Date of Award
Doctor of Philosophy (PhD)
Scott A. Heldt, Ph.D.
Kristin M. Hamre, Ph.D. Michael P. McDonald, Ph.D. Kazuko Sakata, Ph.D. Jeffery D. Steketee, Ph.D.
Zolpidem and benzodiazepines (BZs) potentiate the inhibitory action of gamma-Aminobutyric acid (GABA) by allosterically binding to GABAA receptors (GABAAR). Prolonged use of GABAAR positive allosteric modulators (PAM) can lead to behavioral tolerance, the diminished response to the same drug dose with repeated use, and withdrawal, a group of symptoms that occur due to abrupt end of drug treatment. Zolpidem is a short-acting, non-BZ GABAAR PAM whose potential for tolerance and withdrawal is unclear. Zolpidem demonstrates sedative efficacy similar to BZs and has become a main treatment of insomnia in lieu of BZs. Zolpidem replaced BZs due to lower incidences of tolerance and withdrawal after prolonged treatment and discontinuation. Despite reported lower incidences, some studies find the occurrence of tolerance and withdrawal similar between zolpidem and BZs. Tolerance and withdrawal symptoms are likely caused by drug-induced neuroadaptive changes in central nervous system (CNS)
functioning, and these alterations may be similar between zolpidem and BZ. Past rodent research suggests that long term use of zolpidem and BZs may produce alterations in normal inhibitory GABAergic and excitatory glutamatergic functioning in the cortex, hippocampus, amygdala, and PFC and that these alterations may underlie sedative tolerance and withdrawal symptoms.
The purpose of this project was to examine the molecular mechanisms involved in the tolerance cross-tolerance, and withdrawal of zolpidem and diazepam in C57/BL6J mice after different treatment durations. Elucidating the mechanisms behind zolpidem tolerance and withdrawal is necessary due to the ongoing usage of subunit specific GABAAR PAMs and, to a broader extent, an understanding of GABAARs themselves.
In Study 1, we measured sedative tolerance, cross-tolerance, and GABAAR associated mRNA levels in 4 regions of interest (ROI; the cortex, prefrontal cortex (PFC), hippocampus, and amygdala) after 3 days of intraperitoneal (i.p.) injections of diazepam and zolpidem in comparison to vehicle. We expected that this “short-term” exposure duration to diazepam and zolpidem would not result is tolerance, cross-tolerance, or changes in mRNA levels. Study 2 examined the same measures as in Study 1, in addition to AMPAR subunits, NDMAR subunits mRNA levels in the ROI, and total, surface, and intracellular GABAAR subunits protein expression due to 7 days of i.p. injections of diazepam and zolpidem compared to vehicle. Based on previous research both groups should become tolerant to zolpidem’s sedative effects and show decreases of GABAAR subunits and increases in NMDAR subunits in the ROI. It is also expected that there will be decreases in total α1 and γ2 in the cortex, a decrease of surface α1 in the cortex, and increases in GluR1 in the hippocampus after zolpidem and diazepam treatment. Study 3 measured the same measures as in Study 1 due to 30 days of i.p. injections of diazepam and zolpidem compared to vehicle. It was expected that both groups would become tolerant to zolpidem’s sedative effects and show decreases of GABAAR subunits in the cortex, PFC, and hippocampus.
The development of sedative tolerance and cross-tolerance to the locomotor impairing effects (measure of sedation) of zolpidem was measured by activity in the open field. Spontaneous withdrawal was also measured by activity and anxiety like behavior in the open field. Flumazenil- induced withdrawal was measured by anxiety- like behaviors in the elevated plus maze (EPM), activity, and anxiety like behavior in the open field. Messenger RNA levels were measured by quantitative real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein expression was measured by western blot. The surface and intracellular proteins were separated using bissulfosuccinimidyl suberate (BS3) cross-linking.
Three days of diazepam but not zolpidem resulted in cross-tolerance to zolpidem in Study 1. Three days of zolpidem but not diazepam resulted in a decrease in the mRNA level of the α5 subunit in the hippocampus in Study 1. After 7 days of zolpidem or diazepam, mice were tolerant and cross-tolerant to zolpidem’s sedative effects. Spontaneous withdrawal resulted in anxiety-like behavior and decreased locomotor activity. Flumazenil did induce a robust withdrawal syndrome as measured in the EPM or open field. Seven days of zolpidem and diazepam caused significant decreases in the mRNA expression of α1, α3, β2, and δ GABAAR subunits in the cortex. Diazepam groups had significant decreases in the mRNA expression of α4, β1, γ2 subunits, GAT, and gephyrin in the cortex and significant decreases of α5- and β3-GABAAR
subunits, and the GluN2A subunit in the hippocampus. Seven days of zolpidem resulted in a decrease in total α2 subunit protein level and 7 days of diazepam decreased total γ2 subunit protein levels. Thirty days of diazepam but not zolpidem resulted in cross-tolerance to zolpidem in Study 3. Thirty days of zolpidem but not diazepam resulted in a decrease in the mRNA levels of α1, α2, α3, β1, β2, β3, γ1, and γ2 subunits in the PFC.
These results suggest that there is a window of time in which sedative tolerance to zolpidem is observed. The lack of zolpidem tolerance and minimal mRNA changes due to 3 days of zolpidem treatment may be due to its pharmacokinetic profile, zolpidem may not be in the system long enough to cause any changes. This may mean that sedative tolerance gradually develops and reaches detectable levels at later time points. Sedative tolerance and cross-tolerance to zolpidem is in line with other studies, however the spontaneous withdrawal is unique. Anxiety- like behavior and decreased activity were observed in our studies unlike other studies. The anxiety- like behavior is a common symptom of BZ withdrawal however the decrease in activity that was observed is not. It is unknown why this occurs though it may be due to a carryover sedative effect or more likely a placebo effect. Few studies have examined changes in protein levels. Study 2 found decreased protein expression of α2 and γ2 in the cortex due to zolpidem and diazepam respectively, indicate GABAARs containing those subunits are associated with tolerance and cross-tolerance to zolpidem’s sedative effects. This implies that the sedative effects of zolpidem is mediated by α1-GABAARs, the development of tolerance is mediated by α2-GABAARs due to zolpidem binding to both. There was also a decrease in the intracellular α1 subunit which may indicate degradation of an intracellular pool of α1 subunits or α1-GABAARs. The lack of zolpidem tolerance due to 30 days of zolpidem may be due to increased metabolism displayed as an increase in CYP3A enzymes. It is unclear what effect the decrease in GABAAR subunits in the PFC due to 30 days of zolpidem implicate. These may affect tolerance to zolpidem’s other effects such as amnesia.
Wright, Brittany T. (http://orcid.org/0000-0002-1501-6596), "Repeated Zolpidem Treatment Effects on Sedative Tolerance, Withdrawal, mRNA Levels, and Protein Expression" (2016). Theses and Dissertations (ETD). Paper 406. http://dx.doi.org/10.21007/etd.cghs.2016.0408.