Article Figures & Data
Figures
- Figure 1.
Location of viral injection site and optical probes. A, qRT-PCR amplification curves showing detection of VGluT2 mRNA from individual VTA microdissected neurons expressing mCherry under the regulation of the VGluT2 promoter (65 neurons). B, Expression of ChR2-eYFP at the viral injection site (VTA). VGluT2-eYFP neurons are localized to midline VTA (green), whereas TH-immunoreactive neurons are localized laterally (red). C, D, Example of an optical fiber placement in a ChR2-eYFP mouse. Yellow line indicates the optical fiber. eYFP fluorescence indicates axons from VTA VGluT2 neurons. b, Blood vessel. E, Distribution of implanted fibers. F, G, Example LHb c-Fos immunoreactivity (brown/black puncta; F′, G′, arrowhead) in ChR2-eYFP (F) and control (G) mice. H, Light stimulation of VGluT2-mesohabenular fibers significantly increased c-Fos immunoreactivity in LHb but not paraventricular thalamus (PVT), in VGluT2-ChR2 mice compared with VGluT2-control mice [data are mean ± SEM (error bars)]. *p < 0.05. Scale bars: B, F, G, 0.25 mm; C, 1 mm; D, 0.2 mm; F′, G′, 0.025 mm.
- Figure 2.
Activation of VGluT2-mesohabenular fibers elicits aversion and forms a conditioned place aversion. A, VGluT2::Cre mice were injected with AAV Cre-dependent ChR2-eYFP or eYFP vectors and optical fibers were implanted dorsal to LHb. During Stage 1, light was continuously delivered to mesohabenular fibers when mice entered chamber A and terminated when mice exited chamber A (blue shading). During Stage 2, light was continuously delivered to mesohabenular fibers when mice entered chamber B and terminated when mice exited chamber B (blue shading). B, Percentage of time spent in each chamber over the course of the experiment. ChR2-eYFP mice (solid lines) and eYFP mice (dotted lines). ChR2-eYFP mice spent significantly less time in the chamber where light activation of mesohabenular fibers occurred compared with the chamber in which light activation did not occur. Blue triangle indicates chamber where light was delivered. Chamber–session–group interaction, F(20,340) = 20.26, p < 0.001. C, Difference in time spent between Test 1 and No Light session in each chamber in ChR2-eYFP mice and eYFP mice. The ChR2-eYFP mice selectively spent less time in the mesohabenular light-activated chamber A. Chamber–group interaction, F(2,34) = 4.04, p < 0.05. D, Difference in time spent between Test 2 and Test 1 for each chamber in ChR2-eYFP mice and eYFP mice. The ChR2-eYFP mice selectively spent less time in the mesohabenular light-activated chamber B. Chamber–group interaction, F(2,34) = 26.47, p < 0.001. Data are mean ± SEM (error bars). Sidak-adjusted pairwise comparisons: *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 3.
Aversion elicited by activation of VGluT2-mesohabenular fibers depends on LHb glutamate receptors. Top, VGluT2::Cre mice were injected with AAV Cre-dependent ChR2-eYFP vectors and cannulas were implanted dorsal to LHb. Intra-LHb mix of CNQX/AP-5 or aCSF injections were made 3 min before optical stimulation. Bottom, Percentage of time spent in each chamber over the three tests. Intra-LHb CNQX/AP-5, but not intra-LHb aCSF, blocked mesohabenular light stimulation-elicited aversion. Session–chamber interaction, F(4,20) = 18.24, p < 0.001. Data are mean ± SEM (error bars). Sidak-adjusted pairwise comparisons: *p ≤ 0.05, **p < 0.01.
