Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.

Using patch-clamp analysis of the Drosophila DN1p, we show for the first time that circadian clock control of membrane excitability operates via resting sodium leak conductance through the narrow abdomen (NA) channel, providing timed depolarizing drive to circadian pacemaker neurons. We demonstrate that the sodium leak rhythm depends on rhythmic expression of NCA localization factor 1, linking the molecular clock and membrane excitability. We reveal that both flies and mice, separated by hundreds of millions of years in evolution, utilize antiphase oscillations of sodium and potassium conductances to drive clock control of membrane potential. Thus, the conservation of clock mechanisms between invertebrates and vertebrates extends from core timing mechanisms to the control of membrane excitability in the master clock neurons governing sleep and wake.

While molecular clocks are expressed in a variety of cell types, those in specific circadian clock neurons in the brain exhibit special properties. These so-called “master” circadian pacemakers, such as the mammalian suprachiasmatic nucleus (SCN) and the Drosophila lateral and dorsal neurons, drive robust 24 hr rhythms of sleep and wake behavior (). Unlike generic clock cells, these clock neurons are interconnected via neural networks and, as a result, produce coherent and sustained free running molecular and behavioral rhythmicity under constant conditions (). Although the anatomical features of brain pacemaker networks are highly divergent between mammals and invertebrates such as Drosophila, their ability to control sleep and wake cycles uniformly depends on daily rhythms of membrane excitability (). However, the mechanistic links between the molecular clock and the machinery controlling cellular excitability are not well understood.

Rhythmic regulation of membrane potential and potassium current persists in SCN neurons in the absence of environmental input.

Sequential nuclear accumulation of the clock proteins period and timeless in the pacemaker neurons of Drosophila melanogaster.

Circadian clocks have evolved to align organismal biochemistry, physiology, and behavior to daily environmental oscillations. At the core of these clocks in all multicellular organisms are conserved transcriptional feedback loops (). In Drosophila, the bHLH-PAS transcription factor heterodimer CLOCK (CLK) and CYCLE (CYC) directly binds E boxes (CACGTG) in target promoters of the clock genes, period (per) and timeless (tim), and activates their transcription. PER and TIM proteins feed back to repress CLK/CYC activity. The temporal separation of transcriptional activation and repression and/or mRNA and protein oscillations, in some cases by many hours (), results in robust daily oscillations of per, tim, and other rhythmic transcripts. These molecular clocks, in turn, control a broad range of cellular and physiological responses likely via the rhythmic transcription of clock output genes.

To determine the impact of a day-night change in I(∼0.7 pA.pF) on firing frequency and membrane potential, we simulated sodium leak current modulation using an updated version of a mathematical model of SCN membrane excitability () (see Experimental Procedures ). This model accurately captures the effect of NALCN blockers on the membrane potential of SCN neurons ( Figure 7 H). According to this model, modest daily changes in sodium leak conductance comparable to those observed experimentally can have sizable effects on neuronal firing rates ( Figure 7 I). To explore the contributions of both sodium and potassium leak currents to the daily variation of firing rate in SCN neurons, we simulated concurrent modulation of these two conductances. Beginning from a subjective day firing rate of 7 Hz, reducing sodium leak conductance by the amount suggested by our experimental measurements (∼0.7 pA.pF Figure 7 G) decreases firing rate to 2 Hz. Experimentally, we observed that, during the subjective day, Iis, in fact, positively correlated with firing frequency ( Figure S6 J), suggesting that Isignificantly impacts neuronal physiology. Even lower firing rates that are characteristic of subjective night (0.5 Hz) can be achieved by increasing potassium leak conductance in conjunction with this reduction in sodium leak ( Figures 7 J and S7 ). Thus, elevated sodium leak during the day and elevated potassium leak at night can recapitulate the experimentally observed daily variations in SCN firing rate through relatively modest changes in these leak currents.

To confirm the molecular identity of the sodium leak in the SCN, we generated a forebrain-specific knockout of NALCN with a CamkIIα-cre driver. With this driver, CRE expression mimics the endogenous expression of CamkIIα (enriched in the forebrain, neuron-specific []). CamkIIα expression is also highly enriched in the SCN, and loss of circadian rhythms in mice with a CamkIIα-specific knockout of a core clock gene (Bmal1) is observed (). We first confirmed that the NMDG-evoked hyperpolarization ( Figure 7 A) is greatly reduced in the CamkIIα−Cre;NALCNanimals compared to age-matched sibling controls ( Figures 7 C and 7D, p = 0.005). Consistent with a role of NALCN in controlling the membrane potential and firing frequency of neurons, CamkIIα−Cre;NALCNSCN neurons were hyperpolarized and silent ( Figures S6 E and S6F, p < 0.007). Importantly, a small depolarization current injected into the CamkIIα−Cre;NALCNSCN neurons was able to evoke strong firing ( Figure S7 G), indicating that cells were healthy but required a greater depolarizing input to evoke action potentials. We further confirmed the presence of NALCN current during the interspike interval in organotypic slices containing the SCN with pharmacology: NMDG and Gdsensitive. No additional block by Gdwas observed after NMDG application ( Figures S6 H and S6I). This NMDG-sensitive inward current was greatly reduced in the CamkIIα-Cre;NALCNSCN neurons ( Figures 7 E and 7F, p = 0.002). Taken together, these data indicate that the vast majority of the sodium leak flowing during the interspike interval in SCN neurons is carried by NALCN (I), consistent with other mammalian neurons (). We then assessed Iat different times of day and found that it was significantly larger during the subjective day than subjective night, consistent with a control by the circadian clock ( Figure 7 G, p < 0.001).

Voltage traces from our simulations are shown with (A) g K-leak = 0.04 and g NALCN = 0.22 nS, and (B) g K-leak = 0.06 and g NALCN = 0.123 nS.

Although we demonstrated a rhythmic function for resting sodium leak in Drosophila clock neurons, rhythmic resting sodium conductances have yet to be described in mammalian clock neurons. Previous patch-clamp analyses of dissociated SCN neurons demonstrated the presence of a NALCN-like current (TTX-resistant, NMDG-sensitive, voltage-independent sodium conductance termed I) that is largely responsible for the initial phase of the depolarizing drive during the interspike interval (). To determine whether this activity is rhythmic in mammalian circadian pacemaker neurons, we performed voltage-clamp analysis during subjective day and night from organotypic slices containing the SCN from mice entrained for 2 weeks in LD and then maintained under constant darkness conditions for at least 3 weeks. Rhythms in firing frequency, membrane potential, and input resistance were observed, thus validating the preparation ( Figures S6 A–S6C). In the presence of TTX to block action potentials, the NALCN blockers, NMDG ( Figure 7 A) or Gd Figure S6 D), induce a hyperpolarization, while no additional effect of applying Gdafter sodium replacement with NMDG was observed ( Figure 7 B). Importantly, in hippocampal neurons, the vast majority of current with this pharmacological profile is mediated by NALCN ().

(J) Firing rate as a function of g NALCN and g K leak in a model SCN neuron. Arrows: decreasing g NALCN alone reduces firing rate from 7 Hz to 2 Hz, whereas increasing g K leak reduces firing rate from 7 Hz to 6 Hz. Concurrently decreasing g NALCN and increasing g K leak reduces firing rate from 7 Hz to 0.5 Hz. Results are expressed as mean ± SEM.

(I) The model predicts the magnitude of change in firing rate as a function of magnitude of change in NALCN current density (g NALCN = 0.12 to 0.22 nS). A decrease of 0.74 pA.pF −1 in I NALCN (observed between the subjective day and night [G]) leads to a 5 Hz decrease in firing rate.

(H) Simulations showing the role of TTX-resistant sodium leak in setting the membrane potential using a mathematical model of SCN membrane excitability. Voltage traces from control simulation (g Na = 229 nS, g NALCN = 0.22 nS) and simulated application of TTX (g Na = 0 nS) and NMDG (g NALCN = 0 nS).

(G) Circadian variation of I NALCN : 1.6 ± 0.1 pA.pF −1 , n = 25 during the subjective day (gray columns) and 0.8 ± 0.1 pA.pF −1 , n = 23 during the subjective night (black columns). Asterisks indicate statistical significance (t test, p < 0.001). Green dots represent individual cells.

(F) I NALCN was reduced in CamkIIa-Cre;NALCN fx/fx compared to sibling controls animals (0.5 ± 0.1 pA.pF −1 , n = 6 in CamkIIa-Cre;NALCN fx/fx [red triangle] and 1.4 ± 0.2 pA.pF −1 , n = 5 in sibling controls [black triangle]). Asterisks indicate statistical significance (t test, p = 0.002).

(E) Action potential clamp recordings showing the sodium leak flowing during the interspike interval in SCN neurons from sibling control (left) and CamkIIa-Cre;NALCN fx/fx animals (right). In the presence of TTX and K blockers (blue trace), the sodium leak current flowing during the interspike interval (I NALCN ) was reduced after sodium substitution with NMDG (green trace). The sodium leak current (I NALCN = subtracted = purple trace) was revealed by subtracting the inward current in the presence of NMDG from the inward current present with TTX and K blockers.

(D) Quantification and statistical analysis of the NMDG-evoked hyperpolarization is shown: −15.9 ± 2.0 mV, n = 9 in controls (black triangle) and −4.5 ± 1.7 mV, n = 4 (red triangle). Asterisks indicate statistical significance (t test, p = 0.005).

(B) NMDG hyperpolarizes the cell with no additional effect in the presence of Gd 3+ .

(A) Representative current-clamp recording showing the role of the TTX-resistant sodium leak (difference between green and blue) in setting the membrane potential of mammalian SCN neurons.

Circadian variations of firing frequency (A), membrane potential (B) and input resistance (C) were detected (during the subjective day: 4.9 ± 1.4 Hz, −63 ± 0.9mV, 1.6 ± 0.1GΩ (n = 16), and during the subjective night: 0.8 ± 0.4Hz, −69.3 ± 1.1mV, 1.1 ± 0.1GΩ (n = 11), respectively). Green dots represent individual cells. The sodium leak observed in the SCN neurons shares identical properties to NALCN: blocking the sodium leak with Gd 3+ hyperpolarizes the neurons and decreases firing rate (D). Membrane potential (E) and firing frequency (F) of CamkIIa-Cre;NALCN fx/fx (red triangle) versus sibling controls (black triangle) (0Hz, −81.2 ± 5.3mV in CamkIIa-Cre;NALCN fx/fx , (n = 6) and 2.9 ± 0.9Hz, −63.5 ± 0.9mV in sibling controls (n = 7), respectively). (G) High firing frequency can be restored in CamkIIa-Cre;NALCN fx/fx neurons by injecting a depolarizing current (+25pA). (H) Action potential clamp recordings showing the sodium leak flowing during the interspike interval in SCN neurons. Top panel shows recorded action potentials used as a voltage command to measure currents flowing during the interspike interval (bottom panel). Blue trace represent currents recorded in the presence of TTX and K blockers. The sodium leak current flowing during the interspike interval was revealed after sodium substitution with NMDG (green trace). Subtracted currents (purple trace) were calculated by subtracting the currents recorded with NMDG to the currents recorded without NMDG. The pharmacological profile of this sodium leak is shown in (I) (TTX-resistant in blue, NMDG and Gd 3+ sensitive (in green). Asterisks indicate statistical significance (t test, p < 0.05). (J) During the subjective day, I NALCN is positively correlated to firing frequency and (linear regression: r 2 = 0.68, p = 0.019); individual cells are shown in green dots. Results are expressed as mean ± SEM.

If the oscillation of Nlf-1 transcript is critical to setting NA levels and DN1p membrane excitability, we would predict that Nlf-1 overexpression would increase NA current at evening time points when NA current is typically at trough levels. We observed that, in the evening (ZT8–12) NLF-1 overexpression depolarizes membrane potential, elevates firing rates ( Figure 6 F) and cellular excitability ( Figure 6 G and Table S2 F), and, most importantly, increases NA current ( Figure 6 H) at a time when each of those parameters is near their daily trough in wild-type flies. Indeed, sodium leak current density in the evening in Nlf-1 overexpression flies (∼2pA.pF) is comparable to that seen at peak levels in wild-type flies in the morning. Taken together, these results indicate that Nlf-1 expression is rhythmic and mediates NA activity rhythms. This demonstrates a molecular mechanism linking the core clock to membrane excitability via the rhythmic transcription of a factor important for ion channel function in Drosophila circadian neurons ( Figure 6 I).

To further examine the mechanism by which NLF-1 might regulate NA, we assayed NA protein expression after Nlf-1 knockdown. Nlf-1 knockdown with a broad neuronal driver (elav-G4) also results in strong reductions in rhythmic strength in DD and reduced morning and evening anticipation ( Figure S5 A and Tables S3 and S4 ). Surprisingly, NA protein levels were dramatically reduced in these flies ( Figure S5 B). We also observed lower NA expression (∼50% reduction) when na was driven transgenically in the DN1p of Nlf-1 knockdown flies ( Figure S5 C). In part due to the small soma and limited expression in projections, we could not reliably assess cell membrane or axonal localization. Yet Nlf-1 knockdown does not reduce DN1p na transcript levels ( Figure S5 D). Nlf-1 knockdown in the DN1p phenocopies a na mutant, suggesting that NA current is nearly abolished ( Figure 6 E) yet transgenic NA is reduced by just ∼50%. Thus, we favor the view that strong effects of Nlf-1 knockdown on NA current are only in part due to changes in NA levels.

(A) Morning and evening anticipation are reduced in Nlf-1 knockdown flies (CTRL: elav-G4;; CTRL RNAi#1/+ versus Nlf-1 RNAi expressing flies (Nlf-1 KD: elav-G4;; Nlf-1 RNAi#1/+). (B) Western blot analyses show reduction in NA expression in Nlf-1 RNAi expressing flies (Nlf-1 KD: elav-G4;; Nlf-1 RNAi#1) (2 left lanes) versus control flies (CTRL: elav-G4;; CTRL RNAi#1/+) and na RNAi expressing flies (na KD: elav-G4; na RNAi:U-Dcr2/+) (2 right lanes) versus control flies (CTRL: elav-G4; CTRL RNAi#2/+). Quantitation of total levels is shown (n = 2). (C) Anti-HA immunostaining of DN1ps in CTRL (top: U-CD8-GFP/U-na HA ; Clk4.1M-G4/+) and in Nlf-1 RNAi expressing flies (bottom: U-CD8-GFP/U-na HA ; Clk4.1M-G4/ Nlf-1 RNAi#1). Quantification of total NA HA levels is shown (p = 0.0032). From FACS sorted DN1p neurons, na mRNA levels were indistinguishable in Nlf-1 RNAi expressing flies U-CD8-GFP/+; Clk4.1M-G4/ Nlf-1 RNAi#1) versus controls (U-CD8-GFP/+; Clk4.1M-G4/ CTRL RNAi#1, n = 2) (D). Results are expressed as mean ± SEM.

We then tested whether Nlf-1 is important for NA current levels, which may reflect the proper channel localization to the cell membrane. Knockdown of Nlf-1 expression was confirmed in the DN1p with quantitative PCR ( Figure 6 B). We find that knockdown in the DN1p results in a similar phenotype to that observed for na mutants with cells becoming hyperpolarized and silent ( Figure 6 C). Cellular excitability is also decreased in the Nlf-1 knockdown, as the neurons are less responsive to depolarizing currents ( Figure 6 D and Table S2 E). NA-dependent current was also strongly suppressed after Nlf-1 knockdown ( Figure 6 E).

The role of Nlf-1 extends to PDF neurons. Restricting Nlf-1 knockdown to PDF neurons, using two different pdf-GAL4 drivers (pdf-GAL4 and pdf0.5-GAL4 []), dramatically reduces free running rhythms ( Table S3 ), consistent with the highly enriched Nlf-1 transcript observed in larval PDF+ sLNv neurons () and with the described role of NA in PDF neurons (). In addition, we extended our patch-clamp analysis to the large LNv neurons ( Figure S4 A). Here, we observed clock-dependent rhythms in membrane properties as previously observed ( Figures S4 B and S4C) (). In addition, we found clock-dependent NA current rhythms similar to those we observed for the DN1p, with peak levels in the morning ( Figure S4 D). Thus, our findings in DN1p extend to other circadian neurons.

(A) Schematic and image of the Drosophila brain indicating the location of the l-LNvs and other clock neurons. Representative images of the GFP-expressing l-LNvs in the intact Drosophila brain are shown below. The l-LNvs were labeled by using the Pdf-G4 driving the expression of U-CD8-GFP. Whole-cell access to GFP labeled neurons was confirmed following diffusion of Alexa Fluor 594 biocytin included in intracellular recording solution. Histograms showing rhythms in wild-type (WT) and the decrease and lack of rhythms in firing frequency (B), membrane potential (C) and sodium leak current density (D) in na har (red) and per 01 (blue) when compared to WT (black) l-LNvs neurons (respectively for WT: 1.5 ± 0.3Hz, −57 ± 1.1mV, 0.6 ± 0.1pA.pF -1 n = 18 at ZT0-4 and 0.7 ± 0.2Hz, -55.6 ± 1.1mV, 0.3 ± 0.0pA.pF -1 , n = 13 at ZT8-12; for na har : 0.1 ± 0.0Hz, −66.2 ± 2.0mV, 0.3 ± 0.0pA.pF -1 n = 11 at ZT0-4 and 0.4 ± 0.2Hz, -59.5 ± 2.4mV, 0.4 ± 0.0pA.pF -1 , n = 11 at ZT8-12; for per 01 : 0.4 ± 0.2Hz, −55.4 ± 1.3mV, 0.3 ± 0.0pA.pF -1 n = 11 at ZT0-4 and 0.6 ± 0.2Hz, -55.2 ± 1.2mV, 0.30 ± 0.0pA.pF -1 , n = 12 at ZT8-12). Results are expressed as mean ± SEM. Asterisks indicate statistical significance (p < 0.05) from t test.

The Cellular Excitability and NA Current of the Drosophila l-LNvs Circadian Pacemaker Neurons Are Clock Controlled, Related to Figure 6

To assess the function of NLF-1 in circadian behavior, we knocked down its transcript levels using three independent transgenic dsRNA and shRNA lines in combination with the broad circadian driver tim-GAL4. In contrast to the previously reported weak effect of CG33988 RNAi knockdown on evening behavior (), we found dramatic reductions in rhythmic strength in DD (3/3 lines) and reduced anticipation of lights-on (2/3 lines) and lights-off transitions (3/3 lines) under LD conditions ( Figures 6 A and S3 ). These effects are comparable to those observed in loss-of-function na alleles () and knockdown of na using RNAi ( Figure S3 and Tables S3 and S4 ). Restricting Nlf-1 knockdown to non-PDF clock neurons (tim-GAL4, pdf-GAL80) also caused reduced morning and evening anticipation, as well as reduced rhythmicity ( Tables S3 and S4 ), consistent with prior na rescue studies (). Further restricting Nlf-1 knockdown to the DN1p using Clk4.1M-GAL4 resulted in reduced DD rhythmicity ( Table S3 ).

(A) Nlf-1 RNAi #2 (from VDRC), (B) Nlf-1 RNAi #3 (from NIG, Japan), (C) na RNAi (from VDRC) lines were crossed to the broad circadian driver tim-G4. The Nlf-1 or na knockdown flies (tim-G4/Nlf-1 RNAi #2;U-Dcr2/+ in (A), tim-G4/+;U-Dcr2/Nlf-1 RNAi #3 in (B) and tim-G4/na RNAi;U-Dcr2/+ in (C) were compared to their appropriate genetic controls (respectively Nlf-1 RNAi#2/+ and tim-G4/CTRL RNAi #2;U-Dcr2/+ in (A), Nlf-1 RNAi #3/+ and tim-G4/+;U-Dcr2/ CTRL RNAi #3 in (B), na RNAi/+ and tim-G4/+;U-Dcr2/+ in (C). Morning Index (black) and Evening Index (blue) are measures of morning/evening anticipation, respectively. Asterisks indicate differences statistically significant in comparison to controls (ANOVA, p < 0.02).

(H and I) (H) Sodium leak current density is also increased in the Nlf-1 V5 -overexpressing neurons (red) versus control neurons (black) (1 ± 0.05 pA.pF −1 , n = 4 in Nlf-1 CT and 1.9 ± 0.1 pA.pF −1 , n = 5 in Nlf-1 OX, measured at ZT8–12, p < 0.05). Results are expressed as mean ± SEM. Asterisks indicate statistical significance (p < 0.05 from a t test). A summary cartoon depicting the conserved bicycle model for controlling membrane excitability of circadian pacemaker neurons is shown in (I). In the morning/day, the molecular clock drives high NLF-1 activity, increasing the sodium leak activity, and K conductances are reduced, thus increasing cellular excitability. In the evening/night, the sodium leak is decreased, and, in parallel, K conductances are high, thus silencing the neurons. This dual regulation of the conductances responsible for the membrane properties is critical for driving high-amplitude rhythmic oscillations of cellular excitability.

(F) Representative current-clamp recordings at ZT10 showing that the Nlf-1 overexpressing DN1p neurons (red) are depolarized and more active compared to control DN1p neurons (black).

(E) Sodium leak current density is dramatically reduced in the Nlf-1 knockdown neurons (red) versus control neurons (black) (1.9 ± 0.7 pA.pF −1 , n = 4 in Nlf-1 CT and 0.6 ± 0.2 pA.pF −1 , n = 5 in Nlf-1 KD, measured at ZT0-4, p < 0.05).

(C) Representative current-clamp recordings at ZT2 showing that the Nlf-1 knockdown DN1p neurons (red) are hyperpolarized and silent compared to control DN1p neurons (black).

To identify molecular links between core clocks and membrane excitability, we employed fluorescence-activated cell sorting of GFP-labeled DN1p and performed RNA-Seq at distinct times during the LD cycle. Using empirical JTK_CYCLE (), an updated version of JTK_CYCLE (), to detect rhythmic transcripts at a false discovery rate of 5% (Benjamini-Hochberg adjusted, p < 0.05), we observed robust 24 hr rhythms in CG33988, the fly ortholog of the NCA localization factor 1 (NLF-1), but not in na itself, its regulatory subunits unc79 and unc80 (), nor the NALCN activators such as Src family kinases (), Src42a, and Src64b in flies ( Figure 5 A). NLF-1 has been previously shown to interact with NA orthologs in worms (NCA-1 and -2) and mammals (). NLF-1 protein is expressed in the endoplasmic reticulum and is required for the proper axonal localization of NCA-1 and -2 (). Rhythmic expression of CG33988/Nlf-1 transcript was further confirmed with quantitative PCR ( Figure 5 B), and consistent with clock control, CG33988/Nlf-1 transcript is rhythmic in the DN1p in constant darkness (DD) ( Figure 5 C). Nlf-1 transcript is also highly enriched in the DN1p clock neurons in comparison to whole heads ( Figure 5 D). Chromatin immunoprecipitation experiments indicate that the core clock transcription factor CLOCK rhythmically binds the Nlf-1 genomic locus, suggesting a direct biochemical link to the core clock (). Taken together, this suggests that Nlf-1 is a key mediator of NA rhythms that couples the transcriptional oscillator to membrane potential rhythms.

(A–D) (A) Nlf-1 mRNA shows rhythmic expression using RNA-seq data from FACS-sorted DN1p neurons in LD (for isoform RB, shown in graph, BH corrected p = 0.005). na, Unc79, Unc80, Src64B, and Src42a are not robustly cycling (graph shows isoforms with highest expression: BH = 0.28 for na-RF, 0.2 for Dunc79-RE, 0.85 for Dunc80-RE, 0.71 for Src42a-RA, and 0.07 for Src64B-RJ). Nlf-1 cycles under LD (B) and during the first day of constant darkness (DD1) conditions (C) in DN1ps using qPCR. Based on two independent experiments, an asterisk indicates differences statistically significant one-way ANOVA, Tukey’s post hoc test, LD ZT0 versus ZT12 p = 0.0011, ZT0 versus ZT16, p = 0.000142, ZT4 versus ZT16 p = 0.029, ZT0 versus ZT8 p = 0.022, ZT12 versus ZT20, p = 0.000441, ZT16 versus ZT20 p = 0.000136. DD1 CT0 versus CT8 p = 0.01081, CT0 versus CT12 p = 0.000142, CT12 versus CT16 p = 0.000145 and CT12 versus CT20 p = 0.000459. (D) Nlf-1 expression is enriched in the DN1ps versus whole head (t test, p < 0.02). Results are expressed as mean ± SEM.

UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

We next directly measured voltage-clamped NA-dependent current (I) at different times of day. A voltage ramp protocol (from –113 mV to +87 mV) was used to measure the inward current at −113 mV, in the presence of TTX. Replacing the sodium from the extracellular solution with NMDG reveals the sodium leak current ( Figure 4 A). Consistent with the sodium leak current being driven specifically by NA, the observed current is reduced in the namutant neurons and can be restored by rescuing the expression of NA in the mutant ( Figure 4 A). Measuring Iat different times of day reveals a diurnal modulation of current density: it is higher in the morning and lower in the evening ( Figures 4 B and 4C and Table S1 D). No rhythm is detected in the na Figures 4 B and 4C and Table S1 D) or in permutants ( Figure 4 D, p = 0.21), the latter indicating core clock control. Further, the rhythm in NA conductance was evident even after Clk4.1M-GAL4-driven rescue ( Figure 4 E). Given GAL4 stability, any promoter-driven transcriptional rhythms may not be evident as GAL4 protein rhythms, and thus GAL4-induced transcription of na may not be rhythmic (), suggesting that NA current rhythms do not require na transcript rhythms. Taken together, these results indicate that the clock control of sodium leak current through NA mediates rhythms of resting membrane potential.

(E) Histograms showing the sodium leak current in WT (black), na har (red), and na har ;; U-na/Clk4.1M-G4 (blue) DN1p neurons at different times of day (ZT0–4 versus ZT8–12) (for na har ;; U-na/Clk4.1M-G4, I NA = 2.3 ± 0.3 pA.pF −1 , n = 4 at ZT0–4 and 1.1 ± 0.1 pA.pF −1 , n = 4 at ZT8–12). Results are expressed as mean ± SEM. Asterisks indicate statistical significance (p < 0.05) from a t test.

(D) Histograms showing the NA current in WT (black) and per 01 (red) DN1ps recorded at different times of day ZT0–4 versus ZT8–12 (for per 01 , I NA = 0.7 ± 0.2 pA.pF −1 , n = 8 at ZT0–4 and 0.5 ± 0.1 pA.pF −1 , n = 7 at ZT8–12). Asterisks indicate statistical difference between WT and per 01 , p < 0.05 from t test.

(C) Quantification and statistical analysis are shown. Gray areas represent the dark phase of the LD cycle. Red asterisks indicate statistical significance between WT and na har neurons, and black asterisks indicate statistical significance between different time points in WT neurons (p < 0.05) from a one-way ANOVA, Tukey’s post hoc test.

(B) All recorded WT neurons (black dots) and na har neurons (red dots) are plotted against time of day for sodium leak current (I NA ).

A candidate mediator of resting sodium conductances in clock neurons and circadian behavior is the NARROW ABDOMEN (NA) ion channel (). NALCN, the closely conserved mammalian homolog of NA, has been characterized as a voltage-independent mixed cation channel important for setting RMP and mediating resting leak sodium current (). This current is not blocked by TTX but can be reduced by either Gdor replacement of extracellular sodium with NMDG (). In a 12 hr LD cycle, increases in locomotor activity in advance of lights-on (i.e., morning anticipation) and lights-off (i.e., evening anticipation) are suppressed in namutants (). Although NA expression in the DN1p can rescue morning and, to a lesser extent, evening phenotypes (), it remains unclear whether NA is a rhythmic mediator of resting membrane potential of circadian clock neurons. We therefore examined clock neuron excitability in na mutant DN1p neurons. Strikingly, namutant DN1p neurons were completely silent ( Figures 3 A and 3B ) and remained hyperpolarized throughout the whole day ( Figure 3 C and Table S1 B). No daily rhythm in cellular excitability was detected in na Figure 3 D and Table S2 C; p > 0.35). Positive current injections show that namutant neurons fire fewer action potentials compared to controls, indicating that these neurons are healthy and can still generate action potentials but require more depolarizing current to fire at the same rate as WT neurons ( Figures 3 D and 3E). Wild-type membrane excitability can be restored by inducing NA expression only in the DN1p in the mutant, confirming that these effects are due to na and are likely cell autonomous ( Figure 3 E and Table S2 D). NMDG substitution induces an increase in the input resistance, indicating that NA is open at rest ( Figure 3 F).

(F) Histograms showing that sodium substitution with NMDG induces an increase in the input resistance, indicating that NA is open at rest (black and green columns are before and after NMDG substitution, respectively). Results are expressed as mean ± SEM. Asterisks indicate statistical significance (t test, p < 0.05).

(E) The decrease in cellular excitability can be restored by rescuing the expression of NA only in the DN1p in the mutant: WT (black), na har (red) and na har ;; U-na/Clk4.1M-G4 (blue) DN1p neurons.

(A–C) (A) Representative current-clamp recordings at ZT2 showing that the naDN1p neurons (red) are hyperpolarized and silent compared to WT DN1p neurons (black). Statistical analysis comparing the firing frequency (B) and membrane potential (C) of the WT (black) and na(red) DN1p neurons. Red asterisks indicate statistical significance between WT and naneurons (p < 0.05, from a one-way ANOVA, Tukey’s post hoc test). (Data for WT neurons are also depicted in Figures 1 B and 1C).

To identify ionic conductances responsible for the resting membrane potential (RMP) rhythm, we blocked action potential firing using the voltage-dependent sodium channel blocker tetrodotoxin (TTX, 10 μM) and then applied a cocktail of potassium (K) channel inhibitors (10 mM TEA, 5 mM 4AP, and 2 mM CsCl) to block both voltage-dependent and voltage-independent (leak) K conductances (). We subsequently used N-methyl-D-glucamine (NMDG) substitution of extracellular sodium to block sodium leak currents () at different times of day. As in mammals () and mollusks (), the effect of blocking K leak conductances in Drosophila was dependent on time of day, producing little change in the morning ( Figures 2 A, 2B, and 2D ) but a sizable depolarization in the evening ( Figure 2 C and 2D), indicating that rhythmic resting K conductance is conserved between flies and mammals (). In contrast to K blockade, we discovered that blockade of resting sodium leak produced a larger hyperpolarization in the morning ( Figures 2 A and 2B–2E) than in the evening ( Figures 2 C–2E). Such time-of-day-dependent effects of sodium channel blockade have not been previously reported. Notably, this time-of-day-dependent effect on membrane potential of sodium blockade (Δ∼7 mV morning versus evening) is roughly equal to that of potassium blockade, suggesting that each makes a comparable contribution to daily excitability rhythms. As these rhythms are observed during network silencing from TTX, this suggests that changes in RMP are not driven by synaptic inputs but are intrinsic to the cells. Taken together, our results demonstrate that time-of-day-dependent sodium and K conductances, in the morning and evening, respectively, may underlie RMP rhythms.

(D and E) (D) Averaged changes of the membrane potential by K blockers (10 mM TEA, 5 mM 4-AP, and 2 mM CsCl): −1.2 ± 1.4 mV, n = 5 between ZT0–4 and 7.1 ± 1 mV, n = 5 between ZT8–12 and (E) sodium replacement with NMDG: −17.2 ± 0.8 mV, n = 5 between ZT0–4 and −12.6 ± 1.2 mV, n = 5 between ZT8–12. Results are expressed as mean ± SEM. Asterisks indicate statistical significance (p < 0.05) from t test.

(A–C) (A) Representative current-clamp recording at ZT2 showing the effect of K and sodium conductance blockers on membrane potential. Bars indicate when drugs were applied (blue, TTX 10 μM; red, TEA 10 mM, 4-AP 5 mM, CsCl 2 mM; green, NMDG to replace the sodium from the extracellular solution). The effect of K blockers and sodium replacement on the membrane potential at different times of day are shown in (B) for ZT2 and (C) for ZT10.

Rhythmic regulation of membrane potential and potassium current persists in SCN neurons in the absence of environmental input.

Using whole-cell patch-clamp analysis, a large daily variation in the firing frequency was detected ( Figure 1 B, p < 0.05, and Figure S1 A). The wild-type (WT) DN1ps fire at ∼10 Hz in the morning (Zeitgeber Time, ZT0–4) and are nearly silent in the evening (ZT8–12) ( Table S1 A). The firing frequency in cell-attached configuration was comparable to that observed in whole-cell mode ( Figures S1 B–S1D), suggesting that dialysis did not alter measurements of firing rates. The membrane potential also exhibited a temporal pattern: more depolarized in the morning than in the evening ( Figure 1 C, p < 0.05, and Table S1 B). The neurons show daily rhythmic cellular excitability: more responsive to depolarizing currents in the morning than in the evening ( Figure S1 E and Table S2 A). The input resistance had no significant diurnal rhythm ( Figure S1 F and Table S1 C). The rhythms in firing frequency and membrane potential were not evident in the arrhythmic core clock mutant per, indicating that the canonical clock controls daily changes in intrinsic membrane properties. Compared to WT, the perneurons are hyperpolarized ( Figure 1 D) and show no rhythm in firing frequency ( Figure 1 E, p = 0.41), membrane potential ( Figure 1 F, p = 0.66), or cellular excitability ( Figure S2 A, p > 0.41). The perneurons also require more depolarizing current to fire at the same rates as WT ( Figure S2 B and Table S2 B). Importantly, the high-amplitude daily rhythms in firing frequency observed in WT neurons exceed those previously described in another set of Drosophila circadian neurons (LNvs) and more closely approximate those described in mammalian SCN clock neurons (), indicating that DN1p analysis will be useful to define the mechanisms for clock control of membrane excitability. Given the role of the DN1p in morning and evening behaviors (), these activity measurements suggest that DN1p activity in the morning can drive locomotor activity, while the relative silence of the DN1p in the evening may have a permissive role on other cells controlling evening behavior.

Depolarizing current injections confirms the lack of rhythms in cellular excitability in the per 01 neurons (light red: ZT0, dark red ZT12, p > 0.41) (A) and the decrease in cellular excitability in the per 01 neurons (red) versus wild-type (black) (B). Results are expressed as mean ± SEM. Asterisks indicate statistical significance (t test, p < 0.05).

(A) Four representative whole-cell current-clamp recordings from four different DN1ps obtained at different times of day (from top to bottom: ZT2, 6, 12, 18) and (B) two representative cell-attached current-clamp recordings from 2 different DN1ps obtained at ZT0 and 12 show rhythms in firing frequency. (C) Recording from same cells in both cell-attached mode and whole-cell mode shows similar firing frequency (linear regression: r 2 = 0.86, p = 0.00157, n = 9). (D) Cell-attached recordings confirm rhythms of firing frequency (8.3 ± 1 Hz, n = 5 at ZT0-4 and 2.6 ± 0.9 Hz, n = 5 at ZT8-12, p = 0.003). (E) In whole-cell configuration, depolarizing current injections in wild-type also shows rhythms in cellular excitability. f-I curves of WT measured at ZT0-4 versus ZT8-12 (p < 0.001) (respectively gray and black). (F) All recorded WT neurons (green dots) are plotted against time of day for input resistance. Results are expressed as mean ± SEM.

To elucidate the mechanistic basis of daily changes in membrane excitability in Drosophila clock neurons, we performed whole-cell patch-clamp electrophysiology on the posterior dorsal neurons 1 (DN1p) on explanted brains (). DN1p neurons harbor molecular circadian clocks, and under 12 hr light-12 hr dark (LD) conditions, they contribute to increases in locomotor activity in advance of lights-on (i.e., morning anticipation) and lights-off (i.e., evening anticipation) (). In addition to their established function in circadian behavior, the DN1p are an attractive target for patch-clamp analysis, as we can selectively label and identify DN1p neurons using the Clk4.1M-GAL4 driver in combination with UAS-CD8-GFP () ( Figure 1 A). Furthermore, the DN1p neurons are easily accessible by electrode, as they are located near the brain surface ().

(D–F) (D) Representative current-clamp recordings at Zeitgeber Time 2 (ZT2) showing that the per 01 DN1p neurons (red) are hyperpolarized and silent compared to WT DN1p neurons (black). Histogram showing the decrease in firing frequency (E) and membrane potential (F) and lack of daily rhythm in per 01 (red, 2.2 ± 1.1Hz, −56 ± 2 mV, n = 15 at ZT0–4 and 3.9 ± 1.5Hz, −55 ± 1.9 mV, n = 10 at ZT8–12, p > 0.41) when compared to WT (black) DN1p neurons. Results are expressed as mean ± SEM. Asterisks indicate statistical significance (p < 0.05) from t test performed in WT at ZT0–4 versus ZT8–12.

(A–C) (A) Schematic and image of the Drosophila brain indicating the location of the DN1ps and other clock neurons. Representative images of the GFP-expressing DN1ps in the intact Drosophila brain are shown below. The DN1ps were labeled by using the Clk4.1M-G4 driving the expression of U-CD8-GFP. Whole-cell access to GFP-labeled neurons was confirmed following diffusion of Alexa Fluor 594 biocytin included in intracellular recording solution. All recorded WT neurons are plotted against time of day (in 4 hr bins) to show daily rhythms of firing frequency (B) and membrane potential (C). Gray areas represent the dark phase of the LD cycle. Asterisks indicate statistical significance (p < 0.05) from a one-way ANOVA, Tukey’s post hoc test.

Discussion

Taken together, our work defines a conserved mechanism for the maintenance of circadian oscillations necessary for robust daily behaviors ( Figure 6 I) that we term the “bicycle” model. Membrane oscillations are driven by two cycles with opposite temporal phases analogous to cycling bicycle pedals. During the morning/day, sodium leak mediated by NA/NALCN is elevated while resting K currents are reduced, depolarizing the neuron to promote elevated firing rates. During the evening/night, sodium leak is low and resting K currents are elevated, hyperpolarizing the cell to suppress firing rates. The clock-controlled transcript Nlf-1 drives the rhythm of NA/NALCN current, linking the core clock to ion channel activity.

Cao and Nitabach, 2008 Cao G.

Nitabach M.N. Circadian control of membrane excitability in Drosophila melanogaster lateral ventral clock neurons. Fogle et al., 2011 Fogle K.J.

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Holmes T.C. CRYPTOCHROME is a blue-light sensor that regulates neuronal firing rate. Fogle et al., 2015 Fogle K.J.

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Holmes T.C. Circadian- and light-dependent regulation of resting membrane potential and spontaneous action potential firing of Drosophila circadian pacemaker neurons. While Drosophila has been a well-established model for defining molecular genetic mechanisms, relatively little is known about the specific ionic currents that underlie fly pacemaker neuron excitability rhythms due to the small size of Drosophila soma. Most cellular electrophysiological analyses have focused on the largest cells, the large ventral lateral neurons (). Yet, even in these neurons, the specific ionic currents under clock control have yet to be defined. Using whole-cell, patch-clamp electrophysiology of DN1p pacemaker neurons, we found high-amplitude oscillations of spontaneous firing rates and basal membrane potential that are comparable to those observed in mammalian SCN clock neurons. Moreover, we demonstrate clock control of both resting sodium leak conductance as well as resting potassium conductance. Our data suggest that the patch-clamp analysis of the DN1p will be valuable in defining the ionic currents that mediate clock control of neuronal excitability.

Kuhlman and McMahon, 2004 Kuhlman S.J.

McMahon D.G. Rhythmic regulation of membrane potential and potassium current persists in SCN neurons in the absence of environmental input. Michel et al., 1993 Michel S.

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Ren D. The neuronal channel NALCN contributes resting sodium permeability and is required for normal respiratory rhythm. + and Gd3+ block. In addition, the current is reduced in na mutant flies and in mice with a brain-specific knockout of NALCN. Clock modulation of this sodium current also likely impacts neurophysiology. Loss-of-function na mutants and NALCN knockout result in silent and hyperpolarized neurons. Computational modeling of SCN neurons demonstrates that the modest daily rhythm of sodium leak can significantly impact overall firing rates. Thus, our work defines a molecular mechanism for clock control of membrane excitability. The clock control of membrane potential has largely focused on modulation of resting potassium conductance in the SCN () as well as in Bulla photoreceptors (). Surprisingly, we observed rhythms of sodium leak conductance in the fly DN1ps and l-LNvs, as well as in mammalian SCN, that are mediated by the NA/NALCN channel. This sodium leak exhibits the pharmacological sensitivity previously defined for the NALCN current (), most notably NMDGand Gdblock. In addition, the current is reduced in na mutant flies and in mice with a brain-specific knockout of NALCN. Clock modulation of this sodium current also likely impacts neurophysiology. Loss-of-function na mutants and NALCN knockout result in silent and hyperpolarized neurons. Computational modeling of SCN neurons demonstrates that the modest daily rhythm of sodium leak can significantly impact overall firing rates. Thus, our work defines a molecular mechanism for clock control of membrane excitability.

Lu et al., 2007 Lu B.

Su Y.

Das S.

Liu J.

Xia J.

Ren D. The neuronal channel NALCN contributes resting sodium permeability and is required for normal respiratory rhythm. Humphrey et al., 2007 Humphrey J.A.

Hamming K.S.

Thacker C.M.

Scott R.L.

Sedensky M.M.

Snutch T.P.

Morgan P.G.

Nash H.A. A putative cation channel and its novel regulator: cross-species conservation of effects on general anesthesia. Joiner et al., 2013 Joiner W.J.

Friedman E.B.

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Sehgal A.

Kelz M.B. Genetic and anatomical basis of the barrier separating wakefulness and anesthetic-induced unresponsiveness. Raman et al., 2000 Raman I.M.

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Padgett D. Ionic currents and spontaneous firing in neurons isolated from the cerebellar nuclei. This mechanism may not only be operating in clock neurons but may also be broadly involved in rhythmic changes in brain states. For instance, NALCN is critical to the maintenance of respiratory rhythms (). Both fly and worm na/nca loss of function results in disrupted locomotion as well as altered sensitivity to general anesthetics (). na mutant flies also show altered behavioral state transitions related to sleep and anesthesia (). More generally, the NA/NALCN current shown here has an identical electrophysiological profile to the tonic cation current required for regular firing in neurons of the mouse cerebellar nuclei ().

Our work also demonstrates that, like the core molecular clock, clock control of membrane potential is also widely conserved in neurons important for sleep and wake. We hypothesize that the common ancestor of the mouse and the fly had master circadian pacemaker neurons that drove its daily behavior. Moreover, these clock neurons employed daily anti-phase sodium and potassium conductances to drive their rhythmic activity. Thus, our finding suggests an ancient strategy governing neuronal activity important for driving daily cycles of sleep and wake.