Calcium Dynamics in Hypothalamic Paraventricular Oxytocin Neurons and Astrocytes Associated with Social and Stress Stimuli

Home cage social interaction schematics and behavioral results. A, A schematic of the home cage social interaction paradigm involving 3 separate days of interacting with familiar and unfamiliar juvenile conspecifics. Created with BioRender.com. B–D, Comparisons of social sniff characteristics (i.e., total sniff duration, number of sniff bouts, and length per sniff bout) between male versus female and familiar versus unfamiliar conspecifics. No significant differences were observed.

Fiber photometry recordings of paraventricular oxytocin (OXT^PVN) neurons and astrocytes during home cage social interactions. A, B, Representative single traces of RCaMP2 (red) and GCaMP6f (green) recordings during social interactions. Shaded areas indicate social interactions and exploratory behaviors. C–H, Red traces (±SEM in shaded lines) represent fiber photometry recordings of RCaMP2 signals from OXT^PVN neurons represented as z-scores in female (C, D) and male (F, G) mice during social sniffing of familiar and unfamiliar conspecifics. Light orange boxes indicate the baseline period, and dotted vertical lines indicate the time of sniffing initiation by experimental mice (t = 0 s). Plots to the right of traces show baseline (BL) and peak responses for each trial (gray lines) and average responses (±SEM) in black dots. A significant Ca²⁺ level change in OXT^PVN is observed only in females while interacting with unfamiliar mice. E, H, Plots compare the area under the curve (AUC) and time of peak for Ca²⁺ changes associated with social sniffing of familiar versus unfamiliar conspecifics in males and females. I–N, Green traces (±SEM in shaded lines) represent fiber photometry recordings of GCaMP6f signals from astrocytes represented as z-scores in female (I, J) and male (L, M) mice during social sniffing of familiar versus unfamiliar conspecifics in males and females. No change in astrocyte signal was associated with social interactions regardless of sex or conspecific familiarity. K, N, Plots compare AUC and time of peak for Ca²⁺ changes associated with social sniffing of familiar versus unfamiliar conspecifics in males and females. n = 5–6 mice, 20–23 traces. *p < 0.05, no other comparison was statistically significant.

Looming shadow stress task schematics and behavioral results. A, A schematic of the looming shadow stress task paradigm illustrating the phases of shadow presentation from the monitor placed above the experimental arena. B, Mice exhibit three different response categories (i.e., run, freeze, and no response) during the looming shadow task. Created with BioRender.com. C, D, Colored boxes (left) and pie charts (right) represent the behavioral responses displayed by female (C) and male (D) mice across trials. E, F, Plots (mean ± SEM) with individual trial values compare the latencies and durations of looming shadow-triggered behavioral responses between females and males. No sex differences were found when comparing the latency to run (E), latency to freeze (F), and freeze duration (G).

Fiber photometry recordings of OXT^PVN neurons and astrocytes during looming shadow task. A, Representative RCaMP2 and GCaMP6f traces from a male exemplar mouse showing Ca²⁺ peaks in response to looming shadow presentations. B–G, Traces (±SEM in shaded lines) represent fiber photometry recordings of RCaMP2 signals from OXT^PVN neurons represented as z-scores in female (B, C) and male (E, F) mice during looming shadow. Plots to the right of the traces show the average z-scores of baseline (BL), looming shadow (5–8 s), and poststimulus (8–15 s) periods for each trial (gray lines) and average responses (±SEM) in black dots. Significant increases in OXT^PVN Ca²⁺ levels are observed in both sexes, only during trials with run responses. D, G, Plots compare the area under the curve (AUC) and time of peak for OXT^PVN neuron Ca²⁺ changes associated with run and freeze responses in males and females. H–M, Traces (±SEM in shaded lines) represent fiber photometry recordings of GCaMP6f signals from astrocytes represented as z-scores in female (H, I) and male (K, L) mice during looming shadow. Significant increases in astrocyte Ca²⁺ level are observed in both run and freeze response trials, except for females during freeze trials. J, M, Plots compare the AUC and time of peak for astrocyte Ca²⁺ changes associated with run and freeze responses in males and females. *p < 0.05, **p < 0.01, all other comparisons were not statistically significant.