Perinatal High Fat Diet and Acute Stress alters the Chloride Gradient in Dorsal Motor Nucleus of the Vagus Neurons Controlling Gastric Function
Authors: Carson Kaitlin E. , Browning Kirsteen N. 
Published: May 2022

The US is currently facing an obesity epidemic, though the concurrent rise in maternal obesity rates receives less attention. Maternal obesity is an important factor that influences intrauterine and early postnatal environment and is associated with developmental changes in the offspring, including alterations in metabolic function and susceptibility to anxiety and depression, all of which are associated with abnormal gastrointestinal (GI) tract function. Previous work from this lab has shown that perinatal high fat diet (pHFD) leads to developmental changes to vagal neurocircuits, including changes to GABAA receptor subunits on dorsal motor nucleus of the vagus (DMV) neurons. However, the manner of neuronal recordings (whole‐cell patch clamp) disrupted internal chloride (Cl‐) concentration, preventing accurate assessment of the extent of GABA‐mediated inhibition. KCC2, a Cl‐ transporter, is important in determining GABAergic inhibition and stress outcomes in hypothalamic neurons, however its role in regulating these factors in DMV neurons is unknown. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently collapsing the Cl‐ gradient and leading to altered gastric output.

Sprague‐Dawley rats were fed either a control (14% kcal from fat) or high fat (60% kcal from fat) diet starting on embryonic day 13. After weaning on postnatal day 28, brainstem slices were made to conduct patch clamp recordings on DMV neurons. This was done in both naïve (unstressed) or stressed conditions, in which rats underwent a 10 minute forced swim (FS). To determine the Cl‐ reversal, perforated patch clamp recordings were performed in order to preserve the internal Cl‐ concentration. Then, DMV neurons were voltage clamped from ‐90 to ‐40 mV and the amplitude of the current response to picospritz of the GABAAagonist muscimol (100 μM) was measured. These events were plotted on an I‐V curve, with the x‐intercept representing the reversal of Cl‐. To determine a potential mediator in the shift in the Cl‐ reversal, changes in KCC2 levels following pHFD exposure were evaluated immunohistochemically. Lastly, gastric emptying (GE) assays were performed using the 13C octanoic acid breath assessment.

In both pHFD and control diet + FS DMV neurons, there was a depolarizing shift in the Cl‐ reversal compared to naïve rats (‐43.05 mV and ‐44.58 mV respectively, compared to ‐64.56 mV in naïve conditions; p < 0.0001). It was also found that there is a significant reduction in KCC2 expression in pHFD DMV region compared to controls (2.237% area fluorescence in pHFD compared to 5.335% in control diet; p=0.0147). Lastly, pHFD rats had significantly delayed GE rates compared to those fed a control diet (83.7 minutes for half‐decay in pHFD compared to 66.7 minutes in controls; p < 0.05), resembling previously published data which showed a delay in GE following an acute stress. These results demonstrate that both a pHFD and acute stress lead to depolarizing shifts in the Cl‐ reversal and that this may be through a mechanism involving a reduction in KCC2 expression. As the gastric emptying rates demonstrate, these changes could lead to a maladaptive GI stress response in pHFD rats.

Determining the mechanism in which pHFD and acute stress modulate DMV function, as well as the impact of their intersection on the gastric stress response, will better inform our understanding of the reduced stress resiliency seen in pHFD offspring.