Alcohol withdrawal produces changes in excitability, population discharge probability, and seizure threshold

Abstract

Background: Alcohol withdrawal syndrome (AWS) results from the sudden cessation of chronic alcohol use and is associated with high morbidity and mortality. Alcohol withdrawal-induced central nervous system (CNS) hyperexcitability results from complex, compensatory changes in synaptic efficacy and intrinsic excitability. These changes in excitability counteract the depressing effects of chronic ethanol on neural transmission and underlie symptoms of AWS, which range from mild anxiety to seizures and death. The development of targeted pharmacotherapies for treating AWS has been slow, due in part to the lack of available animal models that capture the key features of human AWS. Using a unique optogenetic method of probing network excitability, we examined electrophysiologic correlates of hyperexcitability sensitive to early changes in CNS excitability. This method is sensitive to pharmacologic treatments that reduce excitability and may represent a platform for AWS drug development.

Methods: We applied a newly developed method, the optogenetic population discharge threshold (oPDT), which uses light intensity response curves to measure network excitability in chronically implanted mice. Excitability was tracked using the oPDT before, during, and after the chronic intermittent exposure (CIE) model of alcohol withdrawal (WD).

Results: Alcohol withdrawal produced a dose-dependent leftward shift in the oPDT curve (denoting increased excitability), which was detectable in as few as three exposure cycles. This shift in excitability mirrored an increase in the number of spontaneous interictal spikes during withdrawal. In addition, Withdrawal lowered seizure thresholds and increased seizure severity in optogenetically kindled mice.

Conclusion: We demonstrate that the oPDT provides a sensitive measure of alcohol withdrawal-induced hyperexcitability. The ability to actively probe the progression of excitability without eliciting potentially confounding seizures promises to be a useful tool in the preclinical development of next-generation pharmacotherapies for AWS.

Keywords: alcohol withdrawal syndrome; electrophysiology; interictal spikes; optogenetics; seizure.

Figure 1.. Spontaneous interictal spike occurrence increases during alcohol WD in CIE exposure paradigm.

A. Exposure to CIE ethanol vapor increased the frequency of spontaneous IS during each WD. This increase was significant compared to ethanol naïve animals during the third and fourth WD periods, and was no longer significantly different at 72 hours after the final WD period. Mixed effects model. sPD = spontaneous population discharges, B = withdrawal baseline, PYR = pyrazole control, WD = withdrawal, P = days post-withdrawal. B. & C. Example of a spontaneous IS recorded across the peri-hippocampal network. See methods section for brain region abbreviations and coordinates.

Figure 2.. oPDT testing is a sensitive measure of CNS hyper-excitability during CIE exposure related WD.

a. Spontaneously-occurring IS shares a network signature with evoked oPD (blue bar indicates timing and duration of the optical stimulus). b. Light dose-response curves allow for determination of oPD threshold. c. The average oPD probability curve, fit to the Boltzmann equation, for each animal at baseline and during each subsequent WD reveals that multiple EtOH exposure and WD cycles progressively increase network excitability. The leftward shift in the oPD curve indicates that less light is required to produce oPDs. d. The I₅₀ during the 3^rd and 4^th WD were significantly different from baseline. Mixed effects model.

Figure 3.. Optogenetically-induced seizure threshold is reduced in WD while severity and duration is increased.

a. An example of an optogenetically-induced seizure in a Thy1-ChR2 mouse following kindling prior to CIE. b. An induced seizure in the same subject following 4 WD cycles. c. The threshold for generating an AD and behavioral seizure in previously kindled animals was reduced following WD from CIE exposure (p = 0.0127, n = 6, paired t-test). The seizure threshold had previously been stable with no significant differences in threshold from one kindling session to the next. d. The duration of behavioral seizure also increased. Prior to CIE exposure animals had behavioral seizure lasting on average 22.83 +/− 3.070s, after CIE, exposure average seizure increased to 236.2 +/− 46.83s. p = 0.006, n = 6, paired t-test. e. The severity of seizure was increased with each animal experiencing the most severe seizure score, stage 6. Only after CIE exposure did any animal have a stage 6 seizure. p = 0.0053, n = 6, paired t-test.