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. Author manuscript; available in PMC: 2018 Jul 13.
Published in final edited form as: Cell. 2017 Jul 6;170(2):284–297.e18. doi: 10.1016/j.cell.2017.06.015

Figure 1. Distinct subpopulations of VP PV neurons project to the LHb and VTA.

Figure 1

(A) PV neuronal localization in VP. Substance P (SP)-immunostaining denotes boundaries of VP. Scale bars: 100 µm; inset, 50 µm.

(B) Schematic of tracing strategy. Cell bodies and efferent fibers in red and presynaptic sites in green using AAV-DIO-mRuby2-T2A-Synaptophysin-eGFP.

(C) Representative image of injection site in VP. Scale bar: 80 µm.

(D and E) Fibers and synaptic puncta in LHb and VTA from VP PV neurons. Scale: 200 µm; inset, 20 µm.

(F) Two possible projection patterns: VP PV neurons send collateralized axons to both the LHb and VTA (top), or independent subpopulations send axons exclusively to either LHb or VTA (bottom).

(G and H) Schematic of viral strategy. EIAV is pseudotyped with a fusion protein containing rabies virus glycoprotein and VSVG transmembrane (RG-EIAV; G). Injection of RG-EIAV-DIO-Flp into target area and AAV-fDIO-eGFP into VP of PV-Cre mice.

(I) Images of injection site and target areas showing cell-type and projection-specific labeling. VP PV neurons selectively project to either the LHb or VTA, respectively, but not to both. Scale: 200 µm.

(J) Light-evoked synaptic responses in LHb or VTA. EPSCs measured at −70 mV, IPSCs at 0 mV. Pie charts indicate whether absolute amplitude was greater for evoked EPSC, IPSC, or no response (individual responses in Figure S4). Top row, LHb neurons; bottom row, VTA neurons distinguished as either GABAergic or dopaminergic. Scales: 50pA, 100ms.

(K) Strategy using PV-Flp×Ai14 mouse for labeling VP PV neurons in projection specific manner. RG-EIAV-fDIO-Cre is injected into either LHb (top) or VTA (bottom).

(L) mRNA labeling of PVVP→LHb neurons (top panel) and PVVP→VTA neurons (bottom panel). Green arrowheads show VGAT+ cells, white arrowheads VGluT2+ cells. Scale: 10 µm. Pie charts indicate percentage of tdTomato-mRNA+ neurons projecting to LHb or VTA colocalizing with probes to VGAT or VGluT2 (n = 154 tdT+ cells for VTA-projecting, n = 24 cells for LHb-projecting).

EIAV, equine infectious anemia virus; RV G, rabies virus glycoprotein; ac, anterior commissure; SNr, substantia nigra reticulata; MHb, medial habenula; CPu caudate putamen.