Glutamate Is a Wake-Active Neurotransmitter in Drosophila melanogaster

Abstract

Introduction: In mammals, there is evidence that glutamate has a role as a wake-active neurotransmitter. So using video-based analysis of Drosophila behavior, we undertook a study to examine if glutamate, which has been previously shown to have an excitatory role in neuromuscular junctions in Drosophila, may have a conserved wake-active role in the adult brain.

Aims and methods: Using 6- to 9-day-old female flies, we examined the effect of perturbations of the glutamatergic signaling on total wakefulness and wake bout architecture. We increased and decreased neuronal activity of glutamatergic neurons in the brains of adult flies using Upstream Activating Sequence (UAS) NaChBac and UAS EKO, respectively. We blocked neurotransmission from glutamatergic neurons in adult flies using the UAS-driven temperature-sensitive dynamin mutation shibirets. We examined the behavior of flies with loss of function mutations of individual subunits of brain-specific ionotropic glutamate receptors.

Results: Increasing the activity of glutamatergic neurons in the adult brain led to a significant increase in wakefulness compared to the control groups both in the daytime and nighttime and decreasing the activity of these same neurons reduced wakefulness in the nighttime. Blocking neurotransmitter release in glutamatergic neurons significantly reduced wake in the nighttime. The ionotropic receptor mutants had significantly less wake in the nighttime than their respective genetic background controls.

Conclusion: The results show the following: glutamate is indeed a wake-active neurotransmitter in Drosophila; there is a major time of day effect associated with loss of glutamatergic neurotransmission; and it is a major wake-active neurotransmitter in the nighttime.

Keywords: Drosophila melanogaster; sleep duration.; synaptic plasticity; wake bout.

Figure 1

Temperature shift affects wake and sleep behavior. A. Average total wake is shown for the 12 h of lights on (daytime) and lights off (nighttime) for WRR females kept at 22°C (pre-shift, open bars) and 29°C (post-shift, black bars). Increasing ambient temperature significantly decreases daytime total wake while increasing nighttime total wake (***p < .0001). B. Average wake bout duration is shown for WRR females in the same conditions described in panel A. Increasing temperature decreases average wake bout duration during the day but increases it at night. ***p < .0001, **p < .001, average values plus SE are shown in both panels.

Figure 2

Effects of increasing glutamatergic neuronal activity. A. The average changes in total wake from baseline temperature (22°C) to restrictive temperature (29°C) are shown for control genotype tubulin-GAL80^ts/+ VGlut^CNSIII GAL4/+ (GAL80;GAL4) (stippled bar), control genotype UAS NaChBac/+ (UAS NACHBAC) (light gray bar), and the experimental genotype UAS NaChBac/tubulin-GAL80^ts; VGlut^CNSIII GAL4 (UAS NACHBAC/GAL80;GAL4) (black bar) for the 12 h of lights on (daytime) and lights off (nighttime). Activating glutamatergic neurons increases wakefulness. B. The change in average wake bout duration following temperature shift is shown. Genotypes and conditions are the same as in panel A. Activating glutamatergic neurons increases wake bout duration both during the day and at night. For all panels, lines indicate specific pair-wise comparisons, otherwise significance markers without lines indicate significant differences versus all other genotypes, ***p < .0001, **p < .005, *p < .05, average values plus SE are shown.

Figure 3

Effects of reduced neuronal activity on sleep and wake following temperature shift. A. The average change in total wake from baseline temperature (22°C) to restrictive temperature (29°C) is shown for control genotype tubulin-GAL80^ts/+ VGlut^CNSIII GAL4/+ (GAL80;GAL4) (stippled bar), control genotype UAS EKO/+ (UAS EKO) (white bar), and the experimental genotype UAS EKO/tubulin-GAL80^ts; VGlut^CNSIII GAL4 (UAS EKO/GAL80;GAL4) (gray bar) for the 12 h of lights on (daytime) and lights off (nighttime). Reducing glutamatergic neuronal activity reduces wakefulness at night. B. The change in average wake bout duration following temperature shift is shown. Reducing glutamatergic neuronal activity reduces the average wake bout duration. Genotypes and conditions are the same as in panel A. Reducing glutamatergic neuronal activity reduces wake bout duration during the daytime. For all panels, lines indicate specific pair-wise comparisons, otherwise significance markers without lines indicate significant differences versus all other genotypes, ***p < .0001, **p < .01, *p < .05, average values plus SE are shown.

Figure 4

Blocking glutamate release with UAS shibire blocks increased wake following temperature shift. A. The average change in total wake from baseline temperature (22°C) to restrictive temperature (29°C) is shown for control genotype VGlut^CNSIII GAL4/+ (GAL4) (white bar), control genotype UAS shibire^ts/+ (UAS shibire) (gray bar), and the experimental genotype VGlut^CNSIII GAL4; UAS shibire^ts (GAL4; UAS shibire) (black bar) for the 12 h of lights on (daytime) and lights off (nighttime). UAS shibire^ts expression suppresses the increase in nighttime wake seen in the controls but does not significantly change the reduction in daytime total wake. B. The change in average wake bout duration following temperature shift is shown. Genotypes and conditions are the same as in panel A. Blocking glutamate release leads to no increase in wake bout duration at night with increased temperature that occurs in control genotypes. For all panels, lines indicate specific pair-wise comparisons, otherwise significance markers without lines indicate significant differences versus all other genotypes, ***p < .0001, average values plus SE are shown.

Figure 5

Insertion mutations of the ionotropic glutamate receptor subunits also have reduced wakefulness during the nighttime but not daytime. A. Average total wakefulness in minutes is shown for the 12 h of lights on (daytime) and lights off (nighttime) for Minos insertions GluRIB^mb02126 (n = 43) and GluRIB^mb03843 (n = 40), and PiggyBac insertion GluRIA^f05411 (n = 38) and their respective w¹¹¹⁸ background strains w^1118a (Minos, white bar) and w^1118b (PiggyBac, stippled bar). All of these insertion mutants have reduced nighttime wakefulness compared to their wild-type control. B. Average wake bout durations for daytime and nighttime are shown for the same strains described in panel A. Insertion mutants have reduced wake bout duration during the nighttime. For all panels, lines indicate specific pair-wise comparisons otherwise significance markers indicate versus genetic background control, ***p < .0001, *p < .05, average values plus SE are shown.

Figure 6

Steps of glutamate neurotransmission are shown and the relations of the experiments to glutamate neurotransmission presented in this paper are indicated. In presynaptic cells, at the restrictive temperature, UAS NaChBac and UAS EKO increased and decreased, respectively, the signal to the presynapse, which increased or reduced the amount of glutamate released into the synaptic cleft. Also in presynaptic cells, at the restrictive temperature, UAS shibire^ts blocks the release of glutamate into the cleft. In postsynaptic cells, the mutations of the ionotropic receptors, GluRIA and GluRIB, reduce the effect of glutamate on post synaptic cells.