IMIPRAMINE BINDING

Imipramine Binding

Summary

The binding characteristics of [3H]-imipramine in slide mounted tissue sections of rat forebrain have been studied to ascertain the optimal binding conditions for labeling the sites prior to autoradiographic localization. The conditions for the experiments and the kinetics of the imipramine binding correspond reasonably well with those used in membrane preparations to initially define the imipramine binding site. Subsequent labeling of sections, using these parameters, allowed the autoradiographic localization of high concentrations of imipramine binding sites in such areas as the cerebral cortex, striatum, and several limbic and visual system structures.

Imipramine Binding

Introduction

Chronic inhibition of reuptake mechanisms by long-term exposure to tricyclic antidepressants may ultimately decrease the density of catecholamine and serotonin receptors with a time course which closely follows the efficacy of antidepressants in relieving the symptoms of depression (1). Several tricyclic antidepressants interact directly with such neurotransmitter receptors as alpha adrenergic (2), muscarinic cholinergic (3), histamine (4,5,6,7), and serotonin (7). These interactions may explain some of the side effects seen with tricyclic antidepressant treatment. Specific high affinity binding sites for imipramine, the prototype of the tricyclic antidepressants, have been defined in the brain (8,9,10) and introduce the possibility that these substances may inhibit reuptake by acting at specific sites of their own. Imipramine binding sites have been identified in membrane preparations from several regions of rat (11) and human (12,13) brain. The present study was undertaken to define the labeling parameters necessary to obtain specific imipramine binding to slide mounted tissue sections. This was accomplished in order to determine the distribution of these sites using quantitative autoradiographic techniques (14,15).

Methods

Male, Sprague-Dawley rats (150-250 grams) were intracardially perfused 0024-3205/83/202355-07503.00/0 Copyright (c) 1983 Pergamon Press Ltd. 2356 Localization of Imipramine Binding Sites Vol. 32, No. 20, 1983 with an ice cold 0.1% formalin solution in isotonic phosphate buffered saline.

The brains were rapidly dissected and mounted onto chucks coated with O.C.T.

Compound (Lab-Tek Products; Naperville, IL) by freezing on dry ice. Sections (i0 microns in thickness) of forebrain containing striatum were cut in a cryostat (-18=C), thaw mounted onto cold subbed slides, and stored overnight in a self-defrosting freezer (-20°C). The slide mounted tissue sections were then labeled with [3H]-imipramine (New England Nuclear; Boston, MA) in 50mM Tris HCI, pH7.4, under various conditions.

The dissociation, association, and saturation kinetics of [3H]-imipramine binding, as well as the pharmacological specificity, were studied by exposing the sections to different incubation or rinse conditions and determining the radioactivity bound. The amount of radioactivity remaining in the sections was measured by wiping the tissue from the slide with a Whatman GF/B glass microfiber filter disc and counting it in a liquid scintillation counter. Total [3H]-imipramine binding was compared to nonspecific binding as determined by the amount of [3H]-imipramine bound in the presence of a 100 micromolar concentration of unlabeled imipramine in the incubation medium.

The dry labeled sections were stored overnight in an airtight box containing desiccant. After allowing the slides to return to room temperature, the boxes were opened and the slides were taped (double stick ...