Alan N. Bateson

Chronic Treatment with Diazepam or Abecarnil Differentially Affects the Expression of GABAA Receptor Subunit mRNAs in the Rat Cortex

Diazepam and abecarnil produce their overt effects by interaction with the GABAA receptor. Chronic treatment with abecarnil, however, does not induce diazepam-like tolerance. This study investigates the effects of chronic diazepam and abecarnil treatment on expression of GABAA receptor alpha 1-6 beta 1-3 and gamma 1-3 subunit isoform mRNAs in rat cortex. Male Sprague-Dawley rats were injected subcutaneously once daily for 7 or 14 days with 15 mg/kg diazepam or 6 mg/kg abecarnil in sesame-oil vehicle, and steady-state levels of GABAA receptor subunit mRNAs were quantified by solution hybridization. The levels of alpha 4- and alpha-, beta 1- and gamma 3-subunit mRNAs were significantly increased after 7 days of diazepam treatment, and this effect was maintained at 14 days. A significant increase in alpha 3-subunit mRNA was apparent only after 14 days of diazepam treatment and a significant decrease in beta 2-subunit mRNA was seen only after 14 days of abecarnil treatment. Gamma 2-Subunit mRNA was significantly decreased after 14 days of either diazepam or abecarnil exposure. A degree of association between a particular drug treatment and changes in the levels of mRNAs arising from a given gene cluster was noted. Our results are consistent with a model of diazepam dependence based on GABAA receptor subunit isoform switching.

Molecular Neurobiology of the GabaA Receptor

Publisher Summary This chapter discusses the molecular neurobiology of the GABAA receptor. The GABAA receptor is responsible for the majority of neuronal inhibition in the vertebrate CNS. The ubiquitous distribution of the GABAA receptor in the mammalian CNS is revealed by the use of [3H]GABA radioligand binding techniques, whereas autoradiographic studies have demonstrated their distinct topographical localization. There is evidence that the receptor is not only expressed in regions of the cell that receive GABAergic input, but also, for example, on Golgi cell somata where no synaptic contacts are found. With the two monoclonal antibodies used in this study, no immunoreactivity is detected on axons, nerve terminals, or glial cells. The distribution of the GABAA receptor at both synaptic and nonsynaptic sites is quite distinct from that of the closely associated glycine receptor, which appears to be mainly associated with synapses. Initial electrophysiological studies of the GABAA receptor demonstrated that its activation by GABA resulted in an increased chloride conductance of the supporting neuronal membrane.

The effect of treatment regimen on the development of tolerance to the sedative and anxiolytic effects of diazepam

Abstract Rationale: Chronic treatment with benzodiazepines results in tolerance to their sedative and anxiolytic effects and there is considerable evidence that different mechanisms underlie the development of tolerance to different behavioural effects. Objective: The purpose of the present experiment was to compare the behavioural effects of chronic treatment with diazepam (15 mg/kg per day) given as daily subcutaneous injections or by osmotic minipump. Both regimens resulted in continual receptor occupancy, but the daily injections also provided a period of higher brain concentrations. Methods: Rats were tested in the holeboard, which provides measures of exploration and locomotor activity, and in the elevated plus-maze and social interaction tests of anxiety. For those in the subcutaneous injection group the tests were 2 h after injection, when brain concentrations were highest. Results: Despite a higher brain concentration in the injected group, both groups showed tolerance to diazepam’s sedative effects, after 7 days of treatment. In contrast, in the elevated plus-maze, there was tolerance to the anxiolytic effects in the pump group after 14 days, but a persisting anxiolytic effect in the injected group at 14 and 28 days. Whilst higher brain concentrations could explain this result in the plus-maze, they cannot account for the pattern observed in the social interaction test, where the injection group showed a significant anxiogenic effect at 28 days. Conclusions: Whereas the mechanism underlying tolerance to the sedative effects of diazepam was insensitive to the different treatment regimens, the results suggest that different adaptive mechanisms were triggered in the two tests of anxiety with a differential sensitivity to the treatment regimen. The adaptive mechanism predominating in the social interaction test was favoured by the injection regimen which produced intermittent peak concentrations. This mechanism seems to be an oppositional one, leading to an anxiogenic response, which was manifest despite high brain concentrations of diazepam at the time of testing.

Structural Requirements for Ligand Interactions at the Benzodiazepine Recognition Site of the GABAA Receptor

Abstract: His101 of the GABAA receptor α1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [3H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant α subunits were coexpressed with the rat β2 and γ2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [3H]Ro 15‐4513 bound to all mutant receptors with equal or greater affinity than to the wild‐type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15‐1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15‐1788 and Ro 15‐4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.

Rapid increase in inducible nitric oxide synthase gene expression in the heart during endotoxemia

Inducible, Ca(2+)-independent nitric oxide (NO) synthase activity in the heart is elevated during endotoxemia and the resulting excess release of NO depresses cardiac contractile function. We show here that this is due to an extremely rapid induction of inducible NO synthase gene expression. Following injection of endotoxin (bacterial lipopolysaccharide) in rats we detected increased inducible NO synthase mRNA levels in the left ventricular wall within 30 min which then peaked at 3 h. This was followed by an increase in myocardial inducible NO synthase enzyme activity and plasma levels of NO metabolites, nitrate and nitrite, which peaked at 6 and 12 h, respectively. The extremely rapid induction of inducible NO synthase may serve to protect the heart against microbial infection and concomitantly alter myocardial mechanical function.

Chronic diazepam exposure decreases transcription of the rat GABAA receptor γ2-subunit gene

Abstract The rate of transcription of the GABAA receptor γ2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABAA receptor γ2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABAA receptor gene.

GABAA receptor gene expression in rat cortex: Differential effects of two chronic diazepam treatment regimes

Diazepam is widely prescribed as an anxiolytic but its therapeutic application is limited because with daily use tolerance develops to certain aspects of its pharmacological profile. We compared the effects of two dosing paradigms on GABAA receptor gene expression and benzodiazepine binding characteristics. Equivalent daily doses of 15 mg/kg/day diazepam were delivered either via constant infusion or daily subcutaneous injection for 14 days. The two distinct treatment regimes produced significantly different changes in GABAA receptor α4‐, β2‐, β3‐ and γ1‐subunit mRNA steady‐state levels. Similar changes in the GABA enhancement of flunitrazepam binding and the BZ3/BZ2 subtype ratio determined ex vivo were produced, however, significant differences were found in [3H]‐Ro 15‐4513 binding between cortical tissue from diazepam injected animals compared with diazepam infused animals. Our data suggest that it is the diurnal fluctuations in receptor occupancy that are responsible for the different effects produced by these two dosing regimes. J. Neurosci. Res. 64:617–625, 2001.

Chronic zolpidem treatment alters GABAA receptor mRNA levels in the rat cortex

The effect of chronic zolpidem treatment on the steady-state levels of gamma-aminobutyric acidA alpha1-6, beta1-3 and gamma1-3 subunit mRNAs in rat cortex has been investigated. Male Sprague-Dawley rats were injected once daily, for 7 or 14 days, with 15 mg/kg of zolpidem in sesame oil vehicle. The levels of the alpha4 and beta1 subunit mRNAs were significantly increased after 7 days of treatment and the level of alpha1 subunit mRNA was significantly decreased after 14 days of treatment, as determined by solution hybridization. These results are compared to the previously determined effects of an equivalent schedule of treatment with diazepam.

Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam.

Abstract: The GABA and benzodiazepine binding sites on GABAA receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug‐naive rats was potentiated ∼1.6‐fold by 100 γM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA poten‐tiation were observed. Diazepam‐induced changes in GABAA receptor δ2‐subunit gene transcription and α1‐, β2‐, and δ2‐subunit steady‐state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Chronic administration of antipanic drugs alters rat brainstem GABAA receptor subunit mRNA levels.

Mental illness, such as panic disorder and depression, display comorbidity as well as common therapeutic treatments. These features point toward a common etiology and/or therapeutic pathway. There is evidence to suggest that some antipanic agents may mediate their effects by altering gamma-aminobutyric acid (GABA) levels or by modulating the activity of the GABAA receptor. Chronic stimulation of GABAA receptors by agonists or modulators results in changes in the pharmacological properties of the receptor concomitant with alterations in the expression of specific GABAA receptor subunits. Therefore, we investigated the hypothesis that long-term exposure to three antidepressant/antipanic drugs (imipramine, phenelzine and alprazolam) would produce changes in the steady-state levels of those subunit mRNAs that are believed to encode the major GABAA receptor subtype. Further, these changes in gene expression would be different to those produced by the non-antipanic anxiolytic (buspirone). We report here that, following a 21 day treatment, imipramine, phenelzine, alprazolam and buspirone differentially altered rat brainstem levels of GABAA receptor alpha 1-, beta 2- and gamma 2-subunit RNAs. These results demonstrate novel actions of antidepressant/antipanic drugs on GABAergic neurotransmission.