Perinatal High Fat Diet Exposure Reduces KCC2 Expression and Alters GABAergic Signaling in the Dorsal Motor Nucleus of the Vagus

Perinatal high fat diet (pHFD) exposure alters the development of vagal neurocircuits controlling gastrointestinal (GI) functions resulting in gastric motor dysfunction. GABAergic synaptic inhibition of vagal efferent motoneurons within the dorsal motor nucleus of the vagus (DMV) is critical in the brain’s ability to modulate gastric motility. This inhibition is maintained by KCC2, a Cl− transporter, who determines intracellular chloride concentration. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently decreasing GABAergic inhibition and delaying gastric emptying rates. Pregnant Sprague-Dawley rats were fed either a control or high fat (14% vs 60% kcal from fat, respectively) diet from embryonic day 13, and offspring were used starting postnatal day 28. In vitro electrophysiological recordings were made from adult DMV neurons in brainstem slices. 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 bicuculline (BIC; 30μM), a GABAA antagonist. To determine the Cl− reversal potential, perforated patch clamp recordings were performed and the response to picospritz application of the GABAA agonist muscimol (100μM) was measured. KCC2 expression was quantified using pharmacology in the in vitro slice preparation, as well as immunofluorescence in brainstem cross-sections. Gastric emptying rates were quantified using the in vivo13C octanoic acid breath test technique. KCC2 transfection and induced expression in the DMV of pHFD rats was achieved using an adeno-associated viral vector. BIC increased firing rate in control DMV neurons (143.1% of baseline), but failed to increase DMV firing rate in DMV neurons from pHFD rats (96.27% of baseline; p=0.0103 using unpaired t-test). When control DMV neurons were treated with VU0240551 (VU; 10μM), a KCC2 blocker, BIC application led to a decrease in the firing rate (3.118 Hz to 1.606 Hz; p=0.0375 via paired t-test), demonstrating that antagonizing KCC2 attenuates GABAergic inhibition at this synapse. In control DMV neurons, the Cl− reversal potential was calculated to be -64.6mV. There was a depolarizing shift in Cl− reversal potential observed in pHFD neurons (-43.0mV; p=0.0007 using unpaired t-test). Application of VU shifted the chloride reversal from -64.6mV to -57.3mV in control neurons (p=0.0416 using a paired t-test). VU application had a reduced impact on pHFD neurons, shifting the reversal from -46.7mV to -45.0mV (p=0.1689 using a paired t-test), suggesting KCC2 downregulation. In fact, 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). When KCC2 expression was virally induced in young adult pHFD rats, GABA became inhibitory (163.7% of baseline firing rate after BIC application vs. 93.25% in pHFD + empty vector (EV); p=0.0019 using unpaired t-test), the Cl− reversal potential was normalized (-63.0mV vs. -49.7mV in pHFD + EV; p=0.0012 using an unpaired t-test), and gastric emptying rates were accelerated (58.7min vs 72.1min in pHFD + EV). Taken together, these results suggest that the loss of KCC2 function is a driver of the gastric pathophysiology seen in the pHFD rats, and that normalizing KCC2 levels can rescue the cellular and gastric phenotype. DK 111667. 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.