Light response behavior and circadian clock entrainment require light signals from the environment, mediated by distinct visual pathways (Keene et al. 2011). As in mammals, sensing light is important in Drosophila larvae. Moderate light is perceived by the Bolwig Organs (BOs), which are composed of 12 pairs of photoreceptors (PRs) adjacent to the mouth hooks (Steller et al. 1987). Four PRs express Rhodopsin5 (Rh5), which provides for the sensation of blue light, whereas the other eight PRs express Rhodopsin6 (Rh6) and sense green light (Sprecher and Desplan 2008). Genetic ablation of BOs or Rh5 mutation disrupts rapid light-induced responses at moderate light intensities (Busto et al. 1999; Hassan et al. 2000; Mazzoni et al. 2005). However, entrainment of circadian rhythms is sufficient with either Rh5 or Rh6 (Keene et al. 2011). Class IV multidendritic neurons that tile the larval body wall also drive light avoidance (Xiang et al. 2010). The axons of larval PRs synapse with neurons that express circadian rhythm genes: four pigment dispersing factor (PDF)-expressing lateral neurons (PDF neurons) and a fifth non-PDF lateral neuron (the 5th LN) (Sprecher et al. 2011; Yuan et al. 2011). Light avoidance may be modulated by the combined action of the lateral neurons and circadian related dorsal neurons (Keene et al. 2011; Collins et al. 2012). Two pairs of prothoracicotropic hormone neurons downstream of PDF neurons have been reported to be involved in switching between light avoidances and preferences (Gong et al. 2010; Yamanaka et al. 2013).
γ-glutamyl transferase (GGT) is an enzyme that is evolutionary conserved from bacteria to mammals and plays an important role in antioxidant defense, detoxification, and inflammatory processes (Castellano and Merlino 2012). GGT is mainly located on the external surface of the plasma membrane where it catalyzes and transfers the γ-glutamyl moiety of GSH, and other γ-glutamyl-containing compounds, to substrates such as water, dipeptides, and amino acids (Tate and Meister 1981). Thirteen homologs of human GGT have been identified (at least six are active), and four have been identified to exist, so far, in rats (Pawlak et al. 1988; Potdar et al. 1997; Yamaguchi et al. 2000; Puente and Lopez-Otin 2004). In Drosophila, four genes have been predicted to code the GGT protein according to sequence alignment (Walker et al. 2006). Of these homologs, Ggt-1 encodes the male accessory gland protein, which may result in relatively high levels of free glutamic acid in accessory gland secretions (Walker et al. 2006).
In this study, we report that Ggt-1 knockdown in larvae produced a higher percentage of green-light avoidance compared with controls after RNAi screening. However, following Ggt-1 knockdown, responses to blue light did not change significantly. Further results showed that Ggt-1 impacted green-light avoidance by regulating the level of glutamate, which was further supported by feeding experiments. Moreover, glutamate did not directly function as a neurotransmitter rather it was transformed to GABA to produce this effect. Thus, our results implicate a neural pathway that regulates green-light avoidance, which was suppressed by GABA signals in wild-type larvae.
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- Acknowledgments and conflict of interest disclosure
- Supporting Information
In vertebrates and invertebrates, light sensing is important for detecting foods, mates, predators, and prey, or simply for circadian entrainment, pupil reflexes, and phototaxis. Drosophila share many key characteristics with mammals. For example, they use glutamate as their main excitatory neurotransmitter and GABA as their main inhibitory neurotransmitter. Light sensing in Drosophila also shares many key characteristics with mammalian light sensing. Like in mammals, photoreceptors used to sense light in Drosophila are also composed of a vitamin A-based chromophore and opsins, which have seven-transmembrane structures (Yau and Hardie 2009). The human eye perceives equal absolute irradiance green and blue as brighter green (Rovamo et al. 1996). Similarly, Drosophila perceives blue and green as having a different brightness (Zhou et al. 2012). Moreover, in Drosophila larval light avoidance, blue light dominates light avoidance, whereas green light produces very weak avoidance. Here, we reveal the molecular basis of differences between green- and blue-light avoidance in Drosophila larvae.
In previous studies, Rh6 mutation or ablation of Rh6 PRs did not affect light avoidance, but Rh5 mutation or ablation of Rh6 PRs diminished light avoidance (Keene et al. 2011; Kane et al. 2013). These results indicate that green-light-sensitive Rh6 PRs are not required for light avoidance. Experiments by Xiang et al. (Xiang et al. 2010) have shown that larvae have weak green-light avoidance. In our experiments, we also observed larval responses to green light, and in green-light-sensitive Rh6 mutants, such responses were almost eliminated.
Using RNAi techniques to screen genes involved in light avoidance behavior, we found that knocking down Ggt-1 enhances larval green-light avoidance, as attested by Ggt-1 null mutants. In mammals, GGT is most abundant in the kidneys (Tate and Meister 1981). In Drosophila, we also found that expression of Ggt-1 in MT (organs analogous to the kidneys in mammals) affects larval green-light avoidance. As in mammals, GGT can hydrolyze GSH to generate glutamate. In Drosophila, we have observed that GGT-1 regulates the levels of glutamate in the brain.
GGT plays an important role in maintaining the homeostasis of GSH through γ-glutamyl cycle in mammals (Zhang et al. 2005) and it is the most abundant non-protein thiol in cells, which has multiple critical biological functions, such as protecting cells from oxidative or other forms of stresses (Dickinson and Forman 2002). In our experiments, increased green-light avoidance is not the consequences of stresses because Ggt-1 null mutants do not show a significant difference in the percentage of light avoidance for blue light compared with control larvae. We also tested Ggt-1 null mutants in the light-dark choice assay, and they showed similar light avoidance to w1118 larvae (Figure S2).
In mammals, GGT can hydrolyze GSH, providing a physiological reservoir of glutamate (Elce and Broxmeyer 1976; Koga et al. 2011), the principal excitatory neurotransmitter of the CNS. Our findings that glutamate levels are decreased in Ggt-1 null mutant indicate that hydrolytic effect of GGT-1 is also a source of glutamate in Drosophila larvae. Because of the large surface of MT bathing the hemolymph and the location of GGT in the outer membrane, it is reasonable to assume that glutamate generated by GGT proteins in MT cells can be directly released and diffused in the hemolymph. A recent study shows that hormones produced by prothoracicotropic hormone neurons can be secreted into and spread in hemolymph and functions at BOs and class IV dendritic arborization neurons to affect larval light avoidance (Yamanaka et al. 2013), which indicates that hemolymph is a medium to spread neuromodulators. This can also explain our results that glutamate generated by MT might spread in hemolymph and be transported to the brain to impact green-light avoidance.
Glutamate is the main excitatory neurotransmitter in mammals. To test whether glutamate functions directly as a neurotransmitter in larval green-light avoidance, Vglut was knocked down to decrease glutamatergic neurotransmission. VGlut is the homolog of Vglut in mammals and only one single vesicular glutamate transporter exists in the Drosophila genome (Daniels et al. 2006). It takes charge in loading glutamate into synaptic vesicles within the synaptic terminal and thus it is important for synaptic output. Our findings show that decreasing glutamatergic neurotransmission does not release the inhibited green-light avoidance in larvae, indicating that glutamate may not function directly in the light avoidance circuit as a neurotransmitter. As in mammals, glutamate can be transformed to GABA by catalytic effects of GAD in Drosophila. Focusing on the possible role of GABA, our findings reveal that GABA levels in the brain indeed decreases in Ggt-1 mutants. Both glutamic acid decarboxylase 1 mutants and GABAA receptor Rdl knockdown lines can elevate green-light avoidance in larvae. Hamasaka et al. (2005) have shown that GABA signals entered the larval optic center, where the axons of BOs project and can be traced back to the lateral midbrain. These observations suggest that GABA signals may be released in this area to inhibit green-light avoidance. Therefore, we hypothesize that, in wild-type larvae, green light signals from BOs project to larval optic center, and are inhibited by GABA signals. More study is needed to test this hypothesis.
Although the biological significance of the inhibition of responses to green light in wild-type larvae is unknown, we speculate that it may be related to larval foraging behavior for food. Green light has a longer wavelength compared with blue light, and therefore reaches deeper layers of food after the food is diluted by larvae. Blue light, however, is easily scattered on the surface of food because of its shorter wavelength. When larvae crawl on the surface of food, they are driven by blue light to dig into the food. After digging into the deeper layers of food, where green light is more abundant, the need to generate robust avoidance responses is no longer required. This phenomenon may be important for preventing larvae from digging too deeply into food.
Our results reveal that green-light avoidance is inhibited by GABA signals. Since our results indicate that green-light avoidance and blue-light avoidance may involve different neural circuits, future studies could further explore the neural circuits generating green-light avoidance in the brain and how the signals from green light are inhibited by GABAergic neurons to better understand light avoidance behavior.
Acknowledgments and conflict of interest disclosure
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- Acknowledgments and conflict of interest disclosure
- Supporting Information
We are grateful to Yongqing Zhang (Institute of Genetics and Developmental Biology, CAS) and Justin Blau (Department of Biology, New York University) for providing the flies. We thank Haiyun Gong, Yanchao Cai, Jing Liu, and Xudong Zhao of the IBP core facility center for technical assistance. We also thank the Bloomington Drosophila Stock Centers, the Vienna Drosophila RNAi Center, Drosophila Genetic Resource Center at Kyoto Institute of Technology, and Center of Biomedical Analysis at Tsinghua University for fly stock. This work was supported by the National Natural Sciences Foundation of China (31030037), the Ministry of Science and Technology of China (2012CB825504), and the Strategic Priority Research Program B of the Chinese Academy of Sciences (XDB02040200), the External Cooperation Program of BIC, Chinese Academy of Sciences (GJHZ201302).
All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.