Integration of Circadian Clock Information in the Drosophila Circadian Neuronal Network

Ventral Lateral Neurons and PDF

The LNvs are the most studied circadian neuronal cluster for their inferred role as master pacemakers that dominate the neuronal network in constant dark conditions (DD), and for their expression of the neuropeptide PDF that is critical to maintaining rhythmic behavior (Grima et al., 2004; Stoleru et al., 2004). Among the two LNv clusters, s-LNvs appear to be the “true” master pacemaker (Menegazzi et al., 2017; Shafer and Taghert, 2009). In light-dark conditions (LD), the s-LNvs regulate morning anticipation, described as increased locomotor activity of flies before the lights are turned on (Grima et al., 2004; Stoleru et al., 2004). DN1as are presynaptic to the s-LNvs. GRASP (GFP Reconstitution Across Synaptic Partners) and electron microscopy experiments reveal that the s-LNvs extend to the DN1s to form an active synapse (Guo et al., 2016; Yasuyama and Meinertzhagen, 2010). The hemibrain connectome suggests that s-LNvs may form connections with DN1pAs, DN2s and LNds in the CNN, though a connection with the LNds is disputed (W. J. Kim et al., 2013; Scheffer et al., 2020). Possible lack of synaptic connections between s-LNvs and other CNN neurons may suggest that these connections were missed due to time-dependent synaptic plasticity, due to communication by s-LNvs to the remainder of the CNN by other means, such as neuropeptide signaling, or that communication occurs through the limited clusters s-LNvs form synapses with.

Both s-LNvs and l-LNvs express PDF, with s-LNvs also expressing sNPF, to modulate the amplitude, synchrony, and the pace of rhythmic behavior (Helfrich-Förster, 1995; Johard et al., 2009; Park et al., 2000). Loss of PDF (pdf  ⁰¹ mutant) causes flies to become arrhythmic in behavior in DD (Renn et al., 1999). PDF acts on other neurons within the CNN, in addition to the s-LNvs, through its receptor, PDFR (Hyun et al., 2005; Lear et al., 2005b; Mertens et al., 2005) (Figure 2a). As with a pdf  ⁰¹ mutant, pdfr mutants (han⁵³⁰⁴ and han³³⁶⁹) advance evening anticipation and eliminate morning anticipation in a LD cycle (Hyun et al., 2005; Lear et al., 2005b; Renn et al., 1999). Reintroduction of PDFR to circadian neurons outside of the LNvs restores morning anticipation and timing of evening anticipation, as well as rhythmic behavior in DD (Lear et al., 2009), indicating that morning anticipation behavior is in fact regulated by a PDF signaling response to LNv instruction. This point is underscored by experiments in which tethered PDF, a PDF variant that is anchored to the cellular membrane of the neuron in which it is expressed, is expressed exogenously in non-LNv circadian neurons, which similarly restores morning and evening anticipation behaviors (and rhythmic behavior in DD) (Choi et al., 2012). The LNv PDF autocrine loop appears to reinforce PDF expression, adding robustness to the oscillating clock (Mezan et al., 2016). Later experiments narrowed the behavior-restoring function of PDF-responsive neuronal clusters in DD conditions to the DN1s (Goda et al., 2019). The role that DN1s appear to play in regulating behavioral rhythms in DD is consistent with observed damping of DN1 clock oscillations that correlate with a damping of behavioral rhythms in the first 1 to 3 days of DD, in which PDF signaling is disrupted (Hyun et al., 2005; Lin et al., 2004; Renn et al., 1999; Roberts et al., 2015; Yoshii et al., 2009). Another PDF-responsive cluster, the LNds, synapse with the LNvs and communicate with them through release of acetylcholine (Duhart et al., 2020). The excitatory effect of acetylcholine on the s-LNvs is modulated by circadian changes in synaptic strength (Duhart et al., 2020). Changes in synaptic strength may also be influenced by the s-LNv’s own response to PDF, which results in increased arborization during the day (Herrero et al., 2020). These findings suggest that timing of evening anticipation in LD conditions depends on the response of LNds to PDF, but may also involve LNv response to acetylcholine released by the LNds in the light-to-dark transition. Thus, one possible model is that DN1s establish rhythmic behavior in DD, while an LNv-LNd interaction accurately times evening anticipation. However, this interpretation of a DN1-LNd relationship is likely incomplete; if four of the six LNds and 5th s-LNv are silenced, flies become arrhythmic. This suggests that LNd neuronal activity is important for maintaining rhythmic behavior in DD (Guo et al., 2014), suggesting possible redundancy between the functions of DN1s and LNds, or DN1 reliance on rhythmic release of glutamate from the LNds (Duhart et al., 2020; Lear et al., 2009). Dynamic changes in neuronal partnerships may also explain the interaction between these three clusters. Under light dark conditions, the s-LNvs pair with LNds or DN1s based on the presence of light, underscoring the contribution of multiple oscillators to circadian behavior (Chatterjee et al., 2018; Lamba et al., 2018). Whether the arrhythmic behavior caused by silencing the LNds is due to a loss of cholinergic or glutamatergic signaling from LNds to the CNN, or a break in information transmission across the LNds through its PDF response is unclear.

Pigment Dispersing Factor Receptor and the Cytosolic Response

PDF expressed by the LNvs is presumably released to bind to PDFR, a G-protein coupled receptor. Within the CNN, PDFR is expressed by DN1as, some DN1ps, DN2s, some DN3s, some LNds, the 5th s-LNv, and the s-LNvs (Hyun et al., 2005; Mertens et al., 2005; Shafer et al., 2008) (Figure 2a). Once activated, PDFR elicits an increase in cyclic adenosine monophosphate (cAMP) and regulated calcium oscillations, which serve as signaling molecules to tune circadian clocks (Im and Taghert, 2010; Klose et al., 2016; Liang et al., 2016, 2017, 2019; Mertens et al., 2005; Palacios-Muñoz and Ewer, 2018; Shafer et al., 2008). Using PDF signaling as a model, we explore the different potential neuronal responses to G-protein coupled receptor signaling.

Glycine Cooperation With PDF

Neurotransmitters act in a complex milieu in the brain. Therefore, it is likely and even expected that different neurotransmitters would cooperate to exert an effect on target neurons and tissues. Indeed, Choi et al. (2012) suggest that PDF activity is coupled to a small molecule neurotransmitter. LNv-expressed glycine has recently emerged as a candidate for added inhibition of downstream neurons (Frenkel et al., 2017). Glycine activates glycine receptor, allowing permeation of chloride to lower the membrane potential, thus likely inhibiting downstream LNds and DN1ps (Frenkel et al., 2017). Thus, a cooperative inhibition by both PDF and glycine may reduce the probability of neuronal firing and vesicle release in neurons downstream of LNvs.

Ion Transport Peptide Cooperation With PDF

Ion transport peptide (ITP) is expressed in the 5th s-LNv and one LNd (Hermann-Luibl et al., 2014; Johard et al., 2009) (Figure 2d). Similar to PDF, ITP is rhythmically expressed, targeting the dorsal neurons in the brain. However, ITP differs from PDF in that it regulates evening activity and suppresses nocturnal activity. Although the receptor for ITP is not yet characterized in Drosophila, it is likely to be a GPCR (Nagai et al., 2014), and likely expressed in evening cells such as the DN1s, given its effect on evening anticipatory behavior.

Diuretic Hormone 31 Cooperation With PDF

Although locomotor behavior reveals nearly identical phenotypes for both pdf  ⁰¹ and pdfr mutants, in which morning anticipation is lost, evening anticipation is advanced, and in DD conditions flies become arrhythmic (Hyun et al., 2005; Lear et al., 2005b; Renn et al., 1999), there are observable differences at the molecular level. When Ca²⁺ oscillations are monitored in a pdfr mutant background, all neuronal clusters within the CNN show synchrony, with notable exception of the DN1s (Liang et al., 2016). When Ca²⁺ oscillations are monitored in a pdf  ⁰¹ mutant background, the LNds (in addition to the DN1s) do not synchronize with the remainder of the CNN neurons (Liang et al., 2017). This suggests that PDFR in the LNds may be involved in mediating a separate signal, and may also explain LNd unresponsiveness in Ca²⁺ oscillation, in response to loss of PDF.

PDFR has the capacity to respond to a second neurotransmitter. Diuretic hormone 31 (DH31) triggers a PDFR-dependent cAMP response in HEK293 cells, though at half the efficacy of PDF (Mertens et al., 2005). Elimination of DH31 causes a loss of morning anticipation similar to a loss of PDF, but does not alter evening anticipation nor cause arrhythmic behavior in DD conditions (Goda et al., 2019). Indeed, double mutant (DH31 and pdf) experiments measuring locomotion as a behavioral output suggest that both DH31 and PDF cooperate to act through the PDFR, with the DH31 Receptor (also a GPCR) unlikely to play a role in locomotion behavior (Goda et al., 2019). DH31 Receptor instead appears to play a role in temperature preference and is expressed in DN1s, DN3s, and l-LNvs (weakly), but not in DN2s, s-LNvs or LNds (Goda et al., 2016, 2018; Johnson, 2005) (Figure 2e). Temperature cycles that oscillate with day/night cycles can entrain the CNN and the circadian clock (Glaser and Stanewsky, 2007; Matsumoto et al., 1998; Sidote et al., 1998; Yoshii et al., 2005) and appear to act through the DN1ps (posterior DN1s) (Yadlapalli et al., 2018), suggesting DH31 as a candidate for temperature entrainment (Goda et al., 2016). Indeed, DH31 interacts with DN2s through PDFR to guide lower temperature preference by flies at nightfall, though all of the three DN subgroups show calcium responsiveness to temperature changes (Goda et al., 2016; Yadlapalli et al., 2018). Thus, given that DH31 peptide is expressed in the DN1s and also promotes wakefulness (Goda et al., 2016; Kunst et al., 2014), it is tempting to think that a DN1 response to changes in temperature communicate this information to the CNN through DH31 by way of PDFR and/or DH31 Receptor. Indeed, wild type flies that are synchronized to both light-dark and warm-cold cycles exhibit an increase in daytime activity and a decrease in nighttime activity in a sleep analysis when compared to flies synchronized monitored at constant temperature (C. Chen et al., 2018). LNd responsiveness to changes in light regimen through the deep brain photoreceptor Cryptochrome (CRY) or its response to the LNvs, and its glutamatergic communication with the DN1s may provide a mechanism of integrating the CNN response to the two zeitgebers (Duhart et al., 2020). Such cooperation between neuropeptides underscores how combinations of neuronal signaling can fine tune the activity of clock neurons and circadian clocks under diverse environmental conditions.

Glutamatergic Influences on the Ventral Lateral Neurons

Glutamate released from DN1s promotes wakefulness (Figure 2g). LNvs respond to glutamate with a decrease in intracellular calcium, which shortens behavioral period under DD conditions (Guo et al., 2016). Downregulation of glutamate receptor, DmGluRA, in the LNvs lengthens free-running period in DD conditions, and reduces locomotor activity at night in LD conditions (Hamasaka et al., 2007). Expectedly, decreasing glutamate by either blocking neurotransmitter release or decreasing the activity of glutaminergic neurons also promotes sleep at nighttime (Zimmerman et al., 2017). Collectively, this evidence suggests the LNvs are in a circuit feedback loop, placing the LNvs, which often enjoy the pinnacle of CNN hierarchy, into a post-synaptic position within the CNN. Strikingly, loss of glutamate signaling in flies restores rhythmicity in constant light conditions (LL) (de Azevedo et al., 2020), clearly pointing to a critical role for glutamate signaling in light-induced arrhythmia. This is reminiscent of flies carrying a hypomorphic allele or genomic deletion of the deep brain light receptor cryptochrome (cry^b and cry⁰¹, respectively) in which flies exhibit rhythmic behavior in LL conditions (Dolezelova et al., 2007; Emery et al., 2000; Helfrich-Förster et al., 2001). Whether glutamate signaling and cry function in parallel pathways or cooperate with each other in response to constant light conditions remains to be explored.

A glutamatergic response closes a neuronal feedback loop to PDF neurons. While PDF transmits information to both DN1s and LNds, DN1s release glutamate to silence the s-LNvs and the three LNds/5th s-LNv cluster as indicated by decreased cytosolic Ca²⁺ (Guo et al., 2016). These studies were conducted using exogenous expression of the P2X2 ATP-responsive cationic channel that can be used to activate neurons. Activation of DN1s by ATP in explanted brains revealed a decrease in s-LNv and LNd Ca²⁺ levels through glutamate. Hemibrain connectome data support such reciprocal connectivity, with synaptic connections observed between DN1s and s-LNvs, and DN1s and LNds. It is tempting to think of the glutamatergic response to PDF signaling as closing a signaling loop across so-called morning cells (LNvs) and evening cells (DN1s), while signaling to PDF-responsive LNds. However, there are additional complicating factors. Although t-PDF expression in DN1s restores rhythmic locomotor behavior in pdf  ⁰¹ flies in DD (Goda et al., 2019), DN1s do not appear to be Ca²⁺ responsive to exogenous PDF added to explanted pdf  ⁰¹ fly brains in DD, in contrast to other neurons (Liang et al., 2017). Thus, the increase in cytosolic Ca²⁺ in DN1s that trigger a glutamatergic signal to the s-LNvs and the 3LNds/5th s-LNv cluster may not be caused by PDF signaling alone. It is possible that other neurotransmitters released by the s-LNvs act on the DN1pAs as the means to trigger a glutamatergic response, while a PDF response by DN1a and DN1pAs elicits a separate response. How the glutamate and PDF neurotransmitters integrate into a feedback loop and how they regulate other clusters in the CNN will be an exciting area of investigation.

Light and Dopamine Input to the Ventral Lateral Neurons, and Morning Arousal

There are a number of excellent reviews that describe light input pathways in detail (Helfrich-Förster, 2002, 2020; Schlichting, 2020; Yoshii et al., 2016); therefore, we will focus on some of the neurotransmitter signaling pathways as they relate to communication into the various molecular clocks in the CNN. CRY protein, expressed in a portion of each neuronal cluster, directly communicates blue-light information to the clock transcription machinery by virtue of the semi-translucent nature of the fly cuticle (Figure 2f). However, cry⁰¹ flies can be entrained to a new LD phase (Dolezelova et al., 2007), suggesting that the visual system also conveys light to the CNN (Schlichting, 2020). Indeed, cooperative input from CRY and the visual system allow fine tuning of evening anticipation under different photoperiods (Kistenpfennig et al., 2018). The l-LNvs are the target neuronal clusters within the CNN for many arousal promoting neurotransmitters (Mazzotta et al., 2020). The HB-eyelet, a light-sensing body in the retina, and photoreceptors from the eye form synaptic connections with the LNvs, which contributes to morning arousal (Damulewicz et al., 2020; Helfrich-Förster et al., 2002; Hofbauer and Buchner, 1989; Veleri et al., 2007). Light information is communicated to the LNvs through excitatory cholinergic and inhibitory histaminergic signals, though how these two neurotransmitters are coordinated is unclear (Schlichting et al., 2016). Acetylcholine released from HB-eyelets and photoreceptors interacts with nicotinic acetylcholine receptors in both the s- and l-LNvs to trigger increases in cytosolic Ca²⁺ and cAMP, which causes depolarization of the neuronal membrane (McCarthy et al., 2011; Muraro and Ceriani, 2015; Schlichting et al., 2016; Wegener et al., 2004). Although the HB-eyelet may also synapse with the DN1s as suggested by the hemibrain connectome, there is no evidence of synapses between retinal photoreceptors and DN1s (or any other non-LNv within the CNN) (Scheffer et al., 2020). However, when the LNvs are silenced, the LNds, 5th s-LNv, DN1a and DN3s respond to a hub in the accessory medulla that receives signals from the fly visual system, suggesting that indirect connections between photoreceptors and other clusters within the CNN must exist (M.-T. Li et al., 2018).

Dopamine promotes wakefulness in both insects and mammals, and triggers an increase of cAMP in the LNvs (Andretic et al., 2005; Crocker and Sehgal, 2010; Fernandez-Chiappe et al., 2020; Kume et al., 2005; Lebestky et al., 2009; Riemensperger et al., 2011; Shang et al., 2011). fumin mutations in the dopamine transporter result in an excess of synaptic dopamine and hyperactive fly behavior, underscoring dopamine involvement in arousal (Kume et al., 2005). However, knockdown of dopamine receptors Dop1R1 and Dop1R2 (relevant in l- and s-LNvs respectively), do not affect daytime arousal despite reducing cAMP response, suggesting a more complex regulation of morning arousal than previously assumed (Fernandez-Chiappe et al., 2020). Instead, some dopaminergic neurons respond to PDF (Potdar and Sheeba, 2018), suggesting that the LNvs may be further upstream in the morning arousal process. Underscoring this possibility, DN1s may be involved in morning arousal as discussed above, possibly through instruction received from the LNvs or elsewhere (Guo et al., 2016, 2018; Kunst et al., 2014; Lamaze et al., 2017, 2018; L. Zhang et al., 2010a; Y. Zhang et al., 2010b). Although the LNvs are designated “morning cells” (Grima et al., 2004; Stoleru et al., 2004), it may be more accurate to think of “morningness” as the activity within a circuit, rather than the responsibility of a specific neuronal cluster.

Examples of CNN Output

Coordinated oscillations within the CNN must ultimately synchronize clocks within the rest of the body. Neuropeptides, such as short neuropeptide F (sNPF) and neuropeptide F (NPF), mediate communication within the CNN and to the body. Although these two peptides are similar in name, their sequences are different and they share no homology. sNPF is transcribed rhythmically in the s-LNvs (Abruzzi et al., 2017; Kula-Eversole et al., 2010) (Figure 2c). Knockdown of sNPF leads to increased nighttime activity, suggesting that this neuropeptide promotes sleep at night (Johard et al., 2009; Shang et al., 2013), in contrast to PDF, which promotes wakefulness. One mechanism of action is that sNPF moderately suppresses l-LNv electrical activity, thereby suppressing their arousal function and helping consolidate sleep into the night (Lebestky et al., 2009; Parisky et al., 2008; Shang et al., 2008, 2013; Sheeba et al., 2008). Although we do not yet have a complete picture of where sNPF receptor is expressed to determine how this receptor responds in the fly, application of exogenous sNPF to the BG2-c6 Drosophila neuronal cell line, which expresses sNPFR1, leads to a cAMP response in a dose-dependent manner (W. Chen et al., 2013), analogous to a PDFR response (Mertens et al., 2005). Application of exogenous sNPF to explanted brains reveals a cAMP response in the insulin producing cells (IPCs) in the brain, suggesting they may express sNPF receptor (Nagy et al., 2019). Knockdown of sNPF receptor by RNAi in this part of the brain induces reproductive arrest, indicating that sNPF signaling to the IPCs is critical for reproductive activity (Nagy et al., 2019) and suggesting possible contributions to circadian influences on courtship and mating behavior (Allada and Chung, 2010; Sakai and Kitamoto, 2006). Within the CNN, both PDF and sNPF suppress basal Ca²⁺ levels in targeted pacemakers with long durations by cell-autonomous actions (Liang et al., 2017). Notably, sNPF released from morning cells appears to be critical for setting the Ca²⁺ phase of PDF-unresponsive DN1s (Liang et al., 2017).

NPF, the Drosophila homolog of mammalian orexigenic peptide Neuropeptide Y (NPY), regulates diverse behaviors including circadian locomotor activity. The l-LNvs and 3LNds/5th s-LNv express NPF (C. He et al., 2013a; Hermann et al., 2012; W. J. Kim et al., 2013), though expression in the PDF-expressing s-LNvs has also been reported (C. He et al., 2013a) (Figure 2b). The pattern of NPF receptor (NPFR1) expression is not fully resolved. NPFR1 Gal4 drivers suggest that the s-LNvs express the receptor, while immunostaining suggests that instead, DN1s and LNds express NPFR1 (C. He et al., 2013a; W. J. Kim et al., 2013). NPF signaling appears to regulate the phasing and amplitude of evening activity in light-dark cycles and the modulation of evening anticipatory behavior; NPF and NPFR1 loss-of-function mutations lead to an elimination of evening anticipatory behavior (C. He et al., 2013a; Hermann et al., 2012; G. Lee et al., 2006). These phenotypes mirror the effect of PDF and PDFR loss-of-function mutations that lead to an elimination of morning anticipatory behavior, suggesting that PDF and NPF may link morning and evening oscillators. NPF is also involved in sleep regulation (overexpression of NPF increases sleep) and homeostasis of sleep (C. He et al., 2013b); as well as promoting wakefulness and feeding behavior (Chung et al., 2017); alcohol sensitivity (Wen et al., 2005); prolonging mating and courtship (W. J. Kim et al., 2013; W. Liu et al., 2019); and in clock-controlled sexual dimorphism through the LNds (G. Lee et al., 2006). All of these behaviors have a circadian component. It will be exciting to identify the circuits that connect these behaviors to the molecular clock in the CNN.