GAD2 Expression Defines a Class of Excitatory Lateral Habenula Neurons in Mice that Project to the Raphe and Pontine Tegmentum

LHb GAD2-expressing neurons express VGluT2. A–D, Expression of the glutamatergic markers Slc17a7 (VGluT1) and Slc17a6 (VGluT2) in the habenula. The LHb expresses predominantly the subcortical transporter VGluT2, while the MHb expresses both transporters. E, Transgenic strategy for intersectional marking of Gad2^Cre and Vglut2^Flp co-expressing neurons. The tdTomato marker labels both the cell bodies and axons of Gad2/Vglut2^tdT mice. F, Standard atlas view at bregma -1.22 showing the location of detailed images for the rostral LHb and EP (also called MGP and GPi; Paxinos and Watson, 1998). G–I, Expression of tdTomato in the rostral habenula of Gad2/Vglut2^tdT mice. Confocal imaging in H shows that tdT expression is limited to rosettes of afferent fibers in the MHbD, whereas I shows both labeled cell bodies and afferent fibers in the LHb. J–K, Expression of tdT and SST in the EP of Gad2/Vglut2^tdT mice. The EP is a likely source of some of the LHb afferent fibers seen in G. L–O, FISH for VGluT2 and GAD2 in the rostral (L) and central (N) habenula. Confocal imaging in M, O shows co-localization of the VGluT2 and GAD2 signals. Arrows indicate examples of discrete co-expressing neurons. In situ hybridization data in A–D are derived from Allen Brain Atlas case numbers: VGluT1, 70436317 and VGluT2, 73818754. DG, dentate gyrus; EP, entopeduncular nucleus; LHb, lateral habenula; MHb, medial habenula (D, dorsal, V, ventral); PV, paraventricular nucleus (of thalamus). The dashed white line in L, N indicates the border of the LHb. Scale bars: 200 μm (G), 100 μm (J), and 200 μm (L, N).

Expression of GABAergic markers in the rat LHb. FISH was performed for a combination of the GABAergic markers VGAT, GAD1 and GAD2, and for GAD1 plus VGluT2. Sections were selected at rostrocaudal levels that had the largest population of cells expressing the markers shown. A–C, Co-expression of GABAergic markers in “typical” GABAergic neurons. A, Standard atlas view at bregma -3.14 showing location of detailed views (Paxinos and Watson, 1998). Expression of GABAergic markers in the dentate gyrus (B) and in the reticular thalamic nucleus (C). D, Expression of GABAergic markers in the rostral habenula, at bregma -2.6. The rostral population of GAD2-expressing neurons present in the mouse are not detected in the rat. E, F, Expression of GABAergic markers in the central habenula, at bregma -3.2. G, H, Expression of GABAergic markers in the caudal habenula, at bregma -3.6. I, J, Expression of VGluT2 and GAD1 in the caudal habenula. GAD1-expressing neurons at all levels co-express VGluT2. DG, dentate gyrus; LHb, lateral habenula, LHbL, lateral subnucleus; LHbM, medial subnucleus; MHb, medial habenula; Rt, reticular nucleus of thalamus; VP, ventral posterior thalamus. The dashed white line in D, E, G indicates the border of the LHb. Scale bars: 200 μm (B, D, E, G, I).

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 Vglut2^Cre (B, C) and Gad2^Cre (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 Gad2^Cre 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 Vglut2^Cre than in Gad2^Cre 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). Vglut2^Cre-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).

Cre-on/Cre-off mapping of LHb GAD2 neuron efferents. A, Cre-on/Cre-off dual labeling strategy with a AAV-Flex-GFP virus activated by Cre recombinase and an AAV-FAS-tdTomato virus silenced by Cre. B, C, Image of the central (B) and caudal (C) LHb in a mouse injected with a mixture of both viruses. Some off-target labeling is observed in the dentate gyrus but these neurons do not project to any of the areas of interest. The injection coordinates were: AP -1.69, ML 0.45, DV 2.70. D, Confocal images of the injected area in C; arrows indicate examples of Gad2^Cre neurons in which the tdTomato reporter is not completely inactivated, and the markers are co-expressed, producing a yellow-green to yellow color. E, Confocal image of LHb efferents in the CGPn. GAD2-negative fibers appear in red; GAD2-expressing fibers appear in green, and also in yellow due to incomplete silencing of the tdT reporter. Examples of dual-labeled (yellow) fibers are indicated by arrows. F–H, LHb fibers in the IP and RMTg, corresponding to bregma -3.80 in a standard atlas. The projections of LHb GAD2-expressing neurons to the RMTg are very sparse (H1, green), while the projections of GAD2-negative neurons densely fill the RMTg (H2, red). I–K, LHb efferents in the DR and MnR, and adjacent areas (bregma -4.72). L–M, LHb efferents in the CGPn and NI area (bregma -5.34). CGPn, pontine central gray; DG, dentate gyrus; DR, dorsal raphe; DRI, dorsal raphe, inferior part; IPC, interpeduncular nucleus, caudal; IPDL, interpeduncular nucleus, dorsolateral; IPDM, interpeduncular nucleus, dorsomedial; LHb, lateral habenula; MHb, medial habenula; mlf, medial longitudinal fasciculus; MnR, median raphe; NI, nucleus incertus; PMR, paramedian raphe; RMTg, rostromedial tegmental nucleus. Scale bars: 100 μm (B, E, G, J, M).

Relationship of the LHb GAD2 efferents to serotonergic neurons of the raphe and cholinergic neurons of the dorsal tegmentum. LHb efferents were traced in Gad2^Cre mice, and serotonergic and cholinergic neurons were identified by immunofluorescence for Tph2 and ChAT, respectively. A, B, Views showing bilateral LHb injections of a Gad2^Cre mouse with a mixture of AAV-Flex-sypGFP (targets synapses) and AAV-Flex-tdTomato (targets soma/axons). The injection coordinates were: AP -1.64, ML ±0.25, DV 2.60. The tdT signal is shown here to outline the extent of the injection but tdT labeling does not appear in the subsequent views. C–F, Efferents of LHb GAD2 neurons in relationship to serotonergic neurons of the DR and MnR. Sections correspond to bregma -4.36 (C) and -4.72 (D) in a standard atlas. G–I, Efferents of GAD2 neurons in relationship to the serotonergic (H) and cholinergic (I) neurons of the mesopontine tegmentum, at the transition from the DR to the LDTg (bregma -4.94). The area of highest synaptic density does not contain serotonergic or cholinergic cell bodies. J–L, Efferents of GAD2 neurons in relationship to the serotonergic (K) and cholinergic (L) neurons of the pontine tegmentum and in the NI (bregma -5.34). CGPn, pontine central gray; DG, dentate gyrus; DR, dorsal raphe; DRI, dorsal raphe, inferior part; DTg, dorsal tegmental nucleus; LHb, lateral habenula; LDTg, laterodorsal tegmental nucleus (V, ventral part); MHb, medial habenula; mlf, medial longitudinal fasciculus; MnR, median raphe; NI, nucleus incertus; PMnR, paramedian raphe; xscp, decussation of the superior cerebellar peduncle. Scale bars: 200 μm (A, E, H, K).

LHb projections to the hypothalamus. A–J, Anterograde tracing of LHb projections to the hypothalamus using a mixture of synapse-targeted AAV-Flex-sypGFP, and cytoplasm/axon-targeted AAV-Flex-tdT. The injected area is shown in Figure 4B–E. A–E, LHb afferents in the central hypothalamus (bregma -2.18 in a standard atlas). Dense synaptic labeling is observed in the LH of Vglut2^Cre (B, D) but not in Gad2^Cre (C, E) mice. F–J, LHb afferents in the caudal hypothalamus (bregma -2.54). Dense synaptic labeling is observed in the PH of Vglut2^Cre (G, I) but not in Gad2^Cre (H, J) mice. 3V, third ventricle; Arc, arcuate hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus; f, fornix (this is labeled “r” in the figure); LH, lateral hypothalamic area; me, medial amygdaloid nucleus; MM, medial mammillary nucleus, medial part; mt, mammillothalamic tract; PH, posterior hypothalamic area; VMH, ventromedial hypothalamic nucleus. Scale bars: 200 μm (B and G).

Retrograde tracing of LHb Gad2 projections to the raphe and pontine tegmentum. GABAergic neurons throughout the neural axis were genetically labeled by interbreeding Gad2^Cre mice with the reporter line Ai6, which conditionally expresses ZsGreen (Materials and Methods). Gad2^ZsGreen mice were then injected with CTB at the specified sites. Numbers indicate CTB-labeled (red), ZsGreen labeled (green), and dual labeled (yellow) neurons counted in the entire section, using confocal microscopy. Only cells with DAPI-labeled nuclei in the plane of the section were counted. Boxes indicate the area shown in the enlargements below the main figures. Arrows in the enlarged views indicate examples of dual-labeled cells. Only one example is indicated, even when multiple dual-labeled cells appear in the field of view. The number of CTB labeled neurons varied between areas approximately in proportion to the size of the injected fields. A, Injection site in the caudal raphe, including DRI and caudal MnR, at bregma -4.96. Injection coordinates: AP -4.96, ML 0.0, DV 4.50. B–D, ZsGreen expression and CTB retrogradely transported from the raphe in the rostral, central, and caudal LHb. E, Injection site in the right lateral pontine tegmentum, including CGPn and LDTg, at bregma -5.52. Injection coordinates: AP -5.70, ML 0.40, DV 4.15. F, G, ZsGreen expression and CTB retrogradely transported from the pontine tegmentum in the central LHb on the ipsilateral (right, F) and contralateral (left, G) side. H, Injection site near the midline of the pontine tegmentum, confined by fiber tracts largely to the right side, including the NI and CGPn. Injection coordinates: AP -5.52, ML 0.03, DV 4.25. I, J, ZsGreen expression and CTB retrogradely transported from the pontine tegmentum in the central LHb on the ipsilateral (right, I) and contralateral (left, J) side. LHb, lateral habenula; MHb, medial habenula; sm, stria medullaris of the thalamus. Scale bars: 50 μm (B, F, I).