Hamasaka et al. (2007) proposed that glutamate inhibits LNv activity via the metabotropic mGluRA glutamate receptor. Quisinostat in vivo They also showed that light avoidance levels are increased in mGluRA mutant larvae, although they did not determine the relevant cells ( Hamasaka et al., 2007). However, our
gene expression profiles from purified larval LNvs revealed that they also express the glutamate-gated chloride channel GluCl ∼2.5-fold more highly than in Elav+ neurons (M. Ruben & J.B., unpublished data). Adult l-LNvs also have functional GluCl channels, although their behavioral role is unknown ( McCarthy et al., 2011). To test whether glutamate regulates light avoidance in LNvs via GluCl or mGluRA, we used RNAi to reduce expression of each receptor. Both transgenes reduce expression of their target (Hamasaka et al., 2007 and Figure S4C). We found that Pdf > GluClRNAi larvae had significantly increased light avoidance at 150 lux, whereas Pdf > mGluRARNAi and control larvae did not avoid light ( Figure 5C). Thus, reducing GluCl INCB018424 in LNvs phenocopies reducing glutamate release from DN1s. Next, we tested the roles of GluCl and mGluRA in regulating circadian behavior. Our data show that Pdf > GluClRNAi larvae had no light avoidance rhythm, with levels of light avoidance
constitutively high ( Figure 5D), whereas Pdf > mGluRARNAi larvae display rhythmic light avoidance ( Figure 5D). Thus, GluCl is required in LNvs for rhythmic
light avoidance. We propose that DN1s rhythmically release glutamate, which is perceived via GluCl in LNvs to mediate rhythmic inhibition of LNv neuronal activity. We have subsequently found that mGluRA helps synchronize LNv molecular clock oscillations (B.C. and J.B., unpublished data). To directly test whether GluCl can inhibit LNv activity, we measured the responses of dissociated larval LNvs expressing the intracellular Ca2+ sensor GCaMP1.6 (Reiff et al., 2005) to directly applied neurotransmitters. ACh produced by Bolwig’s Urease organ is required for larval light avoidance (Keene et al., 2011). Applying ACh to dissociated LNvs increased intracellular Ca2+ levels, as previously reported (Dahdal et al., 2010 and Wegener et al., 2004), as measured by increased GCaMP fluorescence (Figures 5E and 5F). ACh increases intracellular Ca2+ in LNvs by activating nicotinic ACh receptors to produce excitatory postsynaptic potentials, eventually causing depolarization. In turn, this increases cytoplasmic Ca2+ via voltage-gated Ca2+ channels (Dahdal et al., 2010 and Wegener et al., 2004), which is observed as increased GCaMP fluorescence. Given the relative insensitivity of GCaMP1.6 to single action potentials (Pologruto et al., 2004), these Ca2+ transients in LNvs likely reflect bursts of action potentials.
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