The PVN and PVNOT neurons are FOS activated during huddling substates.

(A-B) Experimental design to identify state-specific activity in 18 brain regions. Behavioral states (A). Experimental timeline (B).

(C) Quantification of percent FOS-DAPI colocalized cells across brain regions for each behavior. Regional FOS percentages are z-scored on a per experiment basis. N = 15 mice/1530 ROIs. Each datapoint is an ROI. Asterisks denote regions in which active huddle FOS activity is greater than both quiescent huddle and solo groom. P-values adjusted for multiple comparisons using the Holm method.

(D-F) Representative histology images showing active huddling associated FOS expression in the DMH (D), LS (E), and PVN (F). 3V: third ventricle; mt: mammillothalamic tract; LV: lateral ventricle. Scale bar equals 500 µm.

(G) Schematic of the PVN. Opt: optic tract.

(H) Representative histology images showing mRNA expression of Fos and Oxt in the PVN.

(I) Quantification of percent Fos:DAPI colocalized cells in dorsal and ventral subregions of the PVN during active huddling and quiescent huddling (N = 8 mice). Each datapoint is an ROI.

C: linear model with Tukey’s post-hoc tests. I: linear mixed effect model. Data are mean ±SEM. P < 0.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

PVNOT peaks track rest and arousal behavior states in social and non-social conditions

(A-C) Fiber photometric recordings of PVNOT cells in different social contexts and floor temperature conditions. Scheme of GCaMP AAV injections (top) and histology showing GCaMP-positive neurons in the PVN optic fiber placement (bottom) Scale bar 200 µm. (A). Floor temperature and social context conditions, and behaviors analyzed (B). Scheme of the timeline. Order of the social and floor temperature conditions was pseudo-randomized (C).

(D) Example traces of calcium-dependent (470 nm) and calcium-independent (415 nm) channels, and the post-processed dF/F trace (bottom), from a two-hour recording.

(E-G) Behaviors associated with PVNOT peaks in solo condition. Example ethogram aligned to photometric recording. Soft red shaded areas align with the quiescent state (E). PVNOT peak amplitude (F) and frequency (G) according to behavioral state across solo trials.

(H-J) Behaviors associated with PVNOT peaks in paired condition. Example ethogram aligned to photometric recording. Soft red shaded areas align with quiescent huddling (H). PVNOT peak amplitude (I) and frequency (J) according to behavioral state across paired trials.

(K - M) Effect of floor temperature and quiescence on PVNOT peaks in solo animals. Peak frequency according to floor temperature (Ta) (K). Peak count according to total duration of quiescent bouts (L). Beta coefficients from a model of the effect of floor temperature, total duration of bouts (boutSum), and the Ta*boutSum interaction on peak count (M).

(N - P) Effect of floor temperature and quiescence huddling on PVNOT peaks in paired animals. PVNOT peak frequency according to floor temperature (N). Peak count according to total duration of quiescent bouts (O). Beta coefficients from a model of the effect of floor temperature, total duration of bouts (boutSum), and the Ta*boutSum interaction on peak count (P).

F,G,I,J,K,L,M,N,O,P: linear mixed model. N = 8 mice/50 recordings. M and P show means plus confidence interval; all else shows mean ±SEM. P <.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

PVNOT peaks predict transitions to behavioral arousal and thermogenesis.

(A) Alignment of behavioral state, body temperature, physical activity, and dF/F from an experiment in the paired context. Bouts of active-and quiescent-huddle are color coded. (B -E) Physical activity around the time of PVNOT peaks. Peri-event time histogram of activity before and after PVNOT peaks in solo mice (B). Per-individual means before and after PVNOT peaks in solo mice (C). Peri-event time histogram of activity before and after peaks in paired mice (D). Per-individual means before and after peaks in paired mice (E). (F-I) PVNOT Ca++ dynamics during onset and offset of rest-behavior bouts. Quiescence onset/offset: peak probability (F) and Ca++ baseline average (G). Quiescent huddle onset/offset: peak probability (H) and Ca++ baseline average (I). (J-M) PVNOT Ca++ dynamics during onset and offset of active-behavior bouts. Nesting onset/offset: peak probability (J). Nesting-associated peak frequency according to quiescence phase. “Neither” refers to bouts not adjoining bouts of quiescence (K). Active huddle onset/offset: peak probability (L). Active-huddle-associated peak frequency according to quiescence phase. “Neither” refers to bouts not adjoining bouts of quiescence (M).

(N - Q) PVNOT Ca++ dynamics and core body temperature. Histogram of body temperature during minutes containing a calcium peak (green) vs. baseline (grey) data in solo animals (N). Peri-event time histogram of body temperature before and after PVNOT peaks in solo animals (O). Histogram of body temperature during minutes containing a calcium peak (green) vs. baseline (grey) data in paired animals (P). Peri-event time histogram of body temperature before and after PVNOT peaks in paired animals (Q).

C,E,K,M,N,P: linear mixed model. F,H,J,L: logistic regression. N = 8 mice/50 recordings, except for N-Q, N = 5 mice/24 recordings. O and Q show predicted values of a general additive model ±SEM; all data shows mean ±SEM. P < 0.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

PVNOT peaks observed in virgin females share characteristics with peaks seen in lactating mothers

(A-C) Longitudinal fiber photometry recordings of PVNOT cells in virgin-to-lactating mice. Scheme of GCaMP8s AAV injections (A) and recording timeline (B). Typical histology section showing PVN stained with anti-OT (red) and GCaMP8s expression (green). Scale bar 100 µm. (C).

(D-G) PVNOT Ca++ peaks in virgin females and in mothers during early-stage (PPD 2-7) and late-stage (PPD 8-14) lactation. Example of a dF/F trace from a two-hour recording on PPD 14 (D). Representative peaks from PPD 2-7 and PPD 8-14 (E). Example of a post-processed dF/F trace from a two-hour recording of a virgin female (F). Representative peaks (G).

(H-K) PVNOT Ca++ peak kinetics in virgins and lactating mothers. Average peaks dF/F over time of peaks according to condition (H). Full-width of half maximum (FWHM) (I), peak amplitude (J), and interpeak interval (K) according to female condition. N = 2 females, N = 24 recordings, N = 174 peaks. Full statistical analysis in Table S1.

A computer vision model for tracking thermal features during Ca++ imaging.

(A) SGBS model architecture. Three main modules are involved in thermographic identification of surface temperature features: processing raw FLIR .seq files (Split_seqs); segmentation of anatomical regions (SGBS); overlaying temperatures onto segmented regions for each frame (Post-processing).

(B) Example image showing segmentation of three anatomical thermal features.

(C) Boxplot shows per-minute mean temperatures (BAT, rump) scaled on a per-mouse basis.

(D) Loss values between SGBS and traditional Mask R-CNN.

(E) Cross-correlation analysis of core body temperature (Tb) and thermal features (BAT, rump, dorsal surface). Data are from light-only control animals recorded for two hours (N = 4). A negative value in the time-shift axis means the thermal features are shifted behind relative to Tb.

(F-G) Alignment of SGBS thermal feature data with calcium imaging. Viral strategy (E) and experimental condition (F).

(H-M) Peristimulus time histograms (PSTH) of SGBS thermal feature data in relation to the time of PVNOT calcium peaks (time 0). Dorsal surface PSTH slopes (H) and means (I). BAT surface PSTH slopes (J) and means (K). Rump surface PSTH slopes (L) and means (M). N = 4 mice; N = 12 recordings.

H, J, L slope-lines are fitted values from a linear mixed model. I, K, M are per-frame means; statistics from a linear mixed model. G-L show means ±SEM. P < 0.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

Optogenetic stimulation of PVNOT neurons at a low Tb increases behavioral arousal and thermogenesis.

(A) Viral strategy for ChR2 transfection.

(B) Histograms showing core body temperature (Tb) during light stimulations in ChR2+ and light-only controls. Tb-T0 is the Tb at the onset of light stimulation pulse-trains. Low Tb-T0 (green) refers to stimuli during low Tb, and High Tb-T0 (red) refers to stimuli during high Tb. Blue dotted line denotes the mean Tb at which PVNOT Ca++ occur (from Fig. 3N).

(C) Blue light stimulation (time 0; shaded blue rectangle) increases core Tb during low Tb-T0 in ChR2+ animals compared to light-only animals (top). No effect during high Tb-T0 (bottom).

(D-F) PVNOT stimulation increases arousal and thermogenesis in ChR2+ but not light-only controls. Shown are effects during low Tb-T0 (top row) and high Tb-T0 (bottom row). Optogenetic stimulation (time 0; shaded blue rectangle) triggers increases in physical activity (D), BAT surface temperature (E), and rump surface temperature (F) in ChR2+ compared to light-only control animals.

C-F: linear mixed model on data binned per-minute. Data show means ±SEM. N = 6 mice (3 ChR2+ animals and 3 light-only controls); N = 12 experiments; N = 73 light stimuli. P < 0.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

Graphical summary.

Increased calcium activity in PVNOT neurons signals the end of rest and the onset of thermogenesis and behavioral arousal. Both the frequency of large amplitude peaks and baseline calcium levels increase approximately two minutes before the end of rest (i.e., quiescence and quiescent huddle). Calcium peaks briefly continue into post-quiescent active states, like nesting and active huddling. Optogenetic stimulation of PVNOT neurons during rest triggers increased BAT thermogenesis, core body temperature (Tb) and physical activity. Arrows indicate transitions between the low-Tb rest balance point (downward vertical arrows) and the high-Tb active balance point (upward vertical arrows).

Histology of FOS activity in the DMH, LS, and PVN. Related to Figure 1.

(A-C) Representative histology images showing quiescent huddling associated FOS expression in the DMH (A), LS (B), and PVN (C). 3V: third ventricle; mt: mammillothalamic tract.

Characterization of PVNOT Ca2+ peaks and their associations with behavioral states. Related to Figure 2.

(A-B) Representative coronal brain slice showing the location of the optical fiber and expression of oxytocin peptide (red) and GCaMP8s (green) in the PVN. Arrows indicate some cells co-labeled with GCaMP8s and anti-OT. The third ventricle is designated by “*”. Scale bar 100 μm.

(C-D) Effect of social context on PVNOT peak amplitude (C) and frequency (D).

(E-F) PVNOT empirical cumulative distribution functions (ecdf) of peak counts according to behavioral state in solo (E) and paired (F) conditions.

(G-H) Effect of floor temperature on PVNOT peak amplitude. Relationship between floor temperature and behavior state in solo (F) and paired (G) females.

(I-J) Effect of floor temperature on PVNOT peak frequency. Relationship between floor temperature and behavior state in solo (I) and paired (J) females.

Statistical results are from linear mixed models. N = 8 solo, N = 7 paired, N = 50 recordings. Data shows mean ±SEM. P < 0.05 *, P < 0.01 **, P < 0.001 ***.

PVNOT Ca2+ peaks during behavioral states and transitions. Related to Figure 3.

(A-D) Peak counts for bouts of resting behaviors. Quiescence onset/offset peak counts (A) and peak count per bout (B). Quiescence huddle onset/offset peaks counts (C) and relationship between bout length and peak count (D).

(E-H) Peaks counts for bouts of active behaviors. Nesting onset/offset peaks counts (E) and relationship between bout length and peak count (F). Active huddle onset/offset peak counts (G) and relationship between bout length and peak count (H).

(I-J) Per-individual means of Ca++ dF/F during onset and offset (i.e., near-zero values) of two resting behaviors: quiescence (I) and quiescent huddle (J).

(K-L) Sum of bouts according to the phase of quiescence. “Neither” refers to bouts that did not adjoin bouts of quiescence. Nesting bouts (K). Active huddling bouts (L). Post quiescence nesting and active huddling is relatively rare. I-L: linear mixed model. F,H,J,L: logistic regression. N = 8 mice/50 recordings; all data shows mean ±SEM. P < 0.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

During low-Tb rest state, optogenetic stimulation of PVNOT neurons increases and physical activity and thermogenesis. Related to Figure 6.

(A) Blue light stimulation (time 0; shaded blue rectangle) during 20s vs. 10s stimulations in ChR2+ animals compared to light-only animals.

(B) Cumulative time spent in arousal/awake (left) and rest/quiescent (right) behaviors following 20s PVNOT stimulation. Each point represents a trial, color-coded by behavior. Black bars show mean ± SEM.

(C) Linear regressio line ± SEM of physical activity (log-transformed Z-score) 5 min before and 10 min following 20s PVNOT stimulation.

(D-F) Same analysis as in (C) for surface temperatures (ΔT/T) of BAT (D), rump (E), and dorsal surface (F). Stimulation in ChR2+ animals significantly increased temperatures in the low Tb-T0 group, with opposite or no effect in the high Tb-T₀ group. P < 0.05 *, P < 0.01 **, P < 0.001 ***. Full statistical analysis in Table S1.

PVNOT parvocellular projections to the rostral medullary raphe.

(A-D) PVNOT parvocellular projections to the rostral medullary raphe (rMR). Scheme of injections to label magno- and parvo-cellular OT neurons (A). Representative histology showing cells labeled for OXT-Cre, FluoroGold, and CTB (B). Distribution of PVNOT neurons retrogradely labeled with FluoroGold and CTB (C). Each map was made from one coronal section. Cells double-labeled with FluoroGold and OT-Cre were mostly distributed in the rostral part of the PVN; cells double-labeled with CTB and OT-Cre were in the caudal part of the PVN (C). Fluorescent in situ hybridization of OXTR (red) in the rMR region (D).

Key Resources Table