Here, we show how dopamine levels in adult Drosophila flies cycle in a circadian manner and provide important evidence for a role of Ih current in this phenomenon. Moreover, we provide evidence that Ih current is necessary to consolidate sleep. As HCN channels, responsible for Ih current in mammals, are becoming pharmacological targets for cardiac diseases, foreseeing the consequences of abolishing Ih current is a requirement for searching new therapies.
Even though our data on dopamine levels was obtained from whole heads, various reasons make us confident that dopamine oscillation precisely reflects the behavior of neural dopamine. First, contribution of hypodermic dopamine to whole head dopamine levels has been reported to be no more than 15% 
. Second, pale
transcripts, which encode tyrosine hydroxylase (TH), have been shown to cycle circadianly in whole heads but not in bodies 
, indicating that hypodermal dopamine is not under circadian control. Finally, expression of Ih
in adult heads has been detected in neural tissues 
, suggesting that Ih affects neural dopamine. Our attempts to measure dopamine in brains have shown that time spent at dissecting is critical, as dopamine becomes rapidly oxidized and dopamine values drop to undetectable values for dissecting times over five minutes (not shown). Moreover, because flies used for each time point must be kept on ice while waiting to be dissected, these anesthetizing conditions imply a change in their physiological state that could influence dopamine content, possibly differentially affecting mutant and wild type flies, and thus precluding the use of this technique for obtaining reliable dopamine measures.
Daily fluctuations of dopamine have already been reported in mammals, and also suggested in Drosophila
based on the circadian oscillations of TH and dopamine receptor responsiveness, but a detailed study of dopamine levels at different time points was lacking. Our results show rhythmicity in the daily levels of dopamine in Drosophila
, with a pronounced bimodal pattern at daylight and a smooth increment towards the end of the night. When analyzing the contribution of the light signal versus the circadian control in this rhythmic behavior, through a comparison of LD and DD dopamine values, it turns out that the bimodal pattern during the day is driven by light while dopamine levels at night are under circadian control. A link between light and dopamine is supported by studies in vertebrates showing that dopamine plays critical roles in the light-induced resetting of the peripheral retinal circadian clock 
, but also by a recent report in Drosophila
demonstrating that dopamine is necessary for low light circadian entrainment 
. The circadian control of dopamine levels at night may reflect the importance of keeping adequate dopamine signaling. It has been shown that the Drosophila
dopamine receptor dDA1 responds homeostatically by downregulating its expression when dopamine signaling is increased 
. The physiological role of dropping the levels of dopamine just before the onset of the light phase could be a means of resetting proper amounts of receptors to respond adequately to the changing dopamine levels during the light phase.
How light affects the dopamine outcome is not known, but our results clearly demonstrate the implication of Ih current in maintaining its bimodal pattern. DmIh
is expressed in photoreceptors 
and although lack of this current does not impair gross responses to light, we cannot rule out an effect on the circuitry conveying light reception to inner brain centers. In fact, evidence for an involvement of Ih in early visual processing comes from the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors 
and by investigations that suggest that Ih current in the rod retinal pathway may contribute to shape the retina light response 
Ih current is also involved in maintaining physiological levels of dopamine, because abolishing this current strongly increases dopamine amount in dark conditions. A number of studies have implicated the hyperpolarization-induced, nonselective cation conductance Ih in the firing activity of mammalian dopaminergic neurons, emerging as one determinant of their spontaneous firing rate 
. Our result could be surprising in view of the results on dissociated dopaminergic neurons. When pharmacologically blocking this current, cultured dopaminergic neurons lower their firing rate, presumably leading to reduced dopamine release, an outcome apparently contrary to our results. However, the contribution of Ih current to the final performance of a neuron is not so straightforward. Despite the fact that Ih provides a depolarizing current at sub-threshold potentials, results from several studies have indicated that it has a paradoxical inhibitory effect by activating the Im potassium current 
. Also, the different subcellular localization of the channel endows different electrical properties to the neuron 
. Moreover, DmIh
mutant flies lack Ih current not only in dopaminergic neurons, but in the entire nervous system, possibly affecting input signals to these cells. Finally, dopamine autoreceptors provide important feedback control during dopamine signaling by governing firing rate, synthesis, and release. Therefore, by acting at different cellular and subcellular locations, Ih current could be critical for proper functioning of the dopaminergic negative feedback loop to prevent excessive release of neurotransmitter.
One of the consequences of the anomalous dopamine amount, when abolishing Ih current, is sleep fragmentation. Previous reports have shown that reducing dopamine signaling, through mutations in dopamine receptors, increases the duration of sleep episodes, suggesting more consolidated sleep 
. Conversely, enhanced dopamine signaling is associated with sleep fragmentation 
deficient flies show a dramatic fragmentation of sleep at nighttime, which is consistent with their increased dopamine levels. This trend towards sleep fragmentation is also observed in mutant flies at daytime, suggesting that disrupting dopamine cycling during the light period can also affect sleep consolidation. Results showing that pharmacologically decreasing the amount of dopamine restores sleep consolidation in mutant flies are consistent with this phenotype being dopamine-dependent (). Our evidence suggests that Ih current, possibly through maintaining proper levels of dopamine, have an effect on the consolidation of sleep.
In general, genetic and pharmacological changes in dopamine content affect both total sleep and sleep consolidation in flies 
, and that is what we observe in control flies. Surprisingly, DmIh
flies have elevated dopamine levels and sleep fragmentation, but total sleep is not significantly altered, nor even by 3YI-treatment. Because the lack of Ih current is the basic difference between control and mutant flies, their differential influence on total sleep must rely on the Ih current itself, or on its possible effects on the release of other neuromodulators involved in the regulation of sleep 
. It would be interesting to tackle this issue in future investigations.
A number of reports positively correlate dopamine and locomotor activity 
. Our data showing that when the bimodal pattern of dopamine is lost (in DmIh
mutant flies), more than 50% of the flies also lack the bimodal activity pattern (), are consistent with an association between dopamine oscillations and locomotor activity. Nevertheless, caution should be taken when interpreting these results because each dopamine measure is an average of 20 brains from a mixture of flies displaying the two different locomotor patterns, i.e. bimodal and non-bimodal. Nevertheless, dopamine should be considered as a modulator of activity rather than responsible for a quantitative signal/response effect. In fact, transient activation of TH-expressing dopaminergic cells (using transgenic ion channels) has opposite effects on activity depending on the previous behavioral state right before photostimulation 
. This could explain the variability found in the locomotor activity pattern of flies ( and ), as well as why the bimodal patterns in LD of both dopamine and activity are not exactly coincident (). Nevertheless, dopamine signalling has also been involved in many other behavioral processes, such as courtship, visual, olfactory and appetitive learning, or mechanosensation 
. The emerging picture indicates that sleep/activity, behavioral arousal, and even learning and memory, are influenced by anatomically distinct sets of dopaminergic cells. Moreover, besides sleep/activity, many of these behaviors show circadian patterns, with maximum performance usually attained during the (subjective) night 
. Therefore, variations of dopamine levels may differ at different anatomical localizations, complicating an interpretation aimed at explaining an individual behavior in terms of total dopamine levels. Even so, it is tempting to speculate that the cyclic pattern of total dopamine in DD conditions may reflect dopamine requirements for both wakefulness (maximum during the subjective day) and behavioral arousal (maximum during the subjective night), but further experiments would be needed to established such a relationship.
The locomotor pattern of activity in DD reveals some interesting features of DmIh
mutant flies: 22.6% become arrhythmic and only 24.5% show robust rhythmicity, and when analyzing the circadian parameters of those rhythmic flies, the most striking fact is a shortening of period length. These abnormalities of circadian rhythm could be a consequence of lack of Ih current in the circuitry underlying the final output of the clock, but PDF secretion from LNvs in DmIh
mutant flies does not differ from control flies, suggesting that altered circadian rhythm may be due to factors downstream of the LNv clock, or alterations in other, non-PDF clock cells 
. Further support for normal activity of the LNv morning-oscillator comes from the observation that, contrary to wild type flies, DmIh
mutant flies kept in DD conditions maintain activity in the first half of the subjective day (), which is under LNv control 
. On the contrary, while wild type flies tend to be more active during the subjective evening, mutant flies show a progressive decay of activity during this period, suggesting alterations in the clock circuit responsible for this phenomenon.
Interestingly, similar defects in rhythmicity and period length have been reported for ebony
mutants, and also the central LNv clock seems not to be affected 
. These mutant flies lack N-β-alanyl-dopamine synthetase activity in glial cells, and have elevated levels of dopamine. Although we cannot assume that the abnormalities of circadian rhythm in DmIh
mutant flies are due to elevated dopamine levels, mutual interactions between dopamine and peripheral circadian clocks have been reported in other systems. In the vertebrate retina, dopamine regulates the phase and amplitude of retinal molecular rhythms and participates in light-induced resetting 
. In rodents, activation of the dopamine D2 receptor signaling cascade results in enhancement of clock genes transcription 
, and disruption of dopamine signaling leads to disruption of circadian rhythms in selected forebrains regions and consequent alterations of circadian locomotor behavior 
, with no effect on molecular rhythms in the central suprachiasmatic nucleus (SCN). Moreover, numerous studies have revealed the existence of a methamphetamine-sensitive circadian oscillator, further supporting a role for the mesolimbic-dopaminergic system as a SCN-independent oscillator 
. Thus, dopamine emerges as an important regulator of peripheral brain clocks in vertebrates, a role that may well be conserved in Drosophila
A link between Ih current and dopamine signaling has been suggested by electrophysiological studies in mammalian dopaminergic cells, and by isolated reports involving Ih current in various dopamine-related disorders 
. However, to our knowledge, our work represents the first in vivo
analysis in which this association has been demonstrated and the behavioral consequences have been analyzed in a complete organism, and, interestingly, our results suggest significant evolutionary conservation. Moreover, we have demonstrated that Ih current regulates dopamine circadian and light-dependent oscillations, and provided evidences indicating that cyclic dopamine signaling is essential for normal behavior. Therefore, our data should be considered not only in view of the value of HCN channels as therapeutic targets, but also when approaching functional and pathological studies of dopamine-related processes. In this sense, our data corroborate the usefulness of Drosophila
as a model for these types of studies.