Figure 3. DLS(Pdyn) cells project to and inhibit GABAergic cells in the LHA.

(A) Cell-type-specific and virally mediated anterograde mapping (DIO-mWGA-mCherry) was used to identify outputs of DLS(Pdyn) cells in Pdyn-Cre mice. (B) Representative coronal images showing DIO-mWGA-mCherry expression in the DLS and output regions, with the quantification of the number of cells per imaged region (ANOVA; main effect of region). (C) Representative coronal images (including confocal cross-section images) showing mCherry-expressing cells and immunolabeling for GABA in the LHA, as well as quantification of the percent of overlap across mice in the LHA for mCherry and GABA (significant paired t-test). (D) Pdyn-Cre::Vgat-Flpo mice were injected in the DLS with Cre-dependent ChR2-YFP-expressing virus and Flp-dependent mCherry-expressing virus was injecting in the LHA and ex vivo electrophysiology was performed. (E) Schematic for strategy for ex vivo electrophysiology, with right images showing representative coronal images of ChR2-YFP in the DLS (top left), ChR2-YFP and mCherry in the LHA (top right), and an example recording site (star) and mCherry-positive cell used for patching (bottom right). (F) Top: Example traces in a mCherry-positive LHA cell showing inhibitory post-synaptic current (IPSC) following paired pulses of blue light, their loss with TTX, and isolation of monosynaptic responses (+4AP). Top-middle: Quantifications of the number mCherry-positive cells exhibiting light-evoked IPSCs, their amplitude (pA), and PPR. Bottom-middle: Quantifications of the number mCherry-negative cells exhibiting light-evoked IPSCs, their amplitude (pA), and PPR. Bottom: Capacitance and membrane resistance of each recorded cell (w/ and w/o mCherry). No statistical tests were used in (F). For (C), the data are shown as mean (±SEM); all other data in the figure are shown as individual datapoints with the mean noted. For all statistics (if applicable): *=p<0.05; **p<0.005, ***p<0.0005; ****p<0.00005.