Ethanol inhibits pancreatic projecting neurons in the dorsal motor nucleus of the vagus

Abstract

Alcohol use disorder (AUD) is a rapidly growing concern in the United States. Current trending escalations of alcohol use are associated with a concurrent rise in alcohol-related end-organ damage, increasing risk for further diseases. Alcohol-related end-organ damage can be driven by autonomic nervous system dysfunction, however studies on alcohol effects on autonomic control of end-organ function are lacking. Alcohol intake has been shown to reduce insulin secretions from the pancreas. Pancreatic insulin release is controlled in part by preganglionic parasympathetic motor neurons residing in the dorsal motor nucleus of the vagus (DMV) that project to the pancreas. How these neurons are affected by alcohol exposure has not been directly examined. Here we investigated the effects of acute ethanol (EtOH) application on DMV pancreatic-projecting neurons with whole-cell patch-clamp electrophysiology. We found that bath application of EtOH (50 mM) for greater than 30 min significantly enhanced the frequency of spontaneous inhibitory post synaptic current (sIPSC) events of DMV pancreatic-projecting neurons suggesting a presynaptic mechanism of EtOH to increase GABAergic transmission. Thirty-minute EtOH application also decreased action potential firing of these neurons. Pretreatment of DMV slices with 20 μM fluoxetine, a selective serotonin reuptake inhibitor, also increased GABAergic transmission and decreased action potential firing of these DMV neurons while occluding any further effects of EtOH application, suggesting a critical role for serotonin in mediating EtOH effects in the DMV. Ultimately, decreased DMV motor output may lead to alterations in pancreatic secretions. Further studies are needed to fully understand EtOH's influence on DMV neurons as well as the consequences of changes in parasympathetic output to the pancreas.

Keywords: Dorsal motor nucleus of the vagus; Electrophysiology; Ethanol; GABA; Pancreas.

Figure 1.. Retrograde labeling of DMV pancreas-projecting neurons.

A) Schematic demonstrating labeling of pancreatic nerve endings with application of DiI lipophilic dye directly to pancreatic tissue and sealed with an epoxy encasing to prevent labeling of other proximal gastrointestinal nerve endings. Figure also depicts the translocation of DiI to the cell bodies of pancreatic neurons within coronal slices of the caudal medulla that contain the DMV. B) On the left is an image of a coronal brainstem slice containing the DMV (outlined in white) at 4x magnification on the electrophysiology rig. The panels on the right show DMV neurons in infrared and fluorescence imaged at 40x magnification. The yellow arrow marks the cell body of a pancreatic-projecting neuron visualized in fluorescence within the DMV. DMV, dorsal motor nucleus of the vagus nerve; AP, area postrema; NTS, nucleus of the tractus solitarius.

Figure 2.. Extended EtOH application enhances GABAergic transmission onto DMV pancreatic-projecting neurons

A,B) Bar graph summarizing the effects of 50mM EtOH treatment duration on sEPSC frequency and sEPSC amplitude. C) Example traces from neurons from each treatment group showing the effects of the duration of 50mM EtOH application on sEPSCs compared to naïve cells. D,E) Bar graph summarizing the effects of EtOH treatment duration on sIPSC frequency and sIPSC amplitude. F) Example traces from neurons from each treatment group showing the effects of the duration of 50mM EtOH application on sIPSCs compared to naive cells. G) Bar graph summarizing the effects of 50mM EtOH on the ratio of excitatory to inhibitory current frequency.

Figure 3.. Extended EtOH application reduces action potential firing in DMV pancreatic-projecting neurons.

A) Graph demonstrating the effects of 50mM EtOH treatment duration on action potential firing after injections of current steps. B) Example traces showing the effects of 50mM EtOH application on action potential firing. C) Bar graph summarizing the effects of 50mM EtOH on resting membrane potential. D) Bar graph showing the capacitance of the cells that were recorded from for each condition.

Figure 4.. Fluoxetine pretreatment effects on EtOH modulation of DMV pancreatic-projecting neurons.

A,B) Bar graph summarizing the effects of 20 μM fluoxetine (FX) pretreatment and 50mM EtOH exposure duration on sEPSC frequency and sEPSC amplitude. C) Example traces from neurons from each treatment group showing the effects of FX pretreatment with 50mM EtOH application on sEPSCs compared to naïve cells. D,E) Bar graph summarizing the effects of FX pretreatment and EtOH treatment duration on sIPSC frequency and sIPSC amplitude. F) Example traces from neurons from each treatment group showing the effects of FX pretreatment and the duration of 50mM EtOH application on sIPSCs compared to naive cells. G) Bar graph summarizing the effects of FX pretreatment and 50mM EtOH on the ratio of excitatory to inhibitory current frequency.

Figure 5.. Fluoxetine pretreatment effects on EtOH modulation of action potential firing in DMV pancreatic-projecting neurons.

A) Graph demonstrating the effects of 20 μM fluoxetine pretreatment and 50mM EtOH treatment duration on action potential firing after injections of current steps. B) Example traces showing the effects of fluoxetine pretreatment and 50mM EtOH application on action potential firing. C) Bar graph summarizing the effects of fluoxetine pretreatment and 50mM EtOH on resting membrane potential. D) Bar graph showing the capacitance of the cells that were recorded from for each condition.