1. 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
Time
BehavioralFrequency
Extinction
Start
Extinction
Burst
Extinction
Phenomenon
Extinct Spontaneous
Relapse
6:00
18:00
Dark Cycle Light Cycle
10:00
Ethanol Exposure
No exposure
Acute Injection
Long Term
Exposure
Chronic
Exposure
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)
BloodEthanolContent(Mg/dL)
©2016 Mayo Foundation for Medical Education and Research
A. B.
C. D.
A. B.
A. B.
C. D.
Repeat EtOH
Repeat EtOH
Repeat EtOH
Repeat EtOH
• 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
A. B.
Repeat EtOH
Repeat EtOH