The DISCO device
In this study, we demonstrate a new method for investigating light-based behaviours in Drosophila by merging widely used equipment for locomotion monitoring and a computationally controlled light system into an apparatus named the Drosophila Interactive System for Controlled Optical manipulations (DISCO). We based our device on the commercially available MB5 activity monitor; however, DISCO modifies the existing infrared-based detection of locomotion by adding a robust, yet straightforward, custom set-up for light-controlled interventions. In fact, our objective was to create simple modifications that can be easily adapted to different activity monitors according to the experimental need, while providing a flexible platform for a range of protocols.
We achieved this goal by integrating RBG-LED lights into the motion detector device, while managing the lights’ function by implementing a MATLAB-Arduino system. These steps, which are easily reproducible, allow us to (i) create complex lights on/off protocols with millisecond precision, (ii) investigate light-based behaviours during short- and long-time monitoring, (iii) study the effects of different light frequencies on Drosophila activity, and (iv) develop second-by-second feedback loop protocols where the motion or position of the flies can modulate the light intervention.
Due to such versatility, the DISCO platform can be used in a variety of ways to study light-dependent behaviours in Drosophila. For instance, in addition to the protocols presented here, it may be used to study light-based appetitive and aversive behaviours, as well as for optogenetic studies. Moreover, the use of the platform for long-term experiments allows for the analysis of circadian responses. Due to the easy device assembly and the possibility of testing multiple individual flies, DISCO can be easily employed for high-throughput experimental settings, which can significantly contribute to drug screenings or genomic studies.
Sudden darkness-induced locomotion
The responses of Drosophila to light-dark stimuli are studied in a range of paradigms. In Drosophila larvae, protocols with intermittent light and dark pulses, also called ON/OFF assays, have been used to measure several locomotory parameters. Larvae show an increase in the distance travelled on the lights-off onset while displaying more head swinging and change of direction behaviours on lights-on onset [13, 14]. This behaviour is shown to be independent of the neuronal circuitry underlying circadian rhythmicity [15]. Notably, the response to light is abolished during the transition to mid-larval third instar, where there is a change from foraging to wandering period, as the fly searches for a site for metamorphosis [16].
In adult flies, the presentation of sudden darkness (lights-off) stimuli triggers a jump response, known to be mediated by the pair of giant descending neurons, also called giant fibres, which convey visual and mechanosensory information to the thoracic ganglia neurons that control the legs and wings [17, 18]. Activation of the giant fibres initiates a motor response in which the fly jumps, thrusted by the mesothoracic legs, with no wing control and apparent directionality [19, 20]. Importantly, the probability of eliciting this jump response in wild-type red-eyed lines is reported to be relatively low (34–37%) in comparison with white-eyed lines (58–97%) [18].
In contrast, a visual looming stimulus that mimics approaching objects prompts a well-coordinated series of stereotypic movements as an escape response for both red-eyed and white-eyed flies [21]. These include preparatory leg movements for a directional motion followed by long or short take-offs, where wing extension depends on the required escape speed [12], similar to voluntary flight initiation [19]. Further dissection of the response to looming stimuli indicates that when take-off is not elicited, flies either exhibit running or freezing behaviours, where the probability of freezing is strongly dependent on baseline activity, as flies moving slower were more likely to freeze upon looming stimulation than flies moving faster [22].
However, most studies on visually evoked escape responses focus on the characterisation of the behavioural components within time frames of milliseconds to a few seconds. Thus, we employed DISCO to describe long-term changes in locomotion elicited by a single ‘lights-off’ stimulus presentation for both white-eyed and red-eyed flies. By using infrared-based detection of locomotion, we demonstrate a previously undescribed increase in activity elicited by a 1-s darkness presentation up to 30 s after stimuli. We show that this behaviour is present in white-eyed and red-eyed fly lines, although white-eyed flies are more sensitive to the stimuli. Moreover, we show that Fmr1 null allele flies, a model of fragile X syndrome, do not exhibit the sudden darkness-induced increase in locomotion. We also show that the observed differences in locomotion after lights-off stimuli between fragile X and control flies cannot be accounted for by deficits in baseline locomotion, as the measured activity and speed before stimulation was comparable or higher to the control lines. Previous studies already reported deficits in odorant sensory responses in fragile X flies [23, 24]; however, to the best of our knowledge, this is the first description of deficits in visually evoked motor responses for this model.
Analysis of the response from single flies shows a highly variable response across individual flies and trials. Red-eyed CSORCs exhibited were completely unresponsive (delta values equal to zero) in 59% of stimuli presentations over the trials, while white-eyed w1118 flies failed to respond only 31% of the time. Interestingly, these ratios are similar to the previously reported jump response probability after lights-off [18]. Moreover, paired analyses of movement counts before and after stimuli showed that the behaviour of individual flies is significantly variable across trials, indicating that although the response is robust at a population level, further investigations are necessary to understand the factors driving the response of a single fly. By correlating locomotion values before and after stimuli, we also observed that flies with higher activity before lights-off were more prone to exhibit increased activity after stimuli, which resembles the literature reports using looming stimulation [22].
Additionally, by making use of the flexibility in the LED programming of the DISCO platform, we explored the different protocols of sudden darkness stimulation, showing that the locomotion response can be inhibited by repeated stimuli presentation. In white-eyed w1118 flies, this desensitisation occurred by repeated stimulation with intervals of 5 min, while for the red-eyed CSORC wild-type flies, the decrease in response only occurred by darkness presentation with 1-min intervals. Interestingly, when looking at individual trials, even initial ones, the average of delta movements is often reduced or even negative; however, after grouping averages in a bigger temporal frame (i.e. 30-min intervals), the group effect of increased mobility is more evident. We believe that due to the highly variable responses among individuals and trials, increasing the number of measurements by shortening the intra-trial intervals amplifies the visualisation of the variability of the results.
Taken together, our findings reinforce that Drosophila can respond and desensitise to sudden darkness (lights-off) presentation and confirm previous literature describing white-eyed flies as more sensitive to such stimuli. Nevertheless, these results should be interpreted with caution due to their relatively high variability at the level of individual flies and shorter inter-trial intervals, and further studies are needed to investigate the relationship between this behaviour and the widely studied jump response.
Alcohol preference and circadian rhythm
The relationship between alcohol consumption and circadian rhythm has been widely studied in the literature, demonstrating that not only can ethanol intake alter the circadian clock and its mechanisms, but also a modulation of consumption depending on daytime [25]. For instance, human studies observed higher levels of alcohol consumption in individuals with evening chronotypes [26, 27]. Accordingly, alcohol intake also peaks at night hours in rodents, which naturally show a nocturnal profile of activity [28].
Due to similar circadian clock and reward systems, the fruit fly has also been a useful model to describe the interactions between the circadian clock and ethanol consumption [29]. Reports show that Drosophila exhibits higher sensitivity to alcohol during the mid to late-night phases, both under light-dark cycles and constant darkness [30, 31]. Recovery from the sedative effects was also significantly greater at night phases [30]. Notably, the circadian rhythmicity of sensitivity and tolerance to ethanol sedation is eliminated after mutation of certain circadian genes and under a constant light regime, corroborating the need for the circadian oscillator to modulate alcohol-induced sedation [30, 32]. However, these results were obtained by administering ethanol gas to the flies to assess their behaviour after consumption, while circadian patterns of active ethanol intake remained unexplored.
Thus, to investigate this question and demonstrate the use of DISCO for long-term experiments, we developed a 48-h ethanol preference test, where the lights were programmed into a 12:12 light-dark cycle. The interaction with the food options (with or without 15% ethanol) was inferred from the position of the flies measured by the infrared beams. We observed that ethanol preference happens intermittently throughout the day and was significantly higher in many hours of the dark phases. Correlation analysis of alcohol preference and daytime showed a small but significant trend for increased ethanol consumption during the dark phases. These results relate to results seen in rodents, which indicate higher alcohol consumption during the night [28]. They also implicate that flies tend to consume ethanol during periods of higher sensitivity to its effects. However, one should notice that the correlation analysis employed to test daytime trends in ethanol preference linearises the multimodal nature of the intake behaviour. Thus, further studies with bigger sample sizes are warranted to comprehensively describe if there are specific peaks of ethanol preference throughout the day. Additionally, future assays on DISCO could determine if specific circadian genes can influence the active seeking for ethanol intake, as for sensitivity and tolerance. Finally, although the indirect assessment of food interaction limits the interpretation of the data, these results are the first to indicate a role of light-dark phases on alcohol intake in flies.
Light colour-based place preference
Place preference studies based on avoidance behaviours in Drosophila commonly rely on heat as an aversive stimulus, in which single freely walking flies are placed in a narrow box in complete darkness and conditioned to avoid one-half of the box by instantaneous heat presentation upon entering that area [33]. Alternatively, flies can be tested in a thermal–visual arena, where they can use environmental cues to find a hidden cool tile in an otherwise unappealing warm environment [34, 35]. However, the widespread use of assays based on a single type of aversive stimulus bring limitations when studying the machinery behind integrating different sensorial elements during learning.
With that in mind, we designed a protocol to explore the innate aversion of flies to blue light and their preference for green light [8]. The avoidance behaviour of blue light is shown to be independent of the circadian clock and relies on the TRPA channel painless, which is primary for nociception in flies, making this stimulus a good candidate for aversive-based behaviours [8]. Thus, we implemented a feedback loop protocol using DISCO, where the experimental tubes containing the flies were divided into two zones (i.e., left and right). When the flies crossed to a given zone, either blue or green LEDs were turned on.
As expected, flies learned to avoid the zones illuminated with blue light. Interestingly, CSORC flies show avoidance of the blue zones from the first hour of the training session, while w1118 flies display a slower learning curve. These results are in line with previous research showing that w1118 mutants perform worse than wild-type flies in heat-induced place conditioning tests, as they have lower levels of serotonin and dopamine [36]. To verify if this avoidance behaviour is in fact related to learning, CSORC flies were tested in a 10-h session, where the zones were interchanged at the 5th hour. We observed that after the zones were changed, the flies were unable to avoid the blue zones, indicating that the place preference is dependent on the association made in the initial hours of the session. Accordingly, it was shown that flies can integrate visual cues to learn place preference but changes in these cues disrupt learning [34]. Our results indicate that operant conditioning based on blue light exposure can be used as an additional tool for various studies, from sensory perception to learning and memory.