Light buffers the effectiveness of dopamine-mediated wake promoting effects in Drosophila. Daytime sleep is relatively insensitive to dopamine activation, whereas nighttime sleep in light-dark conditions is sensitive but less so than nighttime sleep in constant darkness conditions. The 10 l-LNvs, a subset of clock neurons, are the only neurons known to be part of the light-mediated wake-promoting circuits in fly brains. We show here that they are downstream targets of dopaminergic neurons. They not only form membrane contacts with dopaminergic neurons but also respond to dopamine by increasing cAMP levels. This presumably reflects the fact that l-LNvs express stimulatory receptors for these neurotransmitters. The response is largely cell-autonomous, because they still respond to dopamine in the presence of TTX (). We also showed that the responses are likely to be specific to dopamine because they are blocked by a dopamine antagonist and can be induced by a dopamine agonist ().
These cells also receive direct synaptic input from octopaminergic neurons (; data not shown for octopamine in the presence of TTX). Dopamine is likely a stronger arousal signal than octopamine in fly brains, at least for flies raised in standard light-dark conditions. An identical stimulation of octopamine neurons in adult brains only mildly suppressed total sleep, an effect that was also considerably smaller than previously reported 6
. This previous study used a sodium channel to constitutively stimulate octopamine neurons 29
. Combined with the fact that feeding flies with octopamine also requires 2–3 days to suppress sleep and the nighttime sleep was still affected even after octopamine was removed 7
, we suggest that chronic activation of octopaminergic neurons may require a reconfiguration of neural circuits to produce strong behavioral effects.
The s-LNvs are neighbors of the l-LNvs and are key pacemaker neurons in Drosophila.
In contrast to the l-LNvs, s-LNvs show very weak responses to dopamine or octopamine in light-dark conditions, likely reflecting the fact that mRNAs for these receptors are much more abundant in l-LNvs than in s-LNvs 16
. This even includes the dopamine dD2R inhibitory receptors, which also explains why the dD2R knockdown did not lead to a detectable cAMP increase in s-LNvs in response to dopamine application (supplemental Fig. 6
Light has a profound impact on animal behavior. For example, extensive light-driven cyclic gene expression has been detected in Drosophila
. The l-LNvs are also reported to increase their firing rate in response to acute light exposure, especially during early morning 13
. Here we show that the 12hr light exposure of standard light-dark housing conditions has a profound impact on l-LNv physiology. Light-dark rearing not only mitigates the stimulating effects of both dopamine and octopamine but also synchronizes cell responses. One possible function for synchronization is that the l-LNv responses are more stable when synchronized (see below). Although l-LNvs from light-dark reared flies are less sensitive to both dopamine and octopamine than those from constant darkness reared flies, the two signaling pathways are differentially regulated.
Octopamine-mediated responses are time-sensitive in constant darkness, and octopamine activation at night is promoted by the clock but inhibited by prior light exposure. The microarray data indicate that transcription of the octopamine receptor OA2 peaks around ZT12, whereas that for OAMB peaks around ZT6 16
. Since imaging analysis showed that maximum nighttime l-LNv responses to octopamine require the clock (), it is possible that the translation or activities of these receptors, or the expression of signaling molecules downstream of these receptors, peaks at night.
In contrast to octopamine, the dopamine-mediated responses of l-LNvs are time-insensitive and are not affected by per01
mutation (–). However, light exposure suppresses the l-LNv dopamine responses at all times of day, nighttime as well as daytime. As downregulation of dD2R is sufficient to mimic the responses of flies reared in constant darkness and D2R-RNAi had no effect in constant darkness, light exposure apparently upregulates dD2R activity to dampen dopamine responsiveness in light-dark conditions. This implies that there are light-stimulated changes in either dD2R gene expression or regulation, such as a modification of the dD2R receptor or its downstream targets. Light may also downregulate stimulatory D1R signaling pathways in concert with the upregulation of dD2R, although our results suggest that expression of dD2R can account for most of the reduction in responsiveness. Given that there are no known inhibitory receptors for octopamine, the l-LNvs must use a different mechanism to effect light-mediated modulation of octopamine responsiveness (supplemental Fig. 7
). For example, light may downregulate stimulatory octopamine receptors. Nonetheless, a common theme is that light inhibits the ability of these two chemicals to stimulate the l-LNvs. The fact that the 12hr light exposure suppresses the ability of dopamine and octopamine to stimulate l-LNvs suggests that they do not simply sum different arousal signals. Rather, they are integrated and perhaps scaled depending on conditions, suggesting a link to behavioral flexibility. Light appears in this scenario to be a dominant signal, as its presence during the day reduces the ability of internal signals to stimulate arousal. However, the l-LNvs use a number of mechanisms including the circadian clock to integrate signals and produce appropriate responses. The surprisingly weak behavioral effects of acute stimulation of octopamine neurons raises the possibility that there are other circumstances (age, nutritional or reproductive status) in which these inputs become more important.
Because animals must maintain a proper quality and quantity of daily wake and sleep time, counter-balancing mechanisms like those described here may also serve the fly brain to preserve sleep stability. For example, the opposing effects of environmental light and dopamine may allow the l-LNvs and perhaps other arousal-sleep relevant neurons to buffer unexpected fluctuations in light intensity and/or dopamine release from presynaptic partners, i.e., the circuit organization allows the activity of sleep-relevant neurons to be maintained within a physiological range with a relatively stable output. We imagine that only exceptional circumstances would take precedence over sleep-wake stability, for example by modulating the ratio of stimulatory and inhibitory dopamine receptors. Our data suggest that modulation could also occur by altering the synchronization of individual cells within a group, for example between different individual l-LNvs. It will not be surprising if additional integration mechanisms will also be important for the l-LNvs to generate appropriate signals to downstream circuits, both to maintain optimal sleep at night and optimal wakefulness during the day, i.e., for sleep-wake homeostasis, as well as for appropriate responses to emergency circumstances.