Perinatal high fat diet and acute stress exposure alters the chloride gradient in dorsal motor nucleus of the vagus neurons controlling gastric function

Perinatal high fat diet (pHFD) reduces stress resiliency in offspring, and alters the development of vagal neurocircuits controlling gastrointestinal (GI) functions, including GABAergic inhibition of dorsal motor nucleus of the vagus (DMV) neurons. However, the manner of neuronal recordings (whole-cell patch clamp) in previous studies prevented accurate assessment of the magnitude of GABA-mediated inhibition. KCC2, a Cl- co-transporter, determines intracellular chloride concentration [Cl-], and KCC2 function is known to be modulated by stress. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently increasing intracellular Cl- concentration, decreasing GABAergic inhibition, resulting in a maladaptive gastrointestinal (GI) stress response.Pregnant Sprague-Dawley rats were fed either a control (14% kcal from fat) or high fat (60% kcal from fat) diet from embryonic day 13. Electrophysiological recordings were made from adult DMV neurons in thin brainstem slices in both naïve (unstressed) and stressed (10 min forced swim; FS) conditions. To assess the GABA-mediated inhibition, cell-attached recordings were made to preserve the internal Cl- concentration, and firing rate was quantified before and after application of bicuculine (BIC; 10μM), a GABA A antagonist. To determine the Cl- reversal potential, perforated patch clamp recordings were performed and the response to picospritz application of the GABA A agonist muscimol (100μM) measured. Changes in KCC2 levels were also evaluated using immunohistochemistry.BIC increased firing rate in control DMV neurons (143.1%), a response that was absent following FS stress (92.86%), suggesting stress attenuates the inhibitory influence of GABA. When treated with VU0240551 (10μM), a KCC2 blocker, BIC decreased the firing rate of control DMV neurons, (3.118 Hz to 1.606 Hz; p=0.0375 via paired t-test). Following pHFD exposure, BIC failed to increase DMV firing rate in unstressed rats (96.27%), and FS had no further effect (84.83%) (p=.0033 using one-way ANOVA comparing all diet and stress groups). In control DMV neurons the Cl- reversal potential was calculated to be -64.56mV. There was a depolarizing shift in Cl- reversal potential following FS (-44.58 mV), which was similar to that observed in pHFD (-43.05 mV) and pHFD+FS (-53.89 mV) DMV neurons (p < 0.0001 using one-way ANOVA comparing all diet and stress groups). Compared to control rats, there was a significant reduction in KCC2 expression in the pHFD DMV (2.237% area fluorescence in pHFD vs. 5.335%; p=0.0147 using unpaired t-test).These results suggest that stress inhibits KCC2 in DMV neurons, decreasing the tonic inhibitory influence of GABAergic synaptic inputs. Furthermore, pHFD exposure decreases KCC2 expression resulting in a loss of GABAergic inhibition, even in unstressed conditions, which subsequently prevents appropriate GI responses to acute stress. NIH DK111667 This is the full abstract presented at the American Physiology Summit 2023 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.