Elsevier

Neuropharmacology

Volume 42, Issue 5, April 2002, Pages 619-625
Neuropharmacology

Tolerance to the sedative effect of lorazepam correlates with a diminution in cortical release and affinity for glutamate

https://doi.org/10.1016/S0028-3908(02)00012-6Get rights and content

Abstract

Benzodiazepines are anxiolytic, anticonvulsant, sedative and hypnotic compounds usually prescribed on a long-term basis. Chronic treatment with these compounds induces tolerance, which has been extensively attributed to modifications in the GABAergic neurotransmission. However, a compensatory increase in the excitatory response, named as an oppositional response, has also been put forward as a means for explaining such tolerance. Changes in the excitatory neurotransmission have been found in withdrawn rats after a long treatment with benzodiazepines but these modifications have not been conclusively studied during tolerance. In this work we studied several parameters of the glutamatergic neurotransmission in rats made tolerant to the sedative effect of 3 mg/kg (i.p.) of lorazepam (LZ). We found a decrease in the affinity of cortical NMDA receptors for 3H-glutamate (KD: 124.4 ± 13.3 nM in tolerant rats, 71.6 ± 10.4 nM in controls, P<0.05) together with a decrease in the in vitro 60 mM K+-stimulated cortical glutamate release (59± 12% vs. 153 ± 38%, tolerant rats vs. controls, P<0.05). We conclude that tolerance to the sedative effect of LZ correlates with a decreased sensitivity for glutamate that may in turn diminish the cortical response to a chemical stimulus. Our findings constitute an evidence against the oppositional model of pharmacodynamic tolerance in this experimental condition.

Introduction

Benzodiazepines are a group of psychoactive drugs that exert a number of pharmacological effects, such as anxiolysis, sedation, hypnosis, anterograde amnesia, muscle relaxation and anticonvulsant activity. They do so by binding to an allosteric site in the GABAA receptor complex and increasing the opening probability of the associated chloride channel, thus potentiating the inhibitory effect of GABA. Tolerance to benzodiazepines’ diverse effects after a prolonged treatment is a well-documented issue. However, this diminution in the response to the drug does not occur simultaneously for every pharmacological property. For example, the anxiolytic effect of chlordiazepoxide is abolished after a single exposure to the plus-maze test, a phenomenon called “one-trial tolerance” (Pereira et al., 1999). Tolerance to the sedative effect of diazepam can be demonstrated after a four-day treatment with i.p. injections (Marin et al., 1996), while rats orally administered with flurazepam for one week become insensitive to the anticonvulsant action of the drug (Chen et al., 1999). Some studies have failed to find a diminution in the anxiolytic response to diazepam after three weeks of treatment (Stock et al., 2000, Srinivasan et al., 1995). Moreover, experiments performed in our laboratory show that tolerance does not develop to the amnesic effect of lorazepam even after 21 days of treatment (unpublished data).

It has been established that tolerance to benzodiazepines is associated with a progressive diminution in the activity of the drug at the GABAA receptor complex. This ionotropic receptor is a pentamer that results from the combination of five different families of subunits α1–6, β1–4, γ1–4, ρ1–2, δ1 and ε1). Numerous studies have demonstrated changes in the cortical and hippocampal expression of these subunits in tolerant animals (Pesold et al., 1997, Tietz et al., 1999, Chen et al., 1999). As different subunit combinations confer to the GABAA receptor different physiological and pharmacological properties, it is suggested that tolerance is the result of the expression of benzodiazepine-insensitive arrangements. Moreover, several authors found a diminution in the binding of benzodiazepines to their allosteric site in cerebral cortex and hippocampus of chronically benzodiazepine treated animals (Miller et al., 1988, Rosenberg and Chiu, 1981, Tietz et al., 1986), while others demonstrated a functional uncoupling of the benzodiazepines and GABA interaction (Hu and Ticku, 1994, Wong et al., 1994, Primus et al., 1996), both these findings probably related to changes in GABAA receptor subunit composition. Taken together, these experimental evidences are in line with the decremental model proposed by Littleton and Little (1994) to explain pharmacodynamic tolerance: there is an adaptive process caused by the continuous exposure to a drug that leads to a diminution in the drug-induced signal.

However, a reduction in the effect of benzodiazepines at the GABAA receptor complex does not seem to be the only mechanism involved in the expression of tolerance (Pratt et al., 1998, Stephens, 1995, Hutchinson et al., 1996). Regarding this issue, a second model to explain pharmacodynamic tolerance postulates that the initial action of a given drug is progressively attenuated by the recruitment of compensatory mechanisms, resulting in a loss of effect. If this oppositional model is considered for the tolerance to benzodiazepines, an increase in excitatory glutamatergic neurotransmission could be the compensatory mechanism involved. This could also explain the withdrawal signs of excitatory nature, such as increased anxiety or a diminution in the convulsive threshold when treatment with benzodiazepines ends (Elliot and White, 2000). The contribution of the oppositional theory to the tolerance to another CNS depressant, ethanol, has been experimentally demonstrated. Ethanol exerts its acute action by potentiating GABAA receptor currents and inhibiting NMDA receptor currents, together with its effect on membrane lipids and on protein-lipid interactions. After a chronic exposure to the drug, a reduction in GABA-mediated inhibition and an enhancement in NMDA-mediated stimulation have been demonstrated. Both this facts could account for the hyperexcitability seen in ethanol withdrawal (Chandler et al., 1998). Regarding benzodiazepines, there is no conclusive evidence of a contribution of the glutamatergic neurotransmission to the expression of tolerance. Tsuda et al., 1998a, Tsuda et al., 1998b have demonstrated that, during the withdrawal period after a 6-day treatment with high doses of diazepam, there is an upregulation of the NMDA receptor. However, it has not been established whether the expression of these receptors is altered in the tolerance to benzodiazepines, as it is in the tolerance and withdrawal to other drugs of abuse such as ethanol and opiates (Morrow et al., 1994, Belozertseva and Bespalov, 1998). So, the aim of the present work was to evaluate parameters of the cortical glutamatergic neurotransmission in rats tolerant to the sedative effect of lorazepam (LZ), a full allosteric modulator of the benzodiazepine site.

Section snippets

Animals

Male adult Wistar rats, weighing 180 to 200 g at the beginning of treatment, were housed in groups of 4 to 5 in a room with constant temperature and a 12-hour light-dark cycle. They received food and water ad libitum. The animals were treated according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press, Washington D.C, 1996). All efforts were made to reduce the number

Identification of the sedative dose and treatment duration

The dose-response curve to the sedative effect of LZ is shown in Fig. 1, panel A. Doses over 0.1 mg/kg significantly reduced the number of lines crossed in the open field test, indicating the sedative effect of the drug (One-way ANOVA: P<0.0001, F=13.37). A dose of 3 mg/kg was chosen for the subsequent experiments, being the lowest dose that exerted the maximum effect. After 21 days of treatment, animals developed tolerance to the sedative action of the drug. This was evidenced by a lack of

Discussion

In our work we explored several parameters of the cortical glutamatergic neurotransmission, such as endogenous content of excitatory amino acids, 3H-glutamate binding to NMDA receptors and in vitro release of endogenous glutamate, in rats tolerant to a sedative dose of LZ. The development of tolerance to benzodiazepines does not occur at the same time for all of their pharmacological properties, as we mentioned above. This is not only dependent on the length of the treatment, but also on the

Acknowledgments

We very much thank Dr. Analía Reinés for her worthy contribution in the revision of this manuscript. This work was supported by a grant of Agencia Nacional de Promoción Científica y Tecnológica (97-00315), a grant of CONICET (PIP 4453/96) and grant “Ramon Carrillo-Arturo Oñativia” from Ministerio de Salud Pública, Argentina.

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