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Antagonist-induced reversal of functional and structural measures of hippocampal benzodiazepine tolerance
1Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA. etietz@mco.edu
Abstract:
One week oral flurazepam (FZP) administration in rats results in anticonvulsant tolerance in vivo, tolerance measured in vitro in hippocampal CA1 pyramidal cells, and regulation of hippocampal gamma-aminobutyric acid(A)-receptor subunit protein expression. A single injection (4 or 20 mg/kg i.p) of the benzodiazepine antagonist flumazenil (FLM) was given 1 day after FZP treatment, and tolerance and subunit protein expression were evaluated 1 day later. In vivo tolerance was measured by a reduced ability of the alpha(1)-subunit-selective agonist zolpidem to suppress pentylenetetrazole-induced seizures. This tolerance was reversed by 20 but not 4 mg/kg FLM. In in vitro hippocampal slices, there was tolerance to the effect of zolpidem to prolong the decay of pyramidal cell miniature inhibitory postsynaptic currents, which was reversed by FLM (4 mg/kg) pretreatment. A reduction in miniature inhibitory postsynaptic current amplitude ( approximately 50%) was also restored by FLM injection. [(3)H]Zolpidem binding measured 0, 2, and 7 days after FZP treatment was significantly decreased in the hippocampus and cortex at 0 days but not thereafter. Changes in alpha(1)- and beta(3)-subunit protein expression were examined via quantitative immunohistochemical techniques. alpha(1)-Subunit protein levels were down-regulated in the CA1 stratum oriens and beta subunit levels were up-regulated in the stratum oriens and stratum radiatum of the CA3 region. Chronic FZP effects on alpha(1)- and beta(3)-subunit protein levels were also reversed by prior FLM injection. FLM's effect on both functional and structural correlates of benzodiazepine tolerance suggests that each of these measures plays an interdependent role in mediating benzodiazepine tolerance.
Insights
Flurazepam (FZP) causes tolerance to its anticonvulsant effects, altering GABA(A)-receptor subunit expression. The antagonist flumazenil (FLM) reversed these changes, indicating an interdependent role in benzodiazepine tolerance.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- Chronic administration of benzodiazepines, such as flurazepam (FZP), can lead to the development of tolerance.
- Tolerance to benzodiazepines involves alterations in gamma-aminobutyric acid type A (GABA(A))-receptor subunit expression and function.
- Understanding the mechanisms underlying benzodiazepine tolerance is crucial for managing treatment efficacy and withdrawal symptoms.
Purpose of the Study:
- To investigate the effects of flurazepam (FZP) on anticonvulsant tolerance, GABA(A)-receptor subunit protein expression, and receptor function in rats.
- To determine the efficacy of flumazenil (FLM), a benzodiazepine antagonist, in reversing FZP-induced tolerance and associated molecular changes.
- To explore the interdependent roles of functional and structural adaptations in mediating benzodiazepine tolerance.
Main Methods:
- Rats were administered oral flurazepam (FZP) for one week, followed by single injections of flumazenil (FLM) at varying doses.
- In vivo anticonvulsant tolerance was assessed using the pentylenetetrazole seizure threshold test and the efficacy of zolpidem, an alpha(1)-subunit-selective agonist.
- In vitro electrophysiological recordings in hippocampal slices measured miniature inhibitory postsynaptic current (mIPSC) decay and amplitude.
- [3H]Zolpidem binding assays and quantitative immunohistochemistry were used to evaluate GABA(A)-receptor subunit protein expression (alpha(1) and beta(3)).
Main Results:
- FZP administration induced significant in vivo and in vitro anticonvulsant tolerance, which was partially or fully reversed by FLM.
- FLM pretreatment restored normal mIPSC decay and amplitude in hippocampal slices.
- FZP treatment led to decreased [3H]zolpidem binding, down-regulation of alpha(1)-subunit protein in CA1, and up-regulation of beta subunit protein in CA3, all of which were reversed by FLM.
- FZP-induced changes in alpha(1)- and beta(3)-subunit protein levels were reversed by prior FLM injection.
Conclusions:
- Flumazenil (FLM) effectively reverses both functional and structural adaptations associated with flurazepam (FZP)-induced benzodiazepine tolerance.
- The findings suggest an interdependent relationship between GABA(A)-receptor subunit expression changes and functional tolerance.
- These results provide insights into the neurobiological mechanisms of benzodiazepine tolerance and potential therapeutic strategies for its management.