Figure 6. L2 neurons are part of the circuit that promotes lLNv bursting. To test the hypothesis of L2 lamina neurons being part of a neuronal circuit that feeds visual information to lLNvs, we acutely activated L2 neurons via genetic expression of the TrpA1 heat-activated channel and perfusion of warmed solution while simultaneously recording electrophysiologically from lLNvs. A–C, Representative recording of a lLNv of the control genotype (L2>+, pdf-DsRed) that underwent a heat pulse. In this case, bursting frequency decays progressively from its basal 12 bursts/min to 10 bursts/min to 4 bursts/min in the 8 min spent perfusing the solutions. D–F, Representative recording of a lLNv of the experimental genotype (L2>TrpA1, pdf-DsRed) with a basal bursting frequency of 8 bursts/min, which increases to 14 bursts/min upon perfusion of warm solution. The effect of activating L2 neurons persists while the bathing solution is cooling and, in this case, after 8 min of recording, bursting has not decreased below its basal yet. G, Top, Bursting frequency decay profiles of the following groups are compared: TRP + HEAT refers to L2>TrpA1, pdf-DsRed with perfusion of warmed solution; TRP + NO HEAT refers to L2>TrpA1, pdf-DsRed without perfusion of warmed solution; and CTROL + HEAT refers to L2>+, pdf-DsRed with perfusion of warmed solution. Bursting frequency was normalized to basal for each neuron and afterward averaged with the others in the same experimental group, Mean ± SEM is shown. Two-way ANOVA statistical analysis taking the time as one factor and the group (GENOTYPE + TREATMENT) as the other indicates that bursting frequency decays in the three groups (F(16, 238) = 6.56, p < 0.01). However, this decay is not the same for all groups (F(2, 238) = 35.23, p < 0.01). Tukey post hoc analysis indicates that the decay in bursting frequency of the activated group is significantly slower than the decay in bursting frequency of either control (p < 0.01). No significant differences are found between the two control groups (p = 0.68). Bottom, Temperature profile in the chamber during the perfusion of warmed external solution. The time indicated in the bottom panel corresponds to those shown in the top panel. Gray area corresponds to the phase when bath temperature is increasing: nTRP + HEAT = 7; nTRP +NO HEAT = 5; nCTROL + HEAT = 5. A–C and D–F correspond to different parts of the same recording. H, GRASP analysis was performed to test for direct synaptic connections between different eye neuronal subtypes and LNvs. Among the neuronal types tested, only Gmr-GAL4 was able to reconstitute GFP in the context of pdf-LexA. The reconstituted signal was localized onto the HB tract (arrow). Genotype: pdf-LexA>LexAop-CD4spGFP11, Gmr>CD4spGFP1-10. Black indicates immunohistochemistry against reconstituted GFP; red, anti-PDF immunohistochemistry. Scale bar, 20 μm. I, Immunohistochemistry showing the two expression domains that reconstituted GFP in H. Genotype: Gmr>CD8GFP. Black indicates anti-GFP immunohistochemistry; red, anti-PDF immunohistochemistry. Scale bar, 20 μm.