Article Figures & Data
Figures
- Figure 1.
Genetic and viral strategy for transsynaptic labeling of IP afferents. A, A genetic strategy for labeling Cre-expressing neurons in the IP of Chrna5Cre mice. Chrna5Cre mice were interbred with the mouse strain Ai6, which conditionally expresses the fluorophore ZsGreen (Materials and Methods). B–D, ZsGreen expression in the rostral, central, and caudal IP of Chrna5Cre, Ai6 compound heterozygous mice, generated as shown in A. E, RV transsynaptic labeling strategy. A helper virus AAV1-N2cG was injected into the IP of Chrna5Cre mice, followed by RV three weeks later (Materials and Methods). The habenula and IP were examined for the expression of nuclear GFP (nGFP) expressed by RV. Blue lines indicate the planes of section for the habenula, IP, and NI (rostral to caudal). F–K, Imaging of nGFP expressed by RV and cytoplasmic tdTomato (tdT) expressed by the AAV helper virus in the rostral (F, I), central (G, J), and caudal (H, K) IP of two injected cases, r1 and r2 (retrograde 1 and 2). Insets in G, J show higher magnification of the boxed area. L, M, Expression of RV nGFP transsynaptic label in the habenula. Coronal sections correspond to bregma −1.7 in a standard atlas (Paxinos and Franklin, 2001). N, Dual-label FISH for RV-expressed GFP mRNA and Rln3 mRNA in the NI of injected Case r1. IP, interpeduncular nucleus; IPC, central part; IPDL, dorsolateral part; IPDM, dorsomedial part; IPI, intermediate part; IPL, lateral part; IPR, rostral part; LHbL, lateral habenula, lateral part; LHbM, lateral habenula, medial part; MHbD, medial habenula, dorsal part; MHbV, medial habenula, ventral part; NI, nucleus incertus; RMTg, rostromedial tegmental nucleus; VTA, ventral tegmental area. Scale bar: 200 μm (B, L, N).
- Figure 2.
RV-mediated retrograde tracing of projections from the VTA and SN to the IP. Inputs to the IP were retrogradely labeled with RV as described in Figure 1 (Case r1), producing a nuclear GFP label in presynaptic neurons. Serial 25-μm sections were examined at 100-μm intervals through the VTA and SN, at levels from bregma −2.8 to bregma −3.6 in a standard atlas (Paxinos and Franklin, 2001), and imaged as Z-stacks. DA neurons were labeled by immunostaining for TH, and all cells were counterstained for nucleic acids with DAPI. TH-positive neurons were counted if the TH immunostaining formed a cytoplasmic circle or semicircle around a nuclear hole, indicating that the labeled neuron was in the plane of section. Areas of interest were drawn manually around the area containing the VTA+SN (outlines), and all of the nGFP and TH-immunoreactive cells in the left hemisphere of these structures were counted. A, RV nGFP and TH labeling in the rostral VTA/SN. In the region of interest outlined, 9/1606 DAPI-labeled cells were labeled with GFP (0.56%). B, C, RV nGFP and TH labeling in the caudal VTA/SN. In the region of interest outlined, 83/2595 DAPI-labeled cells were labeled with GFP (3.2%). Co-localization of nGFP and TH was very rarely observed. In C, the arrow indicates an example of an nGFP-labeled nucleus which may be in a TH-labeled neuron. The arrowhead indicates an nGFP nucleus which overlies TH-labeling but does not appear to be the nucleus of the TH-expressing cell. D, Count of nGFP labeled, TH-positive, and dual-labeled neurons in the left VTA/SN in sections at the designated coordinates in Case r1. IP, interpeduncular nucleus; ml, medial lemniscus; MM, medial mammillary nucleus; PN, paranigral nucleus; SNC, substantia nigra, pars compacta; SNR, substantia nigra, pars reticulata; VTA, ventral tegmental area. Scale bar: 200 μm (A).
- Figure 3.
Cre-mediated tract-tracing of tegmental DA neurons in the Allen Connectivity Atlas. Three cases of Cre-mediated AAV tract tracing from the Allen Connectivity Atlas are shown. A–E, Labeling mediated by THCre, Allen experiment 156314762, with labeled neurons in the bilateral VTA, PN, and RLi, and sparing the SN. F–J, Labeling mediated by Slc6a3Cre (DATCre), Allen experiment 160539283, with signal in the right VTA and SN. K–O, Labeling using a VMAT2Cre driver, Allen experiment 292958638, with expression in the right VTA and SN. The planes of section from top to bottom are: (A, F, K) VTA/SN, incorporating the injected area, close to bregma −3.1 as designated in a standard atlas (Paxinos and Franklin, 2001); (B, G, L) rostral IP, bregma −3.3; (C, H, M) central IP, bregma −3.5; (D, I, N) caudal IP, bregma −3.8; (E, J, O) striatum, bregma 0.7. The reported target coordinates are (bregma, AP, ML, DV): experiment 156314762 (−3.28, 0.36, 4.13); experiment 160539283 (−3.08, 1.25, 4.08); experiment 292958638 (−3.08, 1.25, 4.15). Acb, accumbens nucleus; CPu, caudate/putamen; IP, interpeduncular nucleus; IPC, central part; IPDL, dorsolateral part; IPDM, dorsomedial part; IPL, lateral part; IPR, rostral part; ipf, interpeduncular fossa; LS, lateral septum; PN, paranigral nucleus; RLi, rostral linear nucleus raphe; SN, substantia nigra; VP, ventral pallidum; VTA, ventral tegmental area. Scale bar: 400 μm (A, E).
- Figure 4.
Cre-mediated tract tracing of tegmental DA neurons using synaptically-targeted GFP. DATCre mice were injected with a Cre-dependent AAV expressing synaptically targeted GFP. Sections were stained for TH by immunofluorescence (red), and with the nuclear marker DAPI (blue). The intended target of the injection was at the level shown in B, F. The targeted coordinates for both cases were: AP −3.4, ML 0.5, DV 4.5. A–D, Case a1 (anterograde 1), injected in the central part of the right VTA, with some spread laterally into SN and RRF. E–H, Case a2, injected nearer the midline, labeling the medial part of the VTA bilaterally, as well as the PN and RLi. Arrows in G indicate rare cell bodies in the IP labeled by AAV, indicating that a few IP cells may express enough Cre recombinase to activate the Cre-dependent AAV. The planes of section from top to bottom are: (A, E) injected area, bregma −3.1; (B, F) rostral IP, bregma −3.3; (C, G) central IP, bregma −3.5; (D, H) caudal IP, bregma −3.8. IF, interfascicular nucleus; IP, interpeduncular nucleus; IPC, central part; IPDL, dorsolateral part; IPDM, dorsomedial part; IPL, lateral part; IPR, rostral part; ipf, interpeduncular fossa; ml, medial lemniscus; PN, paranigral nucleus; RLi, rostral linear nucleus raphe; RRF, retrorubral field; SN, substantia nigra; VTA, ventral tegmental area. Scale bar: 200 μm (A).
Tables
Bregma
CoordinateCase r1 cell count Case r2 cell count nGFP TH Both nGFP TH Both −2.8 12 43 0 2 28 0 −2.9 9 116 0 2 65 0 −3.0 24 134 1 15 112 0 −3.1 31 117 0 19 102 0 −3.2 52 192 1 39 110 0 −3.3 54 156 1 38 121 0 −3.4 83 158 1 38 145 1 −3.5 59 101 1 36 138 0 −3.6 49 87 0 33 128 0 Sum 373 1104 5 222 949 1
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