Figure 3. Hippocampus sends monosynaptic excitatory inputs to the anterior hypothalamic nucleus.

(a) Schematic illustration of anterograde tracing experiment. (b) HPC terminals (green) in the hypothalamus, including anterior hypothalamic nucleus (AHN), dorsomedial and central regions of ventromedial hypothalamus (VMHdm/c), premammillary dorsal nucleus (PMD), paraventricular nucleus (PVN), ventrolateral region of ventromedial hypothalamus (VMHvl), shell of ventromedial hypothalamus (VMHsh). DAPI staining (blue). (c) Quantification of HPC terminal intensity (N=2 animals, ~7 sections per ROI, One-Way ANOVA, F(5,35)=33.24, ****p<0.0001, Dunnett’s multiple comparisons test, AHN vs. VMHdm/c, ****p<0.0001, AHN vs. PMD, ****p<0.0001, AHN vs. PVN, ****p<0.0001, AHN vs. VMHvl, ****p<0.0001, AHN vs. VMHsh, ****p<0.0001). (d) Schematic illustration for patch clamp recordings of AHN neurons in coronal brain slices that express ChR2 in HPC terminals. (e) Examples of cell attach recordings. Illumination of blue light (480 nm, 5ms pulse at 15 Hz) triggered firing of AHN neurons. (f) Examples of whole-cell voltage-clamp recordings of AHN neurons. Blue light illumination (5 ms) evoked inward current. (g-h) Summary of light-evoked EPSCs (g) amplitude and latency (h). (i) Light-evoked EPSCs persisted in the presence of GABA A receptor antagonist picrotoxin (PTX, 100 µM) and eliminated by AMPA/kainite receptor antagonist DNQX (20 µM). (j) Summary of eEPSC change after PTX and DNQX application. (k) Light-evoked EPSCs were eliminated by TTX (0.5 µM) and then recovered by a low dose 4-AP (100 µM). (l) Summary of eEPSC changes after TTX and 4-AP application. All results reported are mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p<0.0001. Scale bar=100 µm.

Figure 3—figure supplement 1. Viral expression of the anterograde tracer ChR2-eYFP.

(a) Schematic showing the viral injection target (green dot) in the ventral subiculum. (b) Representative coronal sections of the hippocampus showing the spread of AAV-hSyn-ChR2-eYFP along the anterior-posterior axis of the hippocampus. DAPI, blue; ChR2, green; VS, ventral subiculum; CA1, cornu ammonis 1 of hippocampus. Scale bar = 500 µm.

Figure 3—figure supplement 2. HPC inputs innervate both GABA and non-GABA cells in the AHN.

(a) Schematic showing the reporter allele, RC::FrePe, containing FRT-flanked and loxP-flanked transcriptional stop cassettes. Dlx 5/6 FLPe-mediated stop cassette removal results in mCherry expression in forebrain GABA cells. The RC::FrePe allele is knocked in to the Gt(ROSA)26Sor(R26) locus with CAG (chicken beta-actin and CMV enhancer) promoter elements. (b) Top: HPC terminals (green) in the hypothalamus, including anterior hypothalamic nucleus (AHN), dorsomedial and central regions of ventromedial hypothalamus (VMHdm/c), premammillary dorsal nucleus (PMD), paraventricular nucleus (PVN), ventrolateral region of ventromedial hypothalamus (VMHvl), shell of ventromedial hypothalamus (VMHsh). DAPI staining (blue). Bottom: mCherry expression in GABA cells in the respective three regions from the same brain. (c) Schematic illustration for patch clamp recordings of AHN neurons in coronal brain slices that express ChR2 in HPC terminals. Red: mCherry-positive GABA cells, Gray: mCherry-negative non-GABAergic cells. (d) Examples of cell attached recordings. Illumination of blue light (480 nm, 5 ms pulse at 15 Hz) triggered action potential firing of AHN mCherry+ and mCherry- neurons. (e) A pie chart depicting the percentage of AHN GABA or non-GABA cells that evoked light-evoked action potentials (mCherry-positive GABA cells, N=10; mCherry-negative non-GABAergic cells, N=10). (f, g) Summary of light-evoked EPSC amplitude (f) and latency (g). (h,i) Number of action potentials evoked 1 s before, 1 s during and 1 s after light onset from mCherry-positive GABA cells (h) of the AHN (one-wa RM ANOVA, F (1.119, 10.07) = 8.271, *p = 0.0146) and mCherry-negative non-GABAergic cells (i) of the AHN (one-way RM ANOVA, F (1.088, 9.791) = 7.116, *p = 0.0223). All results reported are mean ± s.e.m. *p < 0.05. Scale bar = 100 µm.