Journal: Cell reports
Article Title: A spatial and projection-based transcriptomic atlas of paraventricular hypothalamic cell types
doi: 10.1016/j.celrep.2025.116904
Figure Lengend Snippet: (A) Experimental workflow for targeted single-nucleus RNA sequencing of parabrachial (PB)-projecting PVH neurons. (B) PB-projecting PVH neurons were classified with the Sim1 + sc/snRNA-seq reference atlas and projected into its UMAP space. The pie chart illustrates the proportion of PB-projecting PVH neurons that map to individual Sim1 + neuron clusters. (C) Three-level Sankey plot showing the mapping percentage of PB-projecting PVH neurons classified using the mouse Sim1 + sc/snRNA-seq reference atlas (left mapping to center; 5% cutoff) with corresponding Sim1 + MERFISH clusters identified via canonical correlation analysis (CCA; right mapping to center). Line thickness represents strength of mapping. (D) Schematic diagram of retrograde cholera toxin subunit B (CTB) injection into the PB and Cre-dependent AAV-EGFP-L10a injection into the PVH of a Mc4r -2A-Cre mouse (left). Representative coronal brain section showing the bilateral PBN CTB injection sites labeled by CTB IF. Scale bar, 250 μm (right). (E) Top panels show low-magnification (left) and high-magnification (right) views of EGFP IF, CTB IF, and Brs3 FISH labeling at ~−0.9 mm. Middle panels display Brs3 FISH, CTB IF, and EGFP IF, respectively, from left to right. Bottom panels display the overlay of Brs3 FISH with CTB IF (left), EGFP IF with CTB IF (center), and EGFP IF and Brs3 FISH (right). Yellow arrows indicate representative triple-labeled neurons. Low-magnification scale bar, 100 μm, high-magnification scale bar = 20 μm. (F) Schematic of Cre-dependent AAV-tetanus toxin light chain (TeTxLC) or AAV-EGFP injection into the PVH of Brs3 -IRES-Cre or wild-type mice (top left). Representative PVH injection site labeled with Cre-dependent AAV-EGFP-TeTxLC at ~−1.0 mm from bregma. Scale bar, 250 μm (top right). Experimental groups include wild-type (Cre-negative) mice injected with Cre-dependent AAV-TeTxLC ( n = 6), Brs3 -IRES-Cre mice injected with Cre-dependent AAV-GFP ( n = 10), and Brs3 -IRES-Cre mice injected with Cre-dependent TeTxLC ( n = 9). Body weights were monitored from the day of surgery (week 0) (lower left), and total body weight gained over 6 weeks post-surgery is depicted (lower right). Data shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (** p < 0.01, **** p < 0.0001). (G) Schematic and representative trace from Arc Npy/Agrp → PVH Brs3 neuron channelrhodopsin-2 (ChR2)-assisted circuit mapping (CRACM) experiment. Light-evoked IPSCs were detected in 8/14 PVH Brs3 neurons. (H) Schematic showing injection of Cre-dependent ChR2 or mCherry into the PVH and optic fiber implants over the PB in Brs3 -IRES-Cre mice (left), with representative brain sections showing bilateral ChR2 expression in the PVH at ~−0.9 mm from bregma (left-center; scale bar, 100 μm), and bilateral fiber tracks in the PB (right-center; scale bar, 250 μm). Food intake (right) was measured over the first 3-h of the dark cycle, with or without photostimulation, in mCherry- ( n = 4) and ChR2-expressing mice ( n = 8). Data shown as mean ± SEM. Statistical analysis was performed using two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test (** p < 0.01).
Article Snippet: Goat polyclonal anti-cholera toxin subunit B , List Biological Laboratories , Cat#: 703; RRID:AB_10013220.
Techniques: RNA Sequencing, Injection, Labeling, Expressing
