Acute ethanol modulation of neurocircuit function in the nucleus of the tractus solitarius

Abstract

The nucleus of the tractus solitarius (NTS) is a brain stem region critical to many physiologic processes and has been implicated in addiction to multiple classes of abused drugs, including alcohol (EtOH). That said, the mechanism by which EtOH modulates NTS neurocircuit activity is not well characterized and has yet to be examined utilizing electrophysiologic methods in mouse models of alcohol use disorders. To begin to address this gap in knowledge, we sought to use whole-cell and cell-attached recordings to determine the mechanism of acute EtOH action on GABAergic and glutamatergic neurotransmission, as well as on action potential firing in the NTS of adult male, EtOH naïve mice. Bath application of EtOH (50mM) significantly enhanced the frequency of spontaneous inhibitory postsynaptic current events, while increasing the amplitude of these events in half of the neurons tested. This finding suggests a presynaptic mechanism of EtOH action on GABAergic transmission in the NTS as well as a postsynaptic mechanism in subsets of NTS neurons. EtOH application was further associated with a significant decrease in action potential firing in most, but not all, NTS neurons tested. EtOH induced a small but significant decrease in spontaneous excitatory postsynaptic current frequency, indicating that EtOH may also inhibit NTS glutamatergic signaling to some degree. Intriguingly, in vivo EtOH exposure (4g/kg IP) enhanced c-FOS colocalization with tyrosine hydroxylase via immunohistochemical methods, indicating that NTS norepinephrine neurons may be activated by acute EtOH exposure. Although future work is needed, the current data indicate that acute EtOH may enhance GABAergic signaling in local NTS circuits resulting in disinhibition of NTS norepinephrine neurons. Such a finding has important implications in understanding the role of the NTS in the development of alcoholism.

Keywords: Alcoholism; Electrophysiology; Mechanisms of addiction.

Figure 1. EtOH enhances GABAergic transmission in the NTS

A) Exemplar traces from one neuron tested showing the effects of 50 mM EtOH on sIPSCs compared to baseline. Top and bottom traces are continuous but separated into two lines due to space constraints. B) Bar graph summarizing the effects of EtOH on sIPSC frequency and amplitude normalized as percent change from baseline. Black circles are individual data points. Dashed line indicates baseline level, normalized to 100%. Asterisk indicates significant difference from baseline. C) Bar graph summarizing the median split of sIPSC amplitude data from B. Data is expressed as change from normalized baseline (100%). Black circles are individual data point. Solid line indicates median. Asterisk indicates a significant difference from baseline. D) Correlation examining the magnitude of EtOH induced changes to sIPSC frequency vs amplitude, expressed as percent changes from normalized baselines.

Figure 2. EtOH has modest effects on glutamatergic transmission in the NTS

A) Exemplar traces from one neuron tested showing the effects of 50 mM EtOH on sEPSCs compared to baseline. Top and bottom traces are continuous but separated into two lines due to space constraints. showing the effects of 50 mM EtOH on sEPSCs compared to baseline. B) Bar graph summarizing the effects of EtOH on sEPSC frequency and amplitude normalized as percent change from baseline. Black circles are individual data points. Dashed line indicates baseline level, normalized to 100%. Asterisk indicates significant difference from baseline.

Figure 3. EtOH predominantly inhibits action potential firing in the NTS

A) Exemplar traces showing the effects of 50 mM EtOH on action potential firing compared to baseline. B) Exemplar traces showing averaged action potential waveform from experiment in A. Determination of action potential peak and half-width are shown. Dotted line shows no change in peak between baseline and EtOH C) Bar graph summarizing the effects of EtOH on action potential frequency and kinetics normalized as percent change from baseline. Black circles are individual data points. Dashed line indicates baseline level, normalized to 100%. D) Bar graph summarizing the median split of action potentiation frequency data from B. Data is expressed as change from normalized baseline (100%). Black circles are individual data points. Solid line indicates group median. Asterisk indicates a significant difference from baseline.

Figure 4. Effects of EtOH on c-FOS/TH colocalization

A) Exemplar images of NTS slices prepared from saline, 2 g/kg EtOH and 4 g/kg EtOH injected mice utilized for immunohistochemistry. White scale bars indicate 50 microns. B) Bar graph summarizing the effects of treatment on total number of c-FOS+ cell counts. Black circles are individual data points. C) Bar graph summarizing the effects of treatment on percent of TH+ neurons colabeled with c-FOS. Black circles are individual data points. Asterisk indicates significant differences from all other groups.