Reduced benzodiazepine tolerance, but increased flumazenil-precipitated withdrawal in AMPA-receptor GluR-A subunit-deficient mice

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Abstract

Pharmacotherapy with benzodiazepines is compromised by rapid sedative tolerance and diverse withdrawal symptoms. To assess the role of AMPA-type glutamate receptor GluR-A subunits in neuroadaptation to subchronic benzodiazepine treatment, GluR-A subunit-deficient mice were rendered tolerant by a high-dose seven-day flurazepam treatment (40 mg/kg, s.c., twice a day for 4 days, 60 mg/kg twice a day for 3 days). The acute effects to flurazepam were not changed in the GluR−/− mice compared with their littermate control mice. GluR-A−/− mice developed less tolerance than their controls as demonstrated in behavioral tests for muscle relaxation and sensory functions. Actually, the knockout mice exhibited slower recovery than their littermates from impaired gait and pelvic position after an acute 40 mg/kg dose of flurazepam. The apparent elimination of flurazepam was similarly increased in the knockout and control mice as assessed by blood and brain concentrations 2 h after acute and chronic treatments, but the active metabolite desalkylflurazepam cumulated similarly in both mouse lines. Withdrawal symptoms, precipitated by flumazenil (20 mg/kg, s.c.) 48 h after discontinuation of the flurazepam treatment, were enhanced in the GluR-A−/− mice. The results stress the importance of the AMPA-receptor system in neuroadaptation to acute and chronic effects of benzodiazepines.

Introduction

Benzodiazepine (BZ) treatment is widely used for quick and efficient anxiolytic and sedative responses. However, when the use is extended to longer periods, adverse effects emerge. These include tolerance to the initial drug effect, dependence as revealed by withdrawal symptoms after treatment discontinuation, and abuse problems (O'Brien, 2005, Rosenbaum, 2005).

γ-Aminobutyric acid type A (GABAA) receptors mediate the main effects of BZs (Korpi et al., 2002). BZs bind to the GABAA receptor and modulate their properties allosterically, which suggests that receptor adaptation could provide possible mechanisms for the development of tolerance and withdrawal symptoms. Many properties of the GABAA receptor complex have been observed to change during long-term BZ treatment. These include changes in the affinity and number of binding sites of BZs, altered receptor subunit composition, uncoupling of the BZ binding-site from the receptor channel function, and changes in the affinity and number of binding sites of GABA [for review, see Bateson, 2002, Hutchinson et al., 1996, Wafford, 2005]. Furthermore, GABAA receptor subunit-specificity has been suggested to have a role in the development of tolerance to BZs by downregulation of α5 subunit-containing receptors in the hippocampal dentate gyrus (Li et al., 2000, van Rijnsoever et al., 2004). Most of these studies have employed low to moderate doses of BZs, to which tolerance is quickly developed.

Other neurotransmitter systems have also been implicated as mechanisms underlying tolerance and dependence to BZs. Especially interesting is the main fast excitatory neurotransmission system in the brain using glutamate as the transmitter. It has been suggested that the BZ-induced increase in inhibitory transmission is counteracted by the glutamatergic system through modified glutamate metabolism and/or modified receptor mechanisms (Allison and Pratt, 2003, Izzo et al., 2001). When mice are chronically co-treated with BZs and an antagonist of N-methyl-d-aspartate (NMDA) receptors, tolerance develops only partially (Steppuhn and Turski, 1993). Development of tolerance to BZs increases mRNA of NMDA receptor NR1 and NR2B subunits, which can be reversed by NMDA receptor antagonist MK-801 (Almirón et al., 2004). Furthermore, withdrawal symptoms are reduced by the administration of NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists in a time-dependent manner: the former is effective when given at the time of withdrawal and the latter when given just prior to the appearance of the symptoms (Steppuhn and Turski, 1993). An increase in the number and activity of AMPA receptors in BZ tolerance is evidenced by increased binding of AMPA-receptor ligand [3H]Ro 48-8587 and by increased electrophysiological responses (Allison et al., 2005, Van Sickle and Tietz, 2002).

Importantly, the GluR-A (GluR-1) subunit of AMPA-type glutamate receptors has been implicated in neuroadaptational mechanisms, such as long-term potentiation (LTP), learning and memory and drug addiction (Bannerman et al., 2006, Carlezon and Nestler, 2002). During hippocampal synaptic plasticity, GluR-A subunit-containing receptors are targeted to the cell membrane, a pivotal event in LTP (Hayashi et al., 2000, Shi et al., 2001). Pharmacological study of the role of GluR-A subunit-containing AMPA receptors in the development of BZ tolerance is hampered by the lack of a selective ligand. Fortunately, a knockout mouse line has been generated in which the hippocampal LTP is abolished but the synaptic transmission remains largely unmodified (GluR-A−/− mice; Zamanillo et al., 1999). This defect in LTP is rescued by genetic re-expression of the GluR-A subunit (Mack et al., 2001). In the GluR-A−/− mice, chronic morphine administration leads to reduced tolerance and withdrawal symptoms, indicating an interaction between sedative opioid and excitatory glutamate neurotransmitter systems and highlighting the role of AMPA receptor mechanisms in the effects of addictive drugs (Vekovischeva et al., 2001).

Here we studied the effects of BZ flurazepam (FZ) at high doses in acute and chronic settings of tolerance and dependence in GluR-A−/− mice (Zamanillo et al., 1999). We used measurements in which the phenomenon of “learning while intoxicated” should play only a minor role in order to find out the significance of GluR-A subunit-containing AMPA receptors in the adaptation of the brain to the effects of high BZ levels.

Section snippets

Subjects

GluR-A−/− mice were produced by deletion of the Gria1 gene as described previously (Zamanillo et al., 1999). The mice were backcrossed to C57BL/6NHsd (Harlan BV., Horst, Netherlands) line seven times. In the experiments, male and female GluR-A−/− mice and their wild-type littermate controls were from heterozygous breeding, and all experiments were carried out at the age of 2.5–4 months. The animals were genotyped using tail-tip biopsy samples at the age of 30 days. The tails were digested

Acute effects of flurazepam

The GluR-A−/− mice were compared to their wild-type GluR-A+/+ littermates in regard to sensitivity to FZ (40 mg/kg) by assessing various behavioral parameters according to the SHIRPA protocol 30 min after the injection of the drug or saline. None of the measured parameters showed a difference between the genotypes (p > 0.05), except for the body temperature that was slightly lower in the knockouts (Fig. 1). The drug effect was clearly detected in all parameters (F1,69 > 9.95, p < 0.01). There were no

Discussion

Behavioral assessment of the effects of acute and subchronic treatment with FZ showed that the GluR-A subunit-deficient mouse line has a reduced capacity to develop tolerance to this drug. This result indicates that glutamatergic system has an important role in the neuroadaptation process opposing the increased neuronal inhibitory tone caused by BZs. However, precipitated withdrawal symptoms after the treatment were stronger in the GluR-A−/− mice, pointing to further differences in the

Acknowledgements

The present work was supported by the Finnish Foundation for Alcohol Studies (TA, ERK) and the Academy of Finland (ERK). The authors would like to thank Rolf Sprengel for providing the knockout mouse line, Jouko Laitila for excellent technical assistance, and Eeva Harju for checking the language of the manuscript.

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