Research reportIncrease in expression of the GABAA receptor α4 subunit gene induced by withdrawal of, but not by long-term treatment with, benzodiazepine full or partial agonists
Introduction
Clinical, behavioral and electrophysiological studies have shown that long-term administration of benzodiazepines results in the development of tolerance to some of the effects of these drugs, a phenomenon that limits their clinical efficacy [13], [20]. Such long-term treatment is also associated with the development of physical dependence [42]. The molecular bases for these phenomena remain unclear.
Tolerance and dependence appear related to the pharmacodynamics, rather than to the pharmacokinetics, of benzodiazepines [13], [17]. Early electrophysiological evidence indicated that long-term administration of benzodiazepines reduces postsynaptic sensitivity to GABA [16]. This effect is accompanied by biochemical alterations, even though there are controversial data in the literature regarding changes in the number of benzodiazepine recognition sites [11], [36], [37], [46]. However, chronic benzodiazepines treatment has been reported to produce a reduction in the ability of GABA to enhance benzodiazepine binding [16], [24], [25], [40]. Nevertheless no one of these single processes clearly explains the phenomenon of benzodiazepines tolerance and dependence.
Recent in vivo studies have suggested that long-term administration of benzodiazepines modifies the expression of genes that encode various subunits of the GABA type A (GABAA) receptor complex (for review see [35]). Such changes in gene expression might alter the sensitivity of GABAA receptors to their pharmacological modulators and thereby underlie the development of tolerance to or dependence on these drugs. Indeed, the subunit composition of GABAA receptors determines their affinity for benzodiazepine receptor ligands as well as the efficacy of these ligands. Thus, classical benzodiazepine agonists (e.g. diazepam), imidazopyridines, imidazoquinolines and pyrazolopyrimidines show no affinity for or efficacy at GABAA receptors that contain α4 or α6 subunits, whereas the benzodiazepine receptor antagonist flumazenil exhibits a pharmacological profile similar to that of a benzodiazepine receptor agonist at GABAA receptors containing either of these subunits [26], [28], [44], [50], [52].
These observations have suggested that the subunit composition of native GABAA receptors plays an important role in defining their physiological and pharmacological function. It has thus been proposed that the α1 subunit mediates the sedative–hypnotic effects of benzodiazepines [34], [41], whereas the anxiolytic effects of these drugs may be mediated by α2 but not α3 containing receptors [32] and be influenced by the presence of the γ2 subunit [8].
Characterization of the physiological, pharmacological and pathological roles of GABAA receptors thus requires an understanding of the mechanisms by which the subunit composition of GABAA receptors is regulated. The expression of specific GABAA receptor subunit genes in neurons is affected by physiological and pharmacological modulators of receptor function. Such regulation of GABAA receptor subunit gene expression by neuroactive steroids, mediated through the stimulation of GABAA receptors and not via a genomic action through the progesterone receptor, has recently been demonstrated to occur in vivo during pregnancy [6], [7], [14] or pseudopregnancy [48] and in vitro [15].
To evaluate if prolonged stimulation of GABAA receptor by benzodiazepines could have an action similar to that observed after treatment with neuroactive steroids [15], we have now investigated the effects of long-term exposure to and withdrawal of full (known to induce tolerance) and partial (known to do not induce tolerance) agonists of benzodiazepine receptors on, both the expression of GABAA receptor subunits and GABAA receptor function in cultured rat cerebellar granule cells.
Section snippets
Drugs
Diazepam and flumazenil were a gift from Hoffmann-La Roche (Basel, Switzerland); imidazenil was supplied by Fidia (Abano Terme, Italy); all other chemicals were obtained from commercial sources.
Animals
The study was carried out in accordance with the Declaration of Helsinki and/or with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the US National Institute of Health.
Cell culture
Primary cultures of cerebellar neurons enriched in granule cells were prepared from cerebella of
Effects of long-term treatment with and withdrawal of diazepam on GABAA receptor gene expression
The effects of treatment of cerebellar granule cells for 5 days with 10 μM diazepam on the abundance of mRNAs encoding α1, α4, γ2L, and γ2S subunits of the GABAA receptor were examined. Such treatment induced a significant decrease (−20%) in the amount of the α1 subunit mRNA but had no effect on the abundance of the α4 subunit mRNA (Fig. 1A). The amounts of γ2L and γ2S subunit mRNAs were reduced by 26 and 24%, respectively, by chronic diazepam treatment (Fig. 1A).
The effects of diazepam
Discussion
Long-term exposure of rat cerebellar granule cells in culture to the benzodiazepine receptor full agonist diazepam reduced the abundance of the GABAA receptor α1, γ2L, and γ2S subunit mRNAs and peptides. In contrast, the same treatment did not affect the abundance of the α4 subunit mRNA and peptide. These changes in GABAA receptor subunits expression were accompanied by a decrease in the efficacy of diazepam with regard to potentiation of the effect of GABA on the receptor-associated Cl−
Acknowledgements
This study was supported by Grant 9905045782 from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (Projects of National Relevance, Article 65DPR 382/80).
We thank Professor Werner Sieghart for providing antibodies to GABAA receptor subunits.
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2008, Brain ResearchCitation Excerpt :On the other hand, some studies suggested that different benzodiazepine targets or different signaling pathways might be involved in these phenomena (Rosenberg and Chiu, 1985; Bateson, 2002). Likewise, various changes in the GABAA receptor function (Gonsalves and Gallager, 1987; Miller et al., 1988, Roca et al., 1990, Hu and Ticku, 1994) and expression (Toki et al., 1996; Impagnatiello et al., 1996; Follesa et al., 2001) were observed after the cessation of chronic benzodiazepine treatment. Hence, the mechanisms that underlie benzodiazepine tolerance and dependence are not clear (Bateson, 2002; Wafford, 2005).