Previously, we have shown that male and female mice exposed to maternal separation and early weaning (MSEW), a model of early life stress, display exacerbated obesity-induced hypertension in response to chronic high fat diet. However, only male mice showed higher sympathetic tone at baseline, along with increased neuronal activation in PVN and pressor response to the acute stimulation of TRVP1 channels with capsaicin, a mechanism known as adipose afferent reflex (AAR). The aim of this study was to investigate whether obese male MSEW mice show 1) increased intracellular calcium in response to capsaicin in neuronal cultures of dorsal ganglia root (DRG), and 2) a role for sex hormones in AAR reactivity in vivo. Six-months-old control and MSEW fed a high fat diet (HFD, 60% kcal fat) were injected with pAAV-CAG-tdTomato (59462-PHP.S, 1012 viral particles); 8 sites; 4 ul/site; bilateral). After 6 weeks, mice were decapitated and DRGs (L5-T12) were collected and placed in ice-cold HBSS solution, dissociated and mixed with Collagenase P (1 mg/ml in HBSS, 15 min, 37 oC). After 0.25% Trypsin-EDTA incubation, neurons were mechanically dissociated, collected by centrifugation and resuspended in DMEM-10% FBS, plated onto the Matrigel Basement Membrane Matrix covered coverslips and maintained at 37 oC for 15-16 hours. Neuronal cultures were incubated in Ca2+ imaging solution with 2 μM Fura-2 AM, 30 min in dark conditions. After de-estirification, intracellular Ca2+ transients were visualized by exciting Fura-2 at 340 ± 20 nm and 380 ± 20 nm using a high-speed filter changer to obtain a ratiometric value independent of indicator concentration. Emitted light was passed through a dichroic filter (400-nm high pass) and an emitter filter (520 ± 30 nm) and was digitized onto the sensor of an EMCCD camera. Background subtraction and normalization to baseline were used to quantify changes in fluorescence. Despite similar resting uncalibrated ratios of calcium levels between groups (~0.6 ratio units, n=3), sensory neurons from MSEW mice tended to display larger responses to capsaicin (50 uM), showing elevated calcium kinetics vs. controls (0.56±0.09 vs. 0.31±0.12 delta ratio, respectively, p=0.063) as well as longer transients, perhaps indicative of reduced clearance or buffering. A separate set of age-matched mice were gonadectomized. After 4 weeks, anesthetized mice were fitted with carotid artery catheter for direct blood pressure measurement in response to the infusion of perigonadal fat with capsaicin (1.5 pmol/μL during 2 minutes in 4 sites, bilaterally). Capsaicin-induced pressor response was exacerbated in intact MSEW male mice compared to intact controls (9.0±0.2 vs. 1.3±0.2 delta mmHg, respectively, n=6, p<0.05). Orchidectomy attenuated delta blood pressure in MSEW mice to 7.0±0.2 mmHg (p<0.05 vs. intact MSEW), while enhanced delta blood pressure in control mice to 7.5±0.2 mmHg (p<0.05 vs. intact control). MSEW did not influence the pressor response to perigonadal fat stimulation with capsaicin either in intact or ovariectomized obese female mice (n=4-6 per group, p>0.05), suggesting that estradiol may not to play a protective role in obese females AAR reactivity. This data indicates that testosterone could buffer the AAR reactivity of obese control mice, while MSEW exerts an opposite effect. Thus, investigating testosterone signaling on calcium handling mechanisms (i.e., buffers, pumps, ER-mediated calcium release) in sensory neurons innervating visceral fat will provide insights on the sex-specific contribution of afferent sympathoexitatory signals to obesity-hypertension in male MSEW mice. This study was supported by NIH R01 HL135158, and SALT pilot - University of Kentucky to ASL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.