.05) increase in the amount of current required for spiking in DD neurons. The lack of significant differences between LD and DD animals after lights-off implies that resting membrane potential is not changed by the light-to-dark transition, but is specifically sensitive to the dark-to-light transition. Expression of dORK-C in LD appears to phenocopy the effects of darkness at ZT 0–4 (Fig. 3E, P > 0.05 for the comparison of UAS-mCD8/+; pdf-GAL4/+ in DD to UAS-mCD8-GFP/+; pdf-GAL4/+; UAS-dORK-C/+ in LD). These data suggest that the resting membrane potential in LNvs is controlled both by the clock and by light and that these modulations of resting membrane potential may be important for clock function.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. C. Griffith, Dept. of Biology, MS008, Brandeis University, 415 South St., Waltham, MA 02454-9110 (E-mail griffith{at}brandeis.edu)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Choi JC, Park D, and Griffith LC. Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system. J Neurophysiol 91: 2353–2365, 2004.
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