Beyond the Paper: A Conversation with Reginald Cannady and Sudarat Nimitvilai-Roberts

Interviewed by Rosalind S.E. Carney, DPhil

Alcohol (ethanol) use disorder is associated with changes in frontal cortical areas, including the anterior cingulate (ACC) and orbitofrontal cortex that contribute to cognitive deficits, uncontrolled drinking, and alcohol relapse. Acute ethanol exposure reduces the intrinsic excitability of lateral orbitofrontal cortex (lOFC) neurons, while chronic exposure and long-term drinking influence plasticity of intrinsic excitability and function of glutamatergic synapses. However, the time course in which these adaptations occur across a history of ethanol drinking was unknown. In their publication, Cannady, Nimitvilai-Roberts, and colleagues found that voluntary ethanol drinking in mice produced distinct time-dependent changes in cellular excitability in the ACC and IOFC that varied by direction and duration. These drinking-induced changes in cellular excitability were specific to action potential firing, but not to the function of excitatory or inhibitory synapses. These findings highlight the importance and sensitivity of alterations in the cellular firing of cortical neurons that occur early in a drinking history and persist during long-term ethanol consumption.

(Left) Reginald Cannady, PhD, Post-doctoral fellow, K99 trainee, Department of Neuroscience, Medical University of South Carolina, Charleston, SC.
(Right) Sudarat Nimitvilai-Roberts, PhD, Post-doctoral fellow, Department of Neuroscience, Medical University of South Carolina, Charleston, SC.

RC: How did you become interested in this particular line of research?

Reginald: “I became interested in neuroscience as an undergraduate working in a neurotoxicology lab at Fayetteville State University. I was always intrigued by how exogenous substances could alter behavior. Through my post-baccalaureate and post-graduate experiences at Duke and UNC-Chapel Hill, I focused on neuropharmacological treatments that targeted ion channels and neurotransmitter receptors that regulate working memory, alcohol, and other drug reward responses, and associative learning in several models and behavioral tasks. As a post-doc, I wanted to study the cellular mechanisms of alcohol addiction, so I joined Professor Patrick Mulholland’s lab at the Medical University of South Carolina, Charleston, SC. The Mulholland lab has a strong interest in the intrinsic properties of neurons. In the past, we have focused heavily on potassium channels that regulate neuronal firing properties. Therefore, it was a natural transition to investigate other aspects of neuronal activity, including the effects of ethanol exposure. While it was known that ethanol exposure leads to adverse effects on the  anterior cingulate cortex (ACC), there was a large gap in our understanding of how the intrinsic firing properties of ACC neurons would be affected.”

Sudarat: “During my doctoral research at the University of Illinois at Chicago, I investigated the effects of dopamine on the dopamine D₂ receptor in the ventral tegmental area using extracellular recording techniques. For my post-doc, I wanted to combine other types of electrophysiological recordings with behavioral analysis. Therefore, I joined the lab of Professor John Woodward, who is in the same department as Patrick. The labs have a history of collaboration, which has resulted in several prior publications on the effects of alcohol exposure on neurons located in the lateral orbitofrontal cortex (lOFC). These prior studies used experimental approaches such as in vivo ethanol exposure, slice electrophysiology, imaging, and behavioral assays. These studies revealed that IOFC lesions in mice enhance ethanol consumption following the induction of ethanol dependence. In addition, chronic ethanol exposure increases the intrinsic excitability of IOFC neurons and alters synaptic transmission, including dendritic spine morphology in the IOFC.”

What were the main findings in this eNeuro paper?

Sudarat: “Whereas it was known that passive ethanol exposure alters several characteristics of IOFC neurons, the effects of voluntary ethanol drinking on these neurons had not been examined. Therefore, we used a two-bottle choice model to induce high levels of ethanol consumption prior to performing patch-clamp recordings. The intrinsic firing properties of IOFC neurons progressively increased during 7 weeks of ethanol consumption. These findings were similar to those we previously reported in mice exposed to repeated cycles of ethanol vapor. However, we did not observe an effect on excitatory or inhibitory synaptic transmission in IOFC neurons following voluntary exposure to alcohol consumption.”

Reginald: “We found that there were temporal changes in the intrinsic firing properties of deep layer ACC neurons after a brief history of voluntary ethanol drinking that was not found following a protracted period of drinking. The number of action potentials that were fired in ACC neurons was higher in mice that voluntarily drank ethanol for only one day. This change was no longer present at 1, 4, and 7 weeks of ethanol exposure. Our findings suggest that ACC neurons may play a role in the initial processing of information regarding ethanol consumption, but this role is not evident during longer periods of ethanol exposure. These findings are in contrast to the observations in the IOFC that Sudarat described.”

Why is this paper an important contribution to the field?

Reginald: “This paper contributes to the growing body of evidence that each cortical subdivision may function independently to process ethanol-specific information. These findings have implications for potential treatment approaches in individuals who experience cognitive deficits related to alcohol abuse. This study also furthers knowledge of how neurons in the ACC are affected by ethanol exposure. The voluntary ethanol drinking model that we used compliments other studies that use a passive ethanol exposure method. In relation to experimental design, our results emphasize the importance of considering the temporal effects of ethanol exposure per brain region. For instance, if we had just examined 4 or 7 weeks of ethanol exposure, we would have missed some of the transient changes in the intrinsic firing properties of ACC neurons. However, having completed several time points in a broad period, we know that ACC neurons are particularly susceptible to the adverse effects of ethanol following acute exposure. Therefore, it is important to consider in future studies that even brief exposure to ethanol does alter brain function.”

Sudarat: “Our findings show that a history of ethanol drinking differentially affects the intrinsic firing properties of cortical neurons in a region- and time-dependent manner. In our model, these changes in intrinsic firing properties can occur without changes in synaptic transmission. The bidirectional effect of ethanol exposure in the ACC is an unexpected finding and contrasts with our results from IOFC recordings. As the ACC and IOFC are involved in cognition and decision making, it will be of interest to the field of addiction research to relate the history of ethanol exposure to potential transient physiological changes in neurons across different brain regions. Ultimately, the goal of this research is to aid in the future development of therapies that would mitigate the addictive properties of alcohol and reduce the symptoms related to alcohol withdrawal.”

“In relation to experimental design, our results emphasize the importance of considering the temporal effects of ethanol exposure per brain region. For instance, if we had just examined 4 or 7 weeks of ethanol exposure, we would have missed some of the transient changes in the intrinsic firing properties of ACC neurons.“

Reginald Cannady, PhD

What were the challenges and advantages of conducting a collaborative project between two labs?

Sudarat: “The history of collaboration between the Woodward and Mulholland labs meant that this experience was not new for us and had the advantages of support from two labs. In terms of experimental challenges, I recorded from the IOFC and Reginald recorded from the ACC of the same mouse each time. Therefore, our experiments served as reciprocal controls, which was advantageous. However, this did make it challenging to find a schedule for recording that suited us for the duration of the research. We each performed our own analysis of the electrophysiological data. As the Woodward and Mulholland labs are adjacent, we easily had opportunities to discuss the results and interact regularly. We recorded every day, so Reginald and I chatted about our data each morning, in addition to frequent meetings with John and Patrick.”

Reginald: “Planning was very important due to the need to be available at the same time. The Mulholland and Woodward labs have a history of successful collaboration. The Charleston Alcohol Research Center fosters a collaborative environment, and I frequently ask Sudarat questions about electrophysiology as she has more expertise than I do. Patrick and John have done a phenomenal job in guiding us and creating a supportive research environment. I had a great time working with Sudarat, and open communication is key to collaborative research. This paper is a great culmination to a lot of hard work and challenges, such as hurricanes and the labs moving to a new building. I am very happy that this project is now published.”

“Our findings show that a history of ethanol drinking differentially affects the intrinsic firing properties of cortical neurons in a region- and time-dependent manner...Ultimately, the goal of this research is to aid in the future development of therapies that would mitigate the addictive properties of alcohol and reduce the symptoms related to alcohol withdrawal.“

Sudarat Nimitvilai-Roberts, PhD

How was your experience with the eNeuro review process

Reginald: “I think the eNeuro review process was exceptional. We were happy that the reviewers were receptive to our work.”

Sudarat: “John suggested that we submit to eNeuro—we have not published there before. The comments were fair and really helped to make the paper more interesting. I liked the consensus review as we did not have to respond to potentially overlapping comments from two reviewers.”

What are your future research goals?

Sudarat: “In John’s lab, I am continuing research on the effects of acute and chronic ethanol exposure on the structure and function of IOFC neurons and IOFC-mediated behaviors. We know that the IOFC has connectivity with brain regions involved in cognitive flexibility and goal-related behaviors. Therefore, we would like to expand our findings to test the effects of ethanol exposure on IOFC connectivity using ex vivo slice electrophysiology, in vivo calcium photometry, and in vivo optogenetics.”

Reginald: “My overall goals are to begin focusing on inhibitory neurons associated with ethanol consumption with more emphasis on mechanisms of reinforcement and ethanol-seeking behaviors. This avenue of research marries together my graduate research and my current training at MUSC. Many studies link disrupted excitatory transmission in the prefrontal cortex and excessive alcohol consumption and alcohol-seeking behavior. However, there are relatively few studies that have examined the contribution of inhibitory neurons to these behaviors. Now that we have the genetic tools that can selectively target inhibitory neuron subtypes, I will investigate the role of fast-spiking interneurons in mediating alcohol consumption and alcohol-seeking behaviors.”

Read the full article:

Distinct Region- and Time-Dependent Functional Cortical Adaptations in C57BL/6J Mice After Short and Prolonged Alcohol Drinking
Reginald Cannady* , Sudarat Nimitvilai-Roberts* , Sarah D. Jennings, John J. Woodward, and Patrick J. Mulholland

*Co-first authors