Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae

Circadian rhythms are ≅ 24 h oscillations displayed by various organisms^1–4, maintained by an endogenous timekeeping mechanism that can be entrained to the environment by external cues called ‘zeitgebers’ (German for “time-givers”) such as light, temperature and even social interactions in mammals^5–7. Through entrainment, organisms anticipate and importantly, adapt to daily environmental oscillations^8,9 in order to regulate physiological phenomena and behaviors associated with locomotion, sleep patterns, hormone release and body temperature among others^10–15. In the absence of entraining stimuli, circadian rhythmicity is self-sustained in a “free-running” state^2,16. Molecular components responsible for the organismal ability to sustain rhythmicity have been extensively characterized and the pacemaker mechanisms display conserved patterns between fruit flies and humans^17,18. Given that many metabolic processes are highly correlated with circadian clocks, the disruption of circadian rhythms may lead to abnormal behavioral rhythms, altered mood, depression, sleep disorders in humans^19–23 and it has been reported to impact type 2 diabetes and cancer^24–26. Likewise, the ability to properly synchronize endogenous clocks with circadian time was shown to positively impact fitness in fruit flies and various other organisms^27–29.

The molecular clock mechanism in Drosophila is composed of interlocking transcriptional feedback loops³⁰ presenting homologous components with the molecular circadian mechanism in mammals¹⁸. In one loop, CLOCK (CLK) and CYCLE (CYC) proteins heterodimerize (CLCK/CYC) in the cytoplasm and translocate to the nucleus where they positively regulate the expression of period (per) and timeless (tim) genes^31–34. Protein products of these genes (PER and TIM) accumulate in the cytoplasm during late day/early evening and heterodimerize to translocate to the nucleus later in the evening^35–39. Here, PER represses the activity of CLK/CYC, hence negatively regulating its own transcription as well as tim transcription^38,40. TIM is bound by Cryptochrome (CRY) in the nucleus, which is the key and the only circadian-dedicated photoreceptor in Drosophila⁴¹ responsible for promoting light-dependent degradation of TIM as a molecular response to light in order to reset the molecular clock^42–45. Eventually, before dawn, both PER and TIM are degraded, releasing CLK/CYC to resume their activity⁴⁶. The rhythmic activity of core molecular clock components characterizes the circadian pacemaker circuitry. While the molecular clock is conserved developmentally, the fruit fly larva displays a relatively simpler neuronal organization than its adult counterpart, and particularly, an accessible clock network consisting of nine neurons per brain hemisphere, thus representing an attractive model to study circadian-dedicated neural circuits. Specifically, larval pacemaker neurons comprise neuropeptide pigment-dispersing factor (PDF)-expressing lateral neurons (PDF-LaNs) as the main pacemaker neurons, a 5^th PDF-negative lateral neuron (5^th-LaN) and two sets of dorsal neurons (DN1 and DN2)^47,48.

Owing to the experimental advantages and considerable homologies with mammals, Drosophila has been extensively used as a model system to study the genetic and cellular mechanisms as well as the fundamental neural circuits of circadian rhythmicity and entrainment of the molecular clock⁴⁹. The main behavioral outputs tackled in Drosophila circadian rhythm studies are locomotor activity and eclosion, since robust rhythms can be observed and recorded in individuals or fly populations^50,51. Although these studies are conducted by using adult flies, also larvae have been used as a circadian rhythm model^52–55. Besides being equipped with a comparable neuronal simplicity, fruit fly larvae also exhibit well-characterized attraction/avoidance behaviors in response to environmental cues such as temperature, chemicals and light^56–58. Moreover, in response to sensory stimuli, larvae are able to make compound decisions, by modulating two alternate moto-programs as runs and turns in order to navigate towards a preferred condition or away from an undesirable stimulus^59–61. As extensively described for photo-navigation of innately photophobic larvae, they employ distinct navigation strategies which are defined by the length, size, direction and frequency of runs and turns, operated by processing of spatial or temporal cues^62–64.

Despite the complexity and level of detail in the described photo-behavioral programs, it remains unknown how the animals modulate navigational decisions in a circadian fashion. We first analyze navigation relevant behaviors as naïve responses at four different circadian times: dawn (CT0), midday (CT6), dusk (CT12) and midnight (CT 18). Intriguingly, none of the behaviors which are critically involved in navigation show circadian modulation in the absence of light stimulation. However, when a light source is introduced, we show that there is a strong modulation of the light-response during the course of the day with the largest difference being between dawn and midnight. We further find that each one of pdf, per and clk mutants show severe deficits in circadian modulated behaviors supporting that a functional molecular clock as well as proper neural signaling of pacemaker neurons is essential for maintaining circadian-modulated visually-guided navigational decision making.

Keeping animals in constant conditions is a commonly followed procedure applied prior to performing behavioral experiments, particularly important for circadian rhythm studies. Accordingly, placing animals in constant darkness (DD) enables the measurement of the direct effect of the light stimulus on the intrinsic molecular clock mechanism and averts the influence of the ‘masking effect’, which is the immediate adaptive response given to an environmental change⁶⁵. Therefore, 2-day-old larvae are kept in DD for 2 days and the navigation of 3^rd instar foraging larvae is measured by using a computer-based tracking system as previously described⁶⁴. This assay allows in-depth characterization of larval visually-guided navigation by dissecting it into distinct navigation strategies, dependent on either spatial or temporal information processing. Thus, our approach provides detailed insight about how larvae adjust their light avoidance behavior in accordance with circadian rhythm, and how this behavior is affected when the pacemaker mechanism is disrupted.

For efficient photo-navigation, Drosophila larvae process both spatial and temporal cues⁶⁴. Through comparison of light information collected by left and right eyes, spatial navigation strategies, as described for taxis⁸², presuppose a direct behavioral response to the stimulus intensity and directional orientation bias. Conversely, temporal information processing, corresponding to kinesis⁸², involve temporal comparison of stimulus intensity and adjustment of turns, as in size and rate, and head-sweeps, as in acceptance or rejection. Previous studies revealed that circadian rhythm regulates the light-dark preference of Drosophila larvae, measured through a light-dark preference assay^66,83 in which only half of the experimental plate is illuminated. Although this assay allows robust measurements of larval light avoidance, it does not expose distinct navigation strategies underlying the avoidance behavior. Moreover, this assay favors the measurement of temporal information processing, since spatial comparison is only limited to the mid-line that separates light and dark sides of the experimental plate. Our data adds a level of understanding of the daily rhythmicity of photophobic navigation in fruit fly larvae by disintegrating this behavior into distinct navigation strategies. Furthermore, we show that the described rhythmic variation is dependent on light information processing itself, absent in no-stimulus conditions.

In stimulus-naïve conditions, navigation strategies dependent on either spatial or temporal information processing are still performed for navigation although without any bias, presumably for food seeking or exploring the environment. Nonetheless, these navigation strategies are not subject to circadian modulation since larval navigation does not show any time-dependent alteration throughout the day. It could be argued that the variable values obtained for mean run change (Fig. 2A.I) and turn size (Fig. 2B.I) might be due to larval intrinsic activity rhythms. Adult flies anticipate the light-dark transition phases through their biological clock and increase their activity according to this anticipation¹⁰. It appears possible that the same activity rhythm pattern might also be true for larvae. However, since these two navigation strategies were the only behavioral parameters to suggest this pattern, further experiments which specifically investigate activity rhythms should be conducted. Additionally, the tendency for higher turn frequency at dawn illustrated by turn rate (Fig. 2B.II) could be indicative of increased activity at this time. Nevertheless, this tendency alone is not sufficient to support that turn rate would represent larval intrinsic activity modulation by circadian rhythm. Therefore, we conclude that the tendencies observed for these temporal navigation strategies performed in the absence of light stimulation reflect behavioral noise rather than circadian rhythm-induced patterns.

When a light stimulus is presented, larvae avoid light with high efficiency especially at dawn, demonstrated by a stronger bias in the heading direction away from the light source (Fig. 3A.II,A.III). However, it is noteworthy that a tendency to perform spatial navigation strategies more effectively at dusk is observed as well, while this performance was less prominent at midday and midnight. On the other hand, a trend for performing temporal navigation strategies comparable to dawn was observed at midday. For both types of navigation strategies, the efficiency in performance was dampened at midnight. Collectively, an intensive response to both spatial and temporal visual input given particularly at dawn might be due to increased sensitivity to light stimulus. Circadian gating of light information could intensify or diminish the behavioral response. Since the least prominent avoidance performance of all navigation strategies are observed at midnight, the opposite might be true for this time-point. Presumably due to least expectancy of environmental threats such as predators, desiccation and DNA-damage by exposure to ultraviolet light, larvae might be rendered relatively less sensitive to visual stimulus, and rather prioritize other sensory modalities such as chemical-sensing, in accord with circadian rhythms facilitating adaptation to environmental conditions⁴⁹. Another possible explanation for the strong modulation difference observed between dawn and midnight might be the resting state of larvae. Under 12 h LD conditions, adult flies restrict their activity to subjective day and display sleep-like resting behavior, described as resting states during which flies are less responsive to sensory stimuli^84,85. Like their adult counterparts, larvae sleep as well, defined by rapidly reversible quiescent states, which is crucial for their development⁸⁶. Although a clear connection between time of the day and resting behavior is currently lacking in larvae, during nighttime animals might be rendered relatively less sensitive to sensory stimuli, as their adult counterpart. Although strong light stimulation disturbs the sleep state⁸⁶, which is supported by our results showing that larvae use navigation strategies to avoid light throughout the day, circadian modulation presumably defines the animal’s sensitivity to light stimulation. As a result, navigation strategies used for light avoidance are seemingly performed more prominently at dawn, likely due to higher arousal by the stimulus, and conversely, less prominently at midnight. Other tested time-points, midday and dusk, showed variable efficiency in performing avoidance behavior. As illustrated by substantial differences between midday and dusk in conducting mean run change and turn rate delta, it could be argued that a tendency to perform spatial navigation strategies (Fig. 3A) more efficiently at dusk is observed while temporal navigation strategies (Fig. 3B) tend to be performed more prominently at midday. This interchangeable efficiency in performance might be explained by the modulatory effect of circadian rhythm being established distinctly on spatial and temporal information processing, leading to variant efficiency in performance. However, this conclusion can only be reached with further elucidation of how this modulation impacts the neural circuitry which regulates larval navigation.

With the exception of DNs, the pacemaker circuitry is a direct synaptic target of photoreceptors (PR)⁶⁷, exposing the central role of light sensing in circadian entrainment. Correspondingly, the two separate PR pathways, delineated by blue-tuned Rhodopsin 5 (Rh5)- and green-tuned Rhodopsin 6 (Rh6)-expressing PRs, show segregated but also overlapping function in light avoidance behavior and circadian entrainment^64,68. Larval main pacemaker neurons, PDF-LaNs, are the only shared downstream targets of Rh5- and Rh6-PRs⁶⁷, in compliance with the finding that either PR-subtype is sufficient to entrain the molecular clock⁶⁸. On the contrary, both PR-subtypes are necessary for efficient navigation due to their distinct roles; Rh6-PRs are required for temporal information processing whereas Rh5-PRs seem to be predominantly necessary for spatial information processing but also for the integration of both types of light information for downstream transmission⁶⁴. Besides PDF-LaNs, Rh5-PRs also project onto other visual projection neurons, being postero-ventro-lateral neuron 09 (PVL09), projection optic lobe pioneer neurons (pOLP), fifth-LaN and non-clock LaNs, whereas Rh6-PRs only project onto local optic lobe pioneer neurons (lOLP) which are visual interneurons characterized by cholinergic and respectively glutamatergic neurotransmitters, presumably of opposed valence (excitatory/inhibitory)⁸⁷. The lOLPs present reciprocal connections with each other and project onto other visual interneurons, creating circuit modulatory motifs⁶⁷. Interestingly, the lOLPs also create such presumed excitatory-inhibitory motifs with PDF-LaNs, possibly tuning their activity according to the received temporal light information. Thus, lOLPs are likely responsible for temporal comparison of light information⁸⁸ for further attuning of the entire visual system. Since spatial and temporal information processing are segregated through distinct PR pathways, it could also be possible that circadian modulation of visual interneurons is distinctly established on corresponding neuronal circuit components. More specifically, it could be envisaged that visual interneurons regulating temporal information processing are first subject to circadian modulation, conveying further into the circuit through their characteristic modulatory motifs. Alternatively, circadian modulation might be simultaneously established on projection visual interneurons and integrated into downstream connections. Elucidating the connectivity characterizing PDF signaling, additional circadian modulators and the expression of corresponding receptors would shed light on the neurons directly targeted by the pacemaker system for circadian tuning of light information processing, as well as other sensory modalities.

Considering the distinct involvement of Rh5- and Rh6-PR pathways in mediating spatial and respectively temporal information processing, we contemplated that PDF signaling could play a role in the performance of these navigation strategies, since PDF-LaNs receive direct synaptic input from both PR-subtypes. Indeed, mutants deficient in PDF signaling present not only an abolished circadian modulation, but also a considerably dampened performance of navigation strategies compared to WT larvae. This dampening is especially noted for dawn, consistent with the involvement of PDF neuropeptide in adult flies, establishing the morning activity peak in LD conditions and in maintaining rhythmicity in DD conditions. The underlying cause for dampened photo-navigation observed in larvae might be disrupted communication between PDF-LaNs and downstream neurons responsible for larval navigation. However, it remains yet to be determined whether light information is directly conveyed via PDF-LaNs or via additional connections made with downstream visual interneurons. Further dissection into the system is necessary in order to characterize the neuronal targets of PDF-LaNs.

Pdf ¹ larvae have disrupted PDF-neuropeptide signaling, which is necessary for synchronizing larval pacemaker circuitry⁷⁴. Nonetheless, these mutants have an intact molecular clock mechanism which still undergoes circadian oscillations. Divergently, we also tested per¹ and clk^Jrk larvae in order to investigate the impact of a disrupted molecular clock mechanism on circadian modulation of light avoidance behavior. Given that cycling of per mRNA and PER is essential for constituting rhythmic behavior, per null mutants show a prominent phenotype of circadian modulation loss. At the molecular level, CYC and CLK might still act as activators for other genes, however, per gene products as the main clock component which dictate rhythmicity is lost. Presumably, the only rhythmicity indicator in per¹ larvae is light-dependent degradation of TIM, induced by CRY. However, under free-running (DD) conditions TIM cannot be degraded and the timing of PER/TIM nuclear translocation defines the rhythmicity of the animal¹⁷. Thus, per¹ larvae have no molecular oscillations which would indicate circadian rhythmicity in DD, in accord with our findings demonstrating disrupted circadian modulation of navigation strategies. Likewise, clk^Jrk larvae are able to regulate heading direction, turns and head-sweeps in relation to light stimulus, however, this avoidance behavior is not subject to circadian modulation due to disrupted molecular clock mechanism. Given that CLK is a component of the heterodimer that acts as an activator of per and tim genes, protein levels encoded by these genes are extremely low and non-cycling in clk mutants^32,33. Therefore, defects observed in circadian modulation of light avoidance behavior similar to per¹ larvae appears to be coherent. Nevertheless, PER and CLK constitute the negative and respectively the positive components of transcriptional feedback loop, which means that at the molecular level these mutants might entail disruption of the molecular clock mechanism in different aspects, consistent with divergent roles described for these mutants^30,52. Notably, these mutants display tendencies in opposite directions in performing temporal navigation strategies (Fig. 5.B,B’). Considering loss of circadian modulation and previous studies revealing arrhythmic behavior of both mutants, we interpret these tendencies as possible indication of arrhythmicity. Overall, results obtained from larvae with mutated clock components demonstrate that alterations in the signaling and the molecular integrity of the clock circuitry lead to loss of circadian modulation of photophobic navigation, substantiating the modulation observed in WT larvae, along with no intrinsic modulation of distinct navigation strategies observed under stimulus-naïve conditions.