Elsevier

Brain Research

Volume 964, Issue 1, 21 February 2003, Pages 91-99
Brain Research

Research report
Compartmentation of alpha 1 and alpha 2 GABAA receptor subunits within rat extended amygdala: implications for benzodiazepine action

https://doi.org/10.1016/S0006-8993(02)04082-9Get rights and content

Abstract

The extended amygdala, a morphological and functional entity within the basal forebrain, is a neuronal substrate for emotional states like fear and anxiety. Anxiety disorders are commonly treated by benzodiazepines that mediate their action via GABAA receptors. The binding properties and action of benzodiazepines depend on the α-subunit profile of the hetero-pentameric receptors: whereas the α1 subunit is associated with benzodiazepine type I pharmacology and reportedly mediates sedative as well as amnesic actions of benzodiazepines, the α2 subunit confers benzodiazepine type II pharmacology and mediates the anxiolytic actions of benzodiazepines. We determined the localization of α1 and α2 subunits within the extended amygdala, identified by secretoneurin immunostaining, to define the morphological substrates for the diverse benzodiazepine actions. A moderate expression of the α1 subunit could be detected in compartments of the medial subdivision and a strong expression of the α2 subunit throughout the central subdivision. It is concluded that the α1 and α2 subunits are differentially expressed within the extended amygdala, indicating that this structure is compartmentalized with respect to function and benzodiazepine action.

Introduction

The continuum between the bed nucleus of the stria terminalis (BST) and the centro-medial amygdala, known as the extended amygdala [2], is the main neuronal substrate for emotions like fear and anxiety or general negative affective states like dysphoria and irritability [6], [7], [12]. Particular sub-compartments of this system are supposed to be affected in specific emotional states. Whilst highly explicit cue information such as tones and touch activates the central nucleus of the amygdala, less explicit information like exposure to a threatening environment is thought to stimulate the BST. The central nucleus of the amygdala and the BST activate hypothalamic and brain stem target areas involved in specific signs of fear or anxiety [5].

Patients suffering from anxiety disorders are commonly treated by the administration of benzodiazepines, although a significant proportion of these patients display reduced sensitivity to these drugs [30], [35]. Benzodiazepines mediate their sedative, amnesic and anxiolytic actions via binding to GABAA receptors [17], [22]. The subunit composition of GABAA receptors, particularly its α subunit content, determines the sensitivity of benzodiazepine binding [3], [28], [40]. Six α subunit isoforms (α1–α6) have been described. Receptors that include an α1 subunit display benzodiazepine type I pharmacology and bind diazepam and other benzodiazepines (e.g. zolpidem) with high affinity. α2, α3 and α5 subunits are associated with benzodiazepine type II pharmacology and α4 or α6 subunits confer benzodiazepine insensitivity [21], [29]. In addition to binding properties, α subunits determine the action of these drugs as indicated in an animal model for anxiety (5-HT1A receptor knock-out mouse) [34] and in models where GABAA receptors have been rendered insensitive to diazepam by knock-in point mutations of specific α subunits [20], [31]. Whereas the sedative and amnesic actions of diazepam are reportedly mediated by the α1 subunit, anxiolytic actions are mediated by the α2 subunit.

The extended amygdala is the main site of action of benzodiazepines in various emotional states. We determined the specific expression pattern of α1 and α2 subunits within this neuronal system. Our overall aim was to resolve whether the extended amygdala is compartmentalized with respect to the localization of GABAA receptor subunits, which is of functional importance for psychiatric disorders and is relevant for the mode of action of benzodiazepines.

Section snippets

Tissue preparation for histological analysis

Eight male Sprague–Dawley rats (purchased from ‘Forschungsinstitut für Versuchstierzucht’, Himberg, Austria) were used for this study. They were deeply anaesthetized with thiopental (150 mg/kg, i.p.). Brains were perfused via the ascending aorta with 50 ml of phosphate-buffered saline (PBS; 50 mM, pH 7.4), followed by 250 ml 4% paraformaldehyde and 0.1% glutaraldehyde in 50 mM PBS, pH 7.4, postfixed for 2 h in the same fixative, rinsed in PBS and transferred to 20% sucrose in PBS for at least

Results

No specific immunostaining was observed in control sections applying respective pre-immune sera or antisera preadsorbed with the immunogenic peptides.

In general, α1-immunoreactive profiles mainly appeared in the form of particles of varying size (Fig. 1A and B) and varicose fibers (Fig. 1C). In addition, α1 immunoreactivity displayed a pericellular staining pattern and band-like terminal pattern in the ventral pallidum and sublenticular substantia innominata due to peridendritic and periaxonal

Localization of α1 and α2 subunits in the extended amygdala

The α1 and α2 subunits of the GABAA receptor are widely distributed throughout the rat forebrain [10], [26]. We found a particularly dense expression of these subunits in compartments of the extended amygdala. A close anatomical relationship between the centro-medial amygdaloid complex and the bed nucleus of the stria terminalis was described as early as 1923 by Johnston [13]. These compartments, together with the intervening cell corridors, are held to comprise a morphological and functional

Conclusion

The α1 and α2 subunits are differentially expressed in the extended amygdala with a predominant expression of the α2 subunit within this system. The α2 subunit reportedly mediates anxiolytic actions of benzodiazepines. It is concluded that the anxiolytic actions of benzodiazepines might be mediated by the α2 subunit through the extended amygdala.

Acknowledgements

We thank Dr. Werner Sieghart for providing the antisera against GABAA receptor subunits, Dr. Rainer Fischer-Colbrie for providing the antiserum against secretoneurin and Dr. Ole Petter Ottersen for reading the manuscript. This work was supported by the Austrian National Bank (to J.M.; 9295), the Austrian Research Foundation (Erwin-Schroedinger Fellowship to W.A.K; J2121) and by NIH Grants (HL/NS-60097) and (HL/NS-60696) to G.F.A.

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