Fig 2: Single-nuclei RNA sequencing of the aging anterior hypothalamus.a. Schematic illustrating workflow of microdissection and age groups pooled into snRNA-seq (n=4). b. UMAP of all nuclei from both experimental groups, processed into 29 clusters. c. UMAP of 5 major cell types identified in dataset. d. UMAP of subset neural clusters, identifying 25 subclusters. e. Cell proportions across each cell type split by age. f. g. Volcano plot of differentially expressed genes in aging vs. young nuclei pools. h-i. UMAP of inhibitory and excitatory neurons with respective clusters. j. Violin plot of Avp expression across Avp-rich clusters from excitatory (e13, e15, e8) and inhibitory (i15, i20, i21, i23) subclusters, with increased Avp expression observed in e15. k. Dot plot of key gene markers in Avp-rich clusters including Sim1 (marker of PVN and SON neurons) and Rgs16 (labels SCN neurons) shows that high e15 expression of Avp coincides with Sim1 expression.

Fig 3: Metabolic and thermoregulatory changes with aging.a-f. Metabolic monitoring of male C57BL/6 mice (n young = 11, n aged = 8) a. Body weight (left) and body composition (right). b-f. Indirect calorimetry over three days: b. VO2, c. RER, d. X+Y locomotor activity, e. water intake, f. food intake. g-h. Postmortem measurements of g. Osmolality and h. AVP levels in plasma. i. Experimental design for inducing osmotic thirst, j. water intake plotted over time (left) and by stimulus (right). k. Body core temperature (Core T) readings via intraperitoneal Anipill probes over 1 day and 1 night cycle (n young = 4, n aged = 3) at 22 °C and 28 ° C environmental temperatures. l. Core T readings during an acute cold challenge at 4 °C (n young = 4, n aged = 3). Data are means ± sem. Student’s t-test (scatterplot, bar graph) or two-way ANOVA (line graph) were used. p-values: ns = p > 0.05; *** = p < 0.005; **** = p < 0.0005.

Fig 4: Chemogenetic stimulation of SONAVP neurons.a. Experimental Design: AVP-IRES-Cre mice received bilateral stereotaxic injections of AAV-hSyn-DIO-hM3D-mCherry followed by intraperitoneal (I.P.) delivery of saline or DCZ in male (c-j) and female mice (k-o). b. Immunohistochemistry of mCherry (red) and AVP (green) overlapping in SON. c. Plasma levels of copeptin, and blood osmolality between AVP-M3D mice (n=5) and saline controls (n=6) post-injection of DCZ. d. Core T plotted overtime (left) and averaged over 1st hour post-injection (right) of AVP-M3D mice receiving either saline, DCZ (0.1mg.kg) or DCZ (1mg/kg). e-h. Indirect calorimetry assessment of AVP-M3D mice receiving either saline, DCZ (0.1mg.kg) or DCZ (1mg/kg): (e) VO2, (f) X+Y locomotor activity, (g) food intake and (h) water intake. i-j. Infrared (IR) temperature readings of (i) brown adipose tissue (BAT T) and (j) tail (Tail T). k-o. Female AVP-M3D mice receiving either saline or DCZ (0.1mg.kg): (k) Core T, (l) VO2, (m) locomotor activity, (n) food intake, and (o) water intake. Data are means ± sem. One-way ANOVA or Student’s t-test (bar graphs), two-way ANOVA (line-graphs) were used. p-values: * = p < 0.05.

Fig 5: Analysis of AVP expressing cell bodies in PVN, SCN, and SON.a-c. Representative immunohistochemistry image of AVP+ cell bodies in young and old male mice, (i) anti-AVP (green), (ii) Nissl (magenta) and DAPI (blue), iii Merge image. a. PVN, b. SCN, c. SON. d. High magnification images of AVP+ neurons in young and old mice, AVP (red) and DAPI (blue). e-j. Quantification of morphological features of AVP neurons in each region (n young = 5, n aged = 6). e. AVP cell body size, f. ROI size, g. AVP cell bodies per ROI, h. AVP signal intensity per ROI, i. AVP cell roundness, and j. AVP signal intensity per cell. k. Representative image of AVP+ (red arrow) and AVP− (white arrow) neurons recorded from slice electrophysiology, anti-AVP (green), biocytin (magenta). l. Representative trace of a baseline recording at 0 A holding current from AVP+ neuron. m. Spontaneous firing rate of AVP+, but not AVP-negative cells (p>0.9999, Mann Whitney test). n. Comparison of membrane capacitance between young and old AVP+ neurons. o. Resting membrane potential. q. A 1s, 0.5 nA current ramp was applied from a manually adjusted membrane potential to −65 mV to determine firing threshold and highest possible firing ability. r. Threshold, s. total action potential (AP) count. t. maximum instantaneous frequency. u. With membrane voltage still adjusted to −65 mv, 10 depolarizing current steps (from 0 to 100 pA, 10 pA increments) were applied to establish the gain in firing rate over increasing stimulation strength for v. maximum AP count, w. gain value defined as the slope of a linear regression fit to the number of evoked APs at successive steps. x. Slope values of w. Data are means ± sem. One-way ANOVA or Student’s t-test were used. p-values: * = p < 0.05.