Alcohol Use Disorder (AUD) is a debilitating condition that affects many people around the world. When people suffering from this condition stop consumption of ethanol for a period of time, they undergo withdrawal symptoms. While symptoms like anxiety have been attributed to ethanol withdrawal, the biological basis for the withdrawal from alcohol has not been well understood. The Himmelsbach Hypothesis attempted to explain that withdrawal was the reaction to the adaptation to ethanol consumption. Despite various studies indicating that this hypothesis has merit, behavioral biomarkers have not yet been quantified and characterized to augment these findings. While specific behaviors, including seizures and anxiety, have been studied, an progression of withdrawal from ethanol consumption has not been interrogated with a long-term temporal resolution. In patients, symptoms like lethargy and anxiety indicate disrupted energy levels. Thus, we hypothesize that mice undergoing ethanol withdrawal have a disrupted energy homeostasis. In this study, naive C57BL6 mice were made ethanol dependent using intermittent ethanol vapor exposure. Specific factors like their Respiratory Quotient and Energy Expenditure were monitored to find important time-dependent changes to reveal the progression of ethanol withdrawal.

A Novel Comparison and Characterization of Ethanol Withdrawal

Anant Naik1, Katheryn Wininger2, David Hinton1,2,3, Phillip Starski2, Doo-Sup Choi1,2,3 1Department of Molecular Pharmacology and Experimental Therapeutics, 2Neurobiology of Disease Program,

3Department of Psychiatry and Psychology, Mayo Clinic, Rochester, Minnesota.

Himmelsbach Hypothesis of Ethanol Withdrawal:

Administration of Alcohol Acute

Alcohol Effects

Opposing Neuroadaptation

Alcohol tolerance

Removal of Alcohol

Withdrawal Syndrome

Recovery from Neuroadaptation

CNS Equilibrium

Alcohol-free state

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Extinction Burst

Extinction Phenomenon

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Acute Injection

Long Term Exposure

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Acute Re-administration

• Ethanol dependence and recording was conducted by adhering to the rigid time-course and serial recordings.

• A loading dose of EtOH of 2 g/kg was administered IP with 68.1 mg/kg Pyrazole prior to vapor chamber exposure.

• 2 g/kg was administered IP for Acute administration

Metabolic Chamber Recording

BAC measurements

Food dispenser

Rearing IR detectors

Air to gas analyzer

Readouts

Figure 1) Withdrawal is generated by an extinction of ethanol. This extinction results in a reduction of blood ethanol. This effect was observed in one cycle of exposure to blood ethanol. Blood was collected via the tail of mice (n=3), and their BAC was compared at the time of removal, or 0 hrs, and 8 hours after this time period. A significant effect was observed (p = 0.001)

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©2016 Mayo Foundation for Medical Education and Research

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Repeat EtOH

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• Pre-clinical comparisons of genotype using paradigm

• Comparison of RQ and EE values with treatment with Taurine or Acamprosate during alcohol withdrawal

• Clinical monitoring of Respiratory Quotient and Energy expenditure and correlates with withdrawal of ethanol

Behavioral and Molecular

Metabolite-based Biomarkers of withdrawal, observing glutamine, adenosine, correlating with metabolic behavior. • Magnetic Resonance

Spectroscopy Imaging • Micro-dialysis

• Mitochondrial Dysfunction and regulation, size, density, orientation, location in neurons and astrocytes

• The Metabolic Chamber was utilized to monitor metabolic activity of the mice after exposure to ethanol. Metabolic chamber too measurements every 6 minutes regarding metabolic activity and 2 minutes for food consumption and rearing.

3. Blood Alcohol Concentration Reduction

2. Methods & Materials

1. Introduction

Figure 2. A) RQ values were averaged over 1 hour. The overall distribution of the control animals (n=10) compared to the long term and the chronic administration of ethanol (n=8) was significant (p < 0.0001). From the distributions, RQ value for the chronic exposure began more similar to long term exposure, but progressively increased to align with the control. B) A significant difference in the eating pattern of the control compared to the long term and chronic intermittent exposure (p < 0.0001). There was no statistically significant difference in total food consumed, though a trend emerged that the long term exposure generally ate less food than both chronic exposure and control animals. No significance was found between the rates of consumption between either of the three groups. C) The overall energy expenditure, in Kcal, was also significantly different (p < 0.0001). Chronic intermittent exposure also resulted in significantly significant difference at particular time points than the control animals (p < 0.05). D) There are no significant differences between the total amount of food consumed.

Figure 3. Rearing was measured as a general marker for activity. A) Rearing was summed every 60 minutes for entire recording period. While substantial differences did not exist between chronic exposure and control mice, rearing substantially increased from 10 hours to 12 hours in the long term exposure (p < 0.05) compared to other groups. This difference did not manifest in a change in overall rearing comparing in other groups, suggesting a trend of net depression at other time points. B) While there was a significant difference between the Long Term and Chronic Exposure (p < 0.05), there wasn't a significant difference between the control and chronic groups, which can be expected from the Figure 3A.

Figure 4. A) Post-chronic exposure acute administration of ethanol resulted in important metabolic changes. The RQ value was substantially greater in mice undergoing the acute administration post-chronic exposure compared (n = 8) to naive mice acutely administered ethanol (n = 4) of the same dose (p = 0.0013). This suppression was found as a general main effect, but also at particular time points, namely 10-17 hours after administration (p < 0.05). B) Energy expenditure did not vary significantly. However, energy expenditure decreased for the post-chronic injection group 5-6 hours. C) In the overall distribution of eating chronic exposure mice consumed food at a greater consumption rate (p = 0.0110). As evident, the rate of eating seemingly increased during the dark cycle of mice. D) Mice that underwent chronic exposure consumed significantly more food than the acute control mice (p = 0.013).

5. Withdrawal Activity Markers

6. Metabolic Markers in Repeat Administration

4. Withdrawal Metabolic Markers

Figure 1. A) There was a marginal difference in rearing in mice re-administered Ethanol, albeit not significant. However, there was a substantial decrease in repeat exposure mice compared to acute control mice at 9-11 hours after the IP injection B) This decline in rearing was also evident in total rearing measured at the endpoint of the recording. A significant decrease in re-exposure mice was observed (p = 0.03).

• There is an attenuation on the progression of withdrawal after repeated exposures to ethanol.

• Rearing significantly increases from 10-12 hours after extinction of ethanol in long-term exposure. This is a known correlate to handling-induced convulsive seizures in mice experiencing withdrawal.

• Acute re-administration of ethanol in mice previously exposed to ethanol chronically equilibrates their metabolism.

• Acutely injected naive mice also experience an increase in rearing at 10-12 hours, indicating another significant time point in acute withdrawal.

• Time dependent changes in the respiratory quotient, energy expenditure, and eating patterns can be used potentially to characterize the progression of EtOH withdrawal

This research was made possible by the support of Dr. Choi’s Lab resources (RO1s and grants from the Samuel C. Johnson Genomics of Addiction Foundation) and lab members, resources by MPET, and the IMPACT program.

• Abulseoud OA, et. al. (2014) Attenuation of ethanol withdrawal by ceftriaxone-induced upregulation of glutamate transporter EAAT2. Neuropsychopharmacology Jun;39(7):1674-84.

• Kim JH et. al. (2011) Functional role of the polymorphic 647 T/C variant of ENT1 (SLC29A1) and its association with alcohol withdrawal seizures. PLoS One Jan 24;6(1):e16331

8. Discussion & Conclusions

10. References

Acknowledgements

9. Future Directions

7. Activity Markers in Repeat Administration

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