Fig. 5.
Dorsal–intermediate lateral septum (LS) γ-aminobutyric acid–mediated (GABAergic) neurons form monosynaptic synapses with ventral tegmental area (VTA) GABAergic neurons. (A) Anterograde tracing of the dorsal–intermediate lateral septum-to-ventral tegmental area pathway with the H129-ΔTK-tdT virus. Experimental time line for tracing the lateral septum-to-ventral tegmental area pathway with the helper (adeno-associated virus [AAV]-DIO-TK-GFP, day 0) and H129-ΔTK-tdT (day 22). The brains were obtained on day 29. (B) Schematic for virus injection and representative images for the starter neurons indicated by white arrows in the dorsal–intermediate lateral septum and tdTomato-positive cells in the ventral tegmental area. The scale bar in the lateral septum represents 20 μm. The scale bar in the ventral tegmental area represents 100 µm. (C) Representative immunofluorescence images of the ventral tegmental area slices stained with tyrosine hydroxylase or calcium/calmodulin-dependent protein kinase II. Scale bar, 100 μm. (D) Statistical results indicating that only 17.6% of tdTomato-positive cells were dopaminergic neurons, whereas 18.6% were glutamatergic neurons. (E) Experimental time line. AAV-DIO-TK-GFP and AAV-DIO-GFP were injected into the dorsal–intermediate lateral septum and the ventral tegmental area, respectively, on day 0. H129-ΔTK-tdT was injected into the dorsal–intermediate lateral septum on day 22. The brains were obtained on day 29. (F) Representative images of the ventral tegmental area. Scale bar, 100 μm. (G) Summary showing that 70.7% tdTomato-positive cells in the ventral tegmental area were GABAergic neurons. (H) Schematic of the AAV-DIO-channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein (eYFP) injection in the dorsal–intermediate lateral septum and the optrode implantation into the ventral tegmental area to simultaneously stimulate the lateral septum axonal terminals in the ventral tegmental area and record the single-unit activities of ventral tegmental area neurons. (I) Example image showing the expression of ChR2-eYFP and the location of the optical electrode in the ventral tegmental area. Scale bar, 200 μm. (J) Example raster and firing-rate histograms of dopaminergic, GABAergic, or glutamatergic neurons after 20-Hz train of light pulses to optogenetically activate the lateral septum GABAergic terminals in the ventral tegmental area. The train begins at 10 s. DAPI, 4′,6-diamidino-2-phenylindole.