Circadian Rhythm Regulation by Pacemaker Neuron Chloride Oscillation in Flies

Abstract

Circadian rhythms in physiology and behavior sync organisms to external environmental cycles. Here, circadian oscillation in intracellular chloride in central pacemaker neurons of the fly, Drosophila melanogaster, is reviewed. Intracellular chloride links SLC12 cation-coupled chloride transporter function with kinase signaling and the regulation of inwardly rectifying potassium channels.

Introduction

Circadian rhythms, driven in part by intrinsic clocks, allow organisms to anticipate cyclic changes in their external environment, such as periods of light and darkness, and to sync their physiology and behavior to these rhythms. The molecular mechanisms underlying circadian rhythms have long been of interest. In 1974, Njus et al. (1) proposed that “the biological clock may be a feedback system involving ions and ion transport channels.” This model was eventually supplanted by the transcriptional-translational feedback loop model, in which mRNA and protein oscillations of core clock transcription factors in central pacemaker neurons drive circadian oscillations in physiology and behavior (2, 3). Research in the fly, Drosophila melanogaster, was fundamental in discovering and characterizing the transcriptional-translational feedback loop model (4–6), which is evolutionarily conserved. Nevertheless, ongoing research has continued to implicate ions and ion transporters and channels in the central clock mechanism.

SLC12 Cotransporters and Intracellular Chloride Oscillations in Mammalian Central Pacemaker Neurons

In 1997, Wagner et al. (7) proposed circadian oscillation of intracellular chloride in mammalian central pacemaker neurons in the suprachiasmatic nucleus. γ-Aminobyturic acid (GABA) is a neurotransmitter, and there is extensive GABAergic innervation of the suprachiasmatic nucleus (8). One mechanism of GABA signaling is through the GABA_A receptor, which is a chloride channel. Typically, mature neurons have low intracellular chloride concentrations, so GABA binding to the GABA_A receptor results in chloride influx, hyperpolarization of the membrane potential, and neuronal inhibition. Increased intracellular chloride could result in chloride efflux upon GABAergic signaling, leading to membrane depolarization and neuronal excitation. Thus Wagner et al. (7) proposed that oscillations in intracellular chloride would result in inhibitory effects of GABA at night and excitatory effects during the day (FIGURE 1). This is consistent with observations that central pacemaker neurons also have circadian oscillations in neuronal excitability, with increased excitability and firing during the day, and decreased excitability and firing at night (9–14).

FIGURE 1.

Initial model explaining the role of intracellular chloride in mammalian suprachiasmatic nucleus (SCN) pacemaker neurons Most cells have the sodium-potassium ATPase (Na⁺/K⁺-ATPase), which uses energy from ATP to pump 3 sodium (Na⁺) out of cells and 2 potassium (K⁺) in. As a result, intracellular sodium and extracellular potassium concentrations are low, creating the chemical gradients for chloride entry via cotransport through sodium-potassium-2-chloride cotransporters (NKCC) and for chloride exit via cotransport through potassium-chloride cotransporters (KCC). In conditions of high NKCC activity and/or low KCC activity, intracellular chloride will accumulate in cells, whereas in conditions of low NKCC activity and/or high KCC activity, intracellular chloride concentrations will be low. In 1997, Wagner et al. (7) proposed that high intracellular chloride during the day would result in excitatory effects of GABAergic signaling onto SCN pacemaker neurons, since chloride efflux through the GABA_A receptor ligand-gated chloride channel would result in membrane depolarization. In contrast, low nighttime intracellular chloride concentrations will result in chloride influx through the GABA_A receptor chloride channel, membrane hyperpolarization, and decreased SCN neuron activity.

Intracellular chloride concentrations are variable and established in part by the balance of chloride influx and efflux through members of the SLC12 cation chloride cotransporter family (15). The SLC12 transporters consist of sodium-coupled cotransporters, the sodium-potassium-2-chloride (NKCC) and sodium chloride (NCC) transporters, as well as the potassium chloride cotransporters (KCCs) (16, 17). Mammals have two NKCCs, NKCC1 and NKCC2. NKCC2 is present in the kidney, whereas NKCC1 is widespread (16, 17). Most cells have sodium-potassium ATPases, which use the energy of ATP to pump three sodium ions out of the cell, and two potassium ions into the cells (18). As a result, intracellular sodium is low and intracellular potassium is high, while extracellular sodium is high, and extracellular potassium is low. The electrochemical gradient for ion movement through NKCCs and NCCs is usually inward, due to the inward sodium gradient, and is outward through KCCs, due to the outward potassium gradient. Thus cells in which there is higher NKCC activity than KCC activity will tend to accumulate chloride, and the converse is true in cells with higher KCC activity than NKCC activity (15) (FIGURE 1).

The finding of Wagner et al. in 1997 (7) stimulated further research into the role of NKCC, KCC, intracellular chloride, and GABA in central pacemaker neurons. A number of papers examined inhibitory versus excitatory effects of GABA on suprachiasmatic neurons, with differing results regarding calculated intracellular chloride concentrations and when and where GABA is excitatory vs. inhibitory (19–29). Some papers addressed the question of chloride entry or exit into pacemaker neurons. For example, Wagner et al. (26) used GABA stimulation to load or deplete intracellular chloride in pacemaker neurons in the suprachiasmatic nucleus and then examined removal or replenishment of chloride. They found that during the night, there was no recovery of chloride after chloride depletion, which could be suggestive of decreased NKCC activity at night, leading to lower nighttime intracellular chloride, although they did note neuron-to-neuron heterogeneity (26). Alamilla et al. (25) used perforated patch experiments to measure the equilibrium potential of GABAergic postsynaptic potentials (E_GABA) and calculated intracellular chloride using the Nernst equation. They observed heterogeneity in different parts of the suprachiasmatic nucleus, but in the dorsal portion, they estimated an intracellular chloride concentration of 32 mM during the day and 12 mM at night (25), consistent with the proposal of Wagner et al. (7) of higher intracellular chloride during the day. In those neurons with a more depolarized E_GABA, corresponding to a higher intracellular chloride, the addition of the NKCC inhibitor, bumetanide, hyperpolarized E_GABA, consistent with chloride influx through the NKCC. In contrast, in neurons with hyperpolarized E_GABA, corresponding to a lower intracellular chloride, there was no effect of bumetanide (25). Choi et al. (22) also found heterogeneity in neuronal responses to GABA in the suprachiasmatic nucleus, although their results differed from those described above, in that they saw the most excitatory effects of GABA at night. However, they also implicated NKCC in their studies, in that bumetanide blocked excitatory responses to GABA, and GABA-induced increases in intracellular calcium were decreased both by bumetanide or NKCC1 knockout. They also observed increased nighttime NKCC1 in the dorsal portion of the suprachiasmatic nucleus (22). Bumetanide-sensitive increases in intracellular calcium in response to GABA were also observed by Irwin and Allen (30), although they also noted heterogeneity in different neurons. McNeill et al. (31, 32) also implicated NKCC in circadian rhythms in Syrian hamsters. Thus, although different papers varied in their findings of the effects of GABA on neuronal excitability in the suprachiasmatic nucleus, evidence was accumulating that NKCC1 could play a role in central pacemaker neurons and that intracellular chloride may vary across the day-night cycle.

In 2017, Klett and Allen (33) revisited the question of intracellular chloride concentrations in central pacemaker neurons using a transgenic chloride sensor expressed in two specific subpopulations of suprachiasmatic nucleus neurons, those expressing vasoactive intestinal peptide or arginine vasopressin, comprising ∼22% of the central clock neurons. This study was consistent with the initial study by Wagner et al. (7), demonstrating higher intracellular chloride concentrations during the day, and lower concentrations at night. It also implicated NKCC in chloride entry, and KCC in chloride exit, using pharmacological approaches with bumetanide and the KCC inhibitor, VU0240551, although a subsequent paper by the same group did not show an effect of bumetanide on intracellular chloride (34). Interestingly, GABA application consistently resulted in chloride influx during both day and night, suggesting hyperpolarizing (inhibitory) effects of GABA across the day-night cycle (33).

The Role of Intracellular Chloride in Drosophila Pacemaker Neurons

Drosophila melanogaster has played a central role in the elucidation of the molecular mechanisms underlying circadian timekeeping (4–6). A common practice in Drosophila research is “collecting virgins.” Females do not mate with males for the first 8 hours or so after eclosing from the pupal case as adults. Therefore, collecting females during this timeframe allows them to be mated with males of a different genotype to generate progeny with a desired genotype. There is a circadian rhythm of adult eclosion (4), with a peak rate of eclosion in the morning hours. However, flies carrying a hypomorphic/null mutation in the gene Ncc69 (35, 36), which encodes the fly NKCC (35, 37) tended to eclose late in the day, as noted while collecting virgins (A. Rodan, unpublished observations). This suggested a possible circadian rhythm defect. Indeed, in conditions of constant darkness, in which the free-running clock is operative, Ncc69 mutants had a prolonged circadian period, as measured by locomotor activity rhythms (38). Interestingly, Nkcc1 knockout mice also have prolonged locomotor activity periods (39). The prolonged period phenotype of Ncc69 Drosophila mutants was rescued by the expression of wild-type Ncc69 in a specific subset of central pacemaker neurons, the small ventrolateral neurons (sLN_vs). In conditions of 12 hours of light and 12 hours of darkness, flies anticipate morning with an increase in locomotor activity before lights on; this “morning anticipation” was abolished in Ncc69 mutants (38).

As discussed above, one role of the NKCC is to allow the influx of intracellular chloride. The investigators therefore examined intracellular chloride concentrations using the transgenic sensor, ClopHensor (40, 41), in sLN_v neurons at different points of the day-night cycle. A first set of studies examining intracellular chloride every 4 hours suggested that chloride increases over the morning hours. Therefore, paired studies were performed measuring intracellular chloride at zeitgeber time (ZT)2 (2 hours after lights on) and ZT6 (6 hours after lights on). Chloride binding to ClopHensor is pH sensitive, but there were no differences in pH (which can also be measured using ClopHensor) at these time points. However, intracellular chloride increased significantly between ZT2 and ZT6 in controls (38), mirroring results in the mammalian suprachiasmatic nucleus showing a nadir in intracellular chloride at ZT0 and an increase over the day with a peak at ZT8 (34). The increase in intracellular chloride between ZT2 and ZT6 did not occur in Ncc69 mutants, resulting in significantly lower intracellular chloride at ZT6 in the Ncc69 mutants compared to controls (38). The long-period phenotype of Ncc69 mutants was abolished by a heterozygous loss-of-function mutation of kcc [encoding the Drosophila KCC (42)] or by kcc knockdown in the sLN_vs, implicating the perturbation in intracellular chloride in the circadian rhythm phenotype (38).

What might be the effect of this morning increase in intracellular chloride? Like mammals, Drosophila have GABA_A chloride channel receptors, of which the major subunit is encoded by the gene Rdl. However, Rdl expression is weak (or nonexistent) in sLN_v neurons, and knockdown of Rdl in the sLN_vs lengthened period by only 20 minutes in constant darkness (43), as opposed to period length increases of up to 3 hours in Ncc69 mutants (38), suggesting that the long-period phenotype of Ncc69 mutants is not due to differences in GABAergic signaling. Drosophila also have additional classes of ligand (neutrotransmitter)-gated chloride channels, including glutamatergic chloride channels, glycine receptors, and histamine-gated chloride channels. However, the knockdown of the glutamatergic chloride channels in sLN_vs had no effect on period length in constant darkness (44), and histaminergic chloride channels are not expressed in sLN_vs (45). A role for glycine signaling from sLN_vs to other classes of Drosophila pacemaker neurons, the DN1_p and LN_d neurons, has been demonstrated, but glycinergic signaling on to the sLN_vs themselves has not been demonstrated, although it was not directly examined (46). However, existing evidence suggests that intracellular chloride in sLN_v neurons may not regulate neuronal excitability via effects on neurotransmitter-gated chloride channels.

Studies in the 1990s showed that decreases in intracellular chloride increased NKCC phosphorylation and activity, suggesting a signaling role for intracellular chloride via kinase regulation (47–49). Piala et al. (50) demonstrated that chloride directly binds to the active site of with no lysine (K) (WNK) kinases, inhibiting autophosphorylation and activation. This is an evolutionarily conserved mechanism of WNK regulation, as demonstrated by studies of Drosophila WNK (51). Therefore, Schellinger et al. (38) hypothesized that the failure to increase intracellular chloride in sLN_v neurons over the course of the morning in Ncc69 mutants could lead to unrestrained WNK signaling to cause a prolonged circadian period. Indeed, the long-period phenotype of Ncc69 mutants was suppressed by knockdown of WNK or its downstream substrate, Fray. Conversely, expression of chloride-insensitive mutants of Drosophila WNK or human WNK3, which is expressed in the mammalian suprachiasmatic nucleus pacemaker neurons (52), was sufficient to increase the circadian period (38). Importantly, overexpression of wild-type Drosophila WNK or human WNK3 was not sufficient to increase the circadian period, indicating the importance of chloride regulation of WNK at the heart of the signaling pathway.

WNKs phosphorylate the paralogous mammalian Ste20 kinases Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress response (OSR1) and their Drosophila homolog, Fray (53), on a conserved T-loop threonine to activate them (54–56). Mutation of this threonine in Fray, T206, to a phosphomimetic glutamate leads to WNK-independent activity (57). Expression of Fray^T206E in the sLN_vs increased period length, similar to the phenotype of chloride-insensitive WNK expression (38). Furthermore, period lengthening was dependent on Fray kinase activity, as sLN_v expression of kinase-dead Fray^D185A,T206E (57) had no phenotype.

The next question was: what is the target of Fray? SPAK/OSR1/Fray kinases bind to targets via an RFxV/I or RxFxV/I motif (58). Examination of known SPAK/OSR1/Fray binding partners in Drosophila (e.g., Ncc69, KCC, and WNK) identified an expanded motif, which was used to perform a proteome-wide search (59) for additional putative binding partners. Of these, the inwardly rectifying potassium channel, Irk1 (also known as Ir) stood out. Prior studies had shown that Irk1 knockdown in the sLN_vs affects the circadian period (60), Irk1 is enriched in the LN_vs and both its mRNA and protein exhibit circadian cycling (61, 62), and OSR1 regulates mammalian inwardly rectifying potassium channels (63). Schellinger et al. (38) demonstrated that Fray^T206E activates Irk1 channel activity in heterologous Drosophila cultured cells in an RFxV-dependent manner. This suggests a model in which unrestrained WNK-Fray signaling in the Ncc69 mutant sLN_vs results in excess Irk1 activity (FIGURE 2). This was tested by expressing Fray^T206E in sLN_v neurons in which endogenous Irk1 was knocked down and replaced with either wild-type Irk1 or Irk1 in which the RFxV motif was mutated, Irk1^V306A. Fray^T206E expression lengthened the circadian period with wild-type Irk1 coexpression, but not Irk1^V306A, indicating that the period-lengthening effect of WNK-Fray pathway activation depends on wild-type Irk1.

FIGURE 2.

Increased intracellular chloride inhibits WNK-Fray activation of the Irk1 potassium channel to regulate circadian period A: at zeitgeber time (ZT)2, intracellular chloride in the Drosophila sLN_v (small ventrolateral neurons) central pacemaker neurons is low due to low activity of the NKCC encoded by Ncc69. Chloride inhibits with no lysine (K) (WNK) kinases. In low chloride conditions, WNK will be active and phosphorylate and activate the downstream kinase, Fray. Fray in turn activates the inwardly rectifying potassium channel Irk1. B: at ZT6, chloride entry via the NKCC encoded by Ncc69 over the course of the morning restrains WNK-Fray signaling and Irk1 activity. Ncc69 mutants remain “stuck” with low intracellular chloride, as seen normally at ZT2. C and D: disruption of this pathway results in disruptions of circadian locomotor rhythms in both light/dark conditions (C) and in constant darkness (D). C: locomotor activity of controls vs. Ncc69^r2 mutants, showing loss of morning anticipation (the anticipatory increase in locomotor activity before lights on, arrow). D: representative actograms showing average activity across 7 days of subjective day (gray) and night (dark) in constant darkness, in which the free-running clock is operative. Ncc69^r2 mutants have a prolonged circadian period. E: resting membrane potential was measured using whole cell recordings of the sLN_v pacemaker neurons and analyzed using linear regression analysis. Resting membrane potential becomes more hyperpolarized from ZT0 (lights-on) to ZT6, is variable between ZT6 and ZT18, and becomes depolarized between ZT18 and ZT24/ZT0. Changes in Irk1 activity due to changes in WNK-Fray signaling could affect resting membrane potential and neuronal excitability. For example, in the presence of unrestrained WNK-Fray signaling, as would occur with loss of the Ncc69-encoded NKCC, Irk1 would remain activated at ZT6, which could keep the resting membrane potential in a hyperpolarized state and prevent subsequent depolarization before morning. A–D are adapted or reproduced from Ref. 38, with permission from Elsevier. E is reproduced from Ref. 66 with permission from Journal of Neuroscience.

Circadian Oscillations in Pacemaker Neuron Excitability

What role might Irk1 be playing in circadian period regulation? As initially suggested by the model of Njus et al. (1), there are intrinsic, clock-controlled circadian rhythms to central pacemaker neurons’ electrical activity and excitability, such as rhythms in the resting membrane potential, in both Drosophila and mammals (9–14, 64–67). In general, there is increased electrical activity or excitability during the day, and decreased activity at night. In flies, manipulations that alter LN_v excitability, such as loss or increased expression of potassium or sodium channels, disrupt circadian rhythms (60, 68–74). Potassium channel activity is a major determinant of resting membrane potentials. Smith et al. (75) demonstrated circadian cycling of the Shaw and Shal voltage-gated potassium channels in LN_v neurons, and expression of dominant-negative Shaw slightly increased circadian period in conditions of constant darkness, although not to the degree seen in Ncc69 mutants. Inwardly rectifying potassium channels also contribute to the resting membrane potential (76). In Drosophila, the resting membrane potential in sLN_v neurons becomes increasingly hyperpolarized between ZT0 and ZT6, from −40 mV at ZT0 to −60 mV at ZT6. Between ZT6 and ZT18, resting membrane potential is more variable, with some neurons depolarized to −35 to −40 mV, and others remaining more hyperpolarized. Between ZT18 and ZT0, resting membrane potential becomes more consistently depolarized (FIGURE 2E) (66). Thus one possibility is that increasing intracellular chloride in sLN_vs at ZT6 inhibits WNK-Fray signaling and Irk1 channel activity, contributing to the loss of hyperpolarization between ZT6 and ZT18, whereas, in Ncc69 mutants, low intracellular chloride leads to WNK-Fray-Irk1 activation and maintains the pacemaker neurons in a more hyperpolarized state. Measurement of resting membrane potential in sLN_vs would directly answer this question but is technically difficult (66).

Roles of Other SLC12 Cotransporters in Drosophila Circadian Rhythms

In insects, there are two clades of genes related to the human SLC12 sodium-dependent cation chloride cotransporters, the NKCC clade and the CCC2/3 clade. The CCC2/3 clade groups phylogenetically with the NKCCs, but the genes it contains do not have direct mammalian orthologs (77). Ncc69 encodes a bona fide NKCC, with sodium- and chloride-dependent transport of the potassium congener, rubidium, similar kinetics to human NKCC1 (eg K_m for sodium, rubidium and chloride), and inhibition by bumetanide (37, 78). Aedes aegypti mosquito CCC2 (AeCCC2) and its Drosophila homolog, which has been called Ncc83 or NKCC, have quite different transport properties, with a chloride-independent electrogenic sodium/lithium conductance, no rubidium uptake, and no inhibition by the prototypical NKCC inhibitors bumetanide or furosemide, or the NCC inhibitor hydrochlorothiazide (37, 79, 80). Interestingly, Ncc83/NKCC has been implicated in circadian rhythms as well. Overexpression of Ncc83/NKCC depolarizes and increases the firing rate in the large LN_v neurons (lLN_vs), which is typical of the daytime state of these neurons, whereas Ncc83/NKCC knockdown resulted in hyperpolarization and decreased firing rate, similar to the nighttime state (81). Unlike sLN_v neurons, lLN_v neurons express GABA_A receptor chloride channels and are receptive to GABA (43, 82). Changes in the GABA reversal potential were consistent with increased intracellular chloride in the Ncc83/NKCC-overexpressing neurons and decreased intracellular chloride in the Ncc83/NKCC knockdown lLN_v neurons, also resembling the day and nighttime states of wild-type neurons (81). These findings could be consistent with the subsequent discovery of a cationic conductance through these transporters. Dysregulation of Ncc83/NKCC in all clock neurons resulted in phenotypes under constant light: while wild-type flies are arrhythmic under these conditions, Ncc83/NKCC-overexpressing or knockdown flies were rhythmic (81). Unlike Ncc69 mutants, there was no effect on behavior in constant darkness, indicating distinct roles for these two transporters.

Open Questions

An open question is how Ncc69-encoded NKCC activity may increase over the course of the morning. One of the best-understood mechanisms for NKCC activation is via phosphorylation on conserved NH₂-terminal serines and threonines by SPAK/OSR1/Fray kinases (57, 83–85). However, sLN_v knockdown of Drosophila WNK and Fray did not recapitulate the long-period phenotype of Ncc69 mutants, suggesting that there is some other mechanism modulating Ncc69 NKCC activity in sLN_vs. Interestingly, the Ncc83/NKCC-encoded transporter also contains the canonical Fray-binding motif, RFxI (80), and knockdown of Drosophila WNK or Fray throughout all of the clock neurons recapitulates the phenotype of knockdown of Ncc83/NKCC (and kcc) in the same neurons, i.e., rhythmicity in constant light conditions (86), pointing to differences in the circadian regulation of the Ncc69-encoded NKCC compared to the Ncc83/NKCC transporter.

In principle, Ncc69 regulation could occur at the level of transcription, translation, posttranslational modifications, trafficking, degradation, or some combination of these. This requires further study, although there is some evidence for translational Ncc69 cycling (62). The light input factor Quasimodo regulates the Ncc83/NKCC transporter (81), and the NCA localization factor-1 chaperone regulates firing frequency and resting membrane potential in DN1_p clock neurons via trafficking of the narrow abdomen sodium leak conductance in flies or its mammalian homolog, NALCN, in the suprachiasmatic nucleus (67). Whether similar mechanisms exist to regulate the Ncc69 NKCC remains to be determined. A related question is how and whether Ncc69 expression, localization, or activity and intracellular chloride are influenced by the core molecular clock, and vice versa (FIGURE 3A). This could be determined by examining intracellular chloride in sLN_vs from flies carrying mutations in core clock genes, such as period or timeless, and, conversely, examining transcriptional, translational, or localization rhythms of PERIOD and TIMELESS in Ncc69 mutants.

FIGURE 3.

How do circadian oscillations in intracellular chloride in Drosophila and mammalian central pacemaker neurons interface with the molecular clock? A: simplified schematics of Drosophila and mammalian transcriptional-translational feedback loops are shown. In Drosophila, heterodimeric CLOCK (CLK)-CYCLE (CYC) activates transcription of period (per) and timeless (tim). The protein products PER and TIM then feedback repress CLK-CYC. A similar loop is operative in mammals. BMAL1 is the ortholog of CYC and CRY (CRYPTOCHROME) replaces TIM. Not shown are the multiple paralogs of mammalian PER and CRY, as well as additional transcriptional-translational feedback loops and other regulatory mechanisms that are operative in both systems. The connections between the core clock and intracellular chloride oscillations are currently unknown. B and C: long-term imaging of chloride every 5 or 10 minutes over 4 days was performed in cultured explants of the suprachiasmatic nucleus in arginine vasopressin-expressing (B) and vasoactive intestinal peptide-expressing neurons (C), using the ratiometric chloride sensor Cl-Sensor. Data were detrended in the Lumicycle Analysis Program (Actimetrics) using the 24 hour rolling average baseline subtraction. Lights on is at zeitgeber time (ZT)24, ZT48, ZT72, and ZT96, with a higher ratio indicating higher intracellular chloride concentrations. Thus intracellular chloride rises during the day and falls at night. B and C are reproduced from Ref. 34, with permission from Nathan J. Klett, Olga Cravetchi, and Charles N. Allen.

In initial studies, the greatest difference between control and Ncc69 mutants in sLN_v intracellular chloride was observed during morning hours (38). These experiments were performed in light-dark conditions and could have been confounded by exposure to light during dissection and imaging, particularly for brains analyzed during nighttime points. This problem was bypassed in a study of clock neuron calcium rhythms by using flies carrying null mutations in cryptochrome, which encodes the Drosophila photosensitive protein that links external light to circadian rhythms (87). This approach could also be used to measure sLN_v chloride in conditions of constant darkness, in which behavioral phenotypes are observed (38). Liang et al. (87, 88) have used light-sheet microscopy to image clock neuron calcium oscillations in vivo at a high sampling frequency, up to 5 Hz. Adopting this technique to image intracellular chloride could lead to a better understanding of chloride oscillations. Klett et al. (34) also achieved long-term imaging of intracellular chloride in subsets of suprachiasmatic nucleus neurons ex vivo, imaging every 5–10 minutes and providing the most convincing data to date for oscillations in intracellular chloride in mammalian central pacemaker neurons (FIGURE 3, B AND C).

Another question is whether and how the findings in Drosophila sLN_v neurons relate to the mammalian central clock. As mentioned above, Nkcc1 knockout mice have a prolonged circadian locomotor period, similar to Ncc69 mutants (39). Whether this is due to changes in pacemaker neuron chloride oscillations or WNK signaling is unknown. Of interest, large-conductance Ca²⁺-activated potassium channels have been implicated in the circadian rhythms of mammalian central pacemaker neuronal excitability and have also been demonstrated to be regulated by mammalian WNKs in other contexts (10, 89–94). Thus the link between intracellular chloride, WNK signaling, and pacemaker neuron membrane ion conductance properties and excitability could exist in mammals, although this requires further study.

Morioka et al. (95) showed that in pupal LN_v neurons, there are circadian oscillations in cytosolic pH. This was not observed in adult sLN_v neurons (38). However, it does raise the intriguing question of whether other types of chloride transporters, such as the SLC4 or SLC26 chloride/bicarbonate exchangers present in both flies and mammals (96–98), may also play a role in cellular chloride oscillations.

Conclusions

There is increasing interest in circadian ion oscillations in both central pacemaker neurons and peripheral cells (39, 87, 88, 95, 99–109). Pracucci et al. (110) examined NKCC1- and KCC2-dependent circadian oscillations in intracellular chloride in cortical pyramidal neurons in the occipital cortex. SLC12 cotransporter rhythms have also been studied in other peripheral cells (39, 99). Intracellular chloride has also been of interest in clock seasonality (86, 111–113) and sleep pressure (114). Understanding of the role of chloride as a signaling ion, particularly in the context of WNK regulation, continues to advance (115–117). Thus, ongoing research is sure to continue to illuminate the complex and fascinating interlocking of the circadian clock and intracellular ions.