Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines

Abstract

Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

Figure 1

Evolution of diazepam anticonvulsant tolerance in diazepam-withdrawn rats. Rats receiving 14 days treatment with vehicle (□) or diazepam (■) were withdrawn for different times before receiving orally a vehicle or a diazepam challenge, 30 min before bicuculline-induced seizure test (25). Values are mean ± SE of 5–6 rats. *, P < 0.0.01 when compared with vehicle challenge groups; #, P < 0.01 when long-term diazepam-treated (■) rats were compared with the corresponding long-term vehicle-treated (□) rats. (ANOVA followed by Duncan‘s multiple comparison test)

Figure 2

Elevated-plus maze test following long-term vehicle-treatment (□) or diazepam-treatment (■) withdrawal. Values are mean ± SEM of 5–6 rats. *, P < 0.01 when compared with the respective vehicle-treated rats. Nonparametric analysis by using the Kruskal-Wallis test followed by the Mann–Whitney U test.

Figure 3

GAD₆₅ and GAD₆₇ mRNAs expression in frontal cortex of rats following long-term vehicle- or diazepam-treatment withdrawal. For the diazepam-withdrawn groups the values are mean ± SE of 4–5 animals. For the vehicle-withdrawn group the values were virtually identical at 12, 48, and 96 h after treatment discontinuation and represent mean ± SE of 12 animals. *, P < 0.05 compared with the corresponding vehicle-treated rats (ANOVA followed by Duncan’s multiple comparison test).

Figure 4

AMPA GluR receptor subunit mRNAs in rat brain, 96 h after long-term diazepam-treatment (■) or vehicle-treatment (□) withdrawal. Data are mean ± SE of 5–6 animals. *, P < 0.05 and **, P < 0.01, compared with respective vehicle-treated group; #, P < 0.01 when GluR2,3,4 mRNA subunits are compared with GluR 1 mRNA subunit. (ANOVA followed by Duncan's multiple comparison test.) ND, not determined

Figure 5

GluR1, GluR2, and NR1 mRNAs expression in frontal cortex or hippocampus following long-term diazepam-treatment withdrawal (Dz.W.) or vehicle-treatment withdrawal (V.W.). For the Dz.W. groups, values are mean ± SE of 4–5 animals. For the V.W. groups, values were identical at 12, 48, and 96 h after treatment discontinuation; therefore, the data of the three groups were combined and represented as mean ± SE of 12 animals. **, P < 0.01 compared with V.W. rats (ANOVA followed by Duncan's multiple comparison test).

Figure 6

GluR1 protein expression in hippocampus and frontal cortex following long-term diazepam (Dz)- or vehicle (V)-treatment withdrawal. (A Upper) Representative western blot of crude synaptic membrane extracts prepared from vehicle- or diazepam-withdrawn rats. 106-kDa and 42-kDa bands represent GluR1 and β-actin immunoreactivities, respectively. Lanes 1 and 4 were loaded with 10 μg of proteins, lanes 2 and 5 with 20 μg of protein, and lanes 3 and 6 with 40 μg of protein. (A Lower) Relative levels of GluR1 receptor subunit expressed as the OD ratio with β-actin. (B) Relative levels of GluR1 receptor subunit. Values are the mean ± SE of at least three separate animals. *, P < 0.05 when Dz was compared with the respective V group (ANOVA followed by Duncan's multiple comparison test).

Figure 7

Photomicrographs of 20-μm sections of layer V of the frontoparietal motor cortex of 96-h vehicle-withdrawn (A) or diazepam-withdrawn (B) rats. Sections were immunogold labeled for the GluR1 receptor subunit, and counterstained with toluidine blue Nissl stain. Note that in the diazepam-treated rat there is an increase in the expression of GluR1 subunits (black particles) on pyramidal cell bodies and apical dendrites, but not in the surrounding neuropile.

Figure 8

Photomicrographs of 20-μm sections to the anterior hippocampus of 96 h vehicle-withdrawn (A and C) or diazepam-withdrawn (B and D) rats. Staining as in Fig. 7. Note the increased expression of immunogold-labeled GluR1 receptor subunits (black particles) in all layers of the CA1 region of the hippocampus in the diazepam-withdrawn rat. C and D are higher magnifications of the boxed areas in A and B, respectively. [Scale bar, 500 μm (Upper) and 20 μm (Lower).]