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. 2020 May 20;7(3):ENEURO.0527-19.2020. doi: 10.1523/ENEURO.0527-19.2020

Figure 4.

Figure 4.

Specific projections of LHb GAD2-expressing and VGluT2-expressing neurons. A, Strategy for Cre-dependent labeling of LHb efferents using a mixture of a synapse-targeted GFP reporter virus, AAV-Flex-sypGFP, and a cytoplasm/axon-targeted tdTomato reporter virus, AAV-Flex-tdT. B–E, Reporter expression in the injected area of the LHb of Vglut2Cre (B, C) and Gad2Cre (D, E) mice. Injection coordinates were: AP -1.64, ML 0.25, DV 2.56, and AP -1.3, ML 0.3, DV 2.6, respectively. The Gad2Cre injection was positioned to maximize labeling of Gad2-expressing neurons. Overall, because of the wider expression of VGlutT2, more cells, and consequently more efferent fibers, are labeled in Vglut2Cre than in Gad2Cre mice. F–H, LHb fibers at the level of the IP and RMTg, corresponding to bregma -1.88 in a standard atlas (Paxinos and Franklin, 2001). Vglut2Cre-driven expression heavily labels synapses in the RMTg (G, circle), but synapses from GAD2-expressing LHb neurons are sparse at this level (H, circle). Arrows indicate fibers of passage (red) to more caudal areas, which have similar levels of labeling. I–M, LHb fibers and synapses at the level of the DR and MnR (bregma -4.36). The area designated RMTgC is identified as part of the anterior tegmental nucleus in standard atlases, but has been shown to be part of the RMTg complex (Quina et al., 2015). N–R, LHb fibers and synapses in the caudal MnR (bregma -4.60). S–W, LHb fibers and synapses in the dorsal tegmentum, including the LDTg and NI (bregma -5.34). Synapses from GAD2-expressing neurons are abundant at this level. CGPn, pontine CG; DG, dentate gyrus; DRI, dorsal raphe, inferior part; DTg, dorsal tegmentum; IPC, interpeduncular nucleus, caudal; IPDM, interpeduncular nucleus, dorsomedial; IPI, interpeduncular nucleus, intermediate; IPR, interpeduncular nucleus, rostral; LDTg, laterodorsal tegmental nucleus; LHb, lateral habenula; MHb, medial habenula; MnR, median raphe; NI, nucleus incertus; PMR, paramedian raphe; RMTg, rostromedial tegmental nucleus (C, caudal part); xscp, decussation of the superior cerebellar peduncle. The dashed white line in B, D indicates the border of the LHb. Scale bars: 200 μm (B, G, J, O, T) and 100 μm (L, Q, V).