Figure 5. AgRP neurons presynaptically inhibit tonically-active BNSTGABA neuron afferents to PVHCrh neurons.
a, Schematic for BNSTvGAT→PVH neuron CRACM (n = 18 PVHCrh+ neurons, n = 9 PVHCrh− neurons/2 mice). b, Representative traces from BNSTvGAT→PVHCrh+ (upper) and BNSTvGAT→PVHCrh− (lower) recordings. c, Schematic of BNST or LH injection of Cre-dependent AAV-hM4Di-mCherry into vGAT-IRES-Cre mice. d, Time-line of chemogenetic inhibition of BNSTvGAT or LHvGAT neurons. e, Inhibition of BNSTvGat neurons increased corticosterone (BNST: n = 4 vGATmCherry, n = 4 vGAThM4Di. Two-tailed unpaired t-test- *P = 0.0131; LH: n = 5 vGATmCherry, n = 7 vGAThM4Di. Two-tailed unpaired t-test- P = 0.2837). f, Schematic of BNSTvGAT→PVH terminal inhibition. g, Representative eOpn3 expression in the BNST (upper) and location of optic fibers in the PVH (lower). h, Time-line for terminal inhibition of BNSTvGAT→PVH projections. i, Inhibition of BNSTvGAT→PVH projections increased corticosterone (n = 3 vGATmCherry, n = 5 vGATeOpn3. Two-tailed unpaired t-test- *P = 0.0076). j, Schematic of BNST→PVH terminal recordings with optogenetic stimulation of AgRP→PVH terminals. k, Representative images of synaptophysin-GCaMP6f expression in BNST neurons and terminals and ChrimsonR-tdTomato expression in AgRP neuron terminals. l, Heatmaps of BNST→PVH fiber photometry recordings from two mice. m, n, Optogenetic stimulation of AgRP→PVH terminals for 10 sec at 20 Hz (m) (n = 3 mice) and 10 min at 20 Hz (n) (n = 4 mice) reduced activity of BNST→PVH terminals. o, Representative images of BFP-labelled PVHCrh neurons, synaptophysin-GCaMP6f-expressing BNST neuron terminals and AgRP immunofluorescence (* = PVHCrh neurons apposed by BNST and AgRP neuron terminals). p, Schematic of AgRP neuron action in the PVH. Left = presynaptic disinhibition of PVHCrh neurons by GABA/presynaptic GABA-B-Rs. Right = monosynaptic inhibition of PVHMc4r neurons by GABA/postsynaptic GABA-A-Rs. Scale bar (g, k) = 200 μm. Scale bar (o) = 10 μm. Data represent = mean ± sem.