Injection of dye into neurones in rat and human post-mortem brain, in combination with acetylcholinesterase histochemistry: permanent preparations

Abstract

We describe a protocol for the intracellular injection of dye into neurones in thick sections of fixed, post-mortem rat and human brain tissue. To render the sections with the intracellularly injected neurones permanent, they are sectioned again, and the resulting subsections are either immunocytochemically treated or stained histochemically for acetylcholinesterase (AChE) activity. The resultant preparations can be stored at room temperature for prolonged periods. Background staining produced by accumulation of erythrocytes in blood vessels is greatly reduced or virtually eliminated by exposure of the sections to ultraviolet radiation prior to the intracellular injection. The pattern of AChE staining is not affected by this procedure. The ability to stain sections according to a histochemical AChE procedure after the intracellular injection of dyes into striatal neurons opens the possibility to study the relationship of neuronal dendritic trees with the striosome/matrix compartmental boundaries in post-mortem (human) brain tissue of Huntington's disease patients.

Introduction

The geometry and the details of a dendritic tree reflect important physiological functions of the parent neurone and through this, of the neuronal circuitry in which this neurone participates. For example, the extent of branching of a dendritic tree and the locations of the endings of different types of afferent fibres on the spines and shafts of the dendritic branches determines the way in which summation of electrical impulses in the neurone as a whole occurs (Rall et al., 1967; Rall, 1974). Thus, studies of the morphology of dendritic trees of neurones may contribute to a better understanding of their physiological function. In human pathology, studies have been conducted investigating dendritic geometry in post-mortem brains in search for a better understanding of the onset and course of neurological disorders (e.g. Graveland et al., 1985; Ferrante et al., 1991). In our particular application, we are ultimately interested in visualising the dendritic trees of striatal neurones in post-mortem human brains of patients suffering from Huntington's disease. In the same material, we want to delineate simultaneously striosomal compartments in the striatum via acetylcholinesterase (AChE) histochemistry, because this marker remains detectable throughout the course of Huntington's disease whereas other markers strongly diminish (Ferrante et al., 1986; Ferrante et al., 1991).

Several methods are available to study dendritic geometries. The classical Golgi silver-impregnation procedure suffers from the disadvantage that neurones become stained in an indiscriminate and virtually uncontrollable way. By contrast, intracellular injection of a fluorescent dye, Lucifer Yellow (LY; Stewart, 1981) into neurones in lightly fixed brain slices followed by fluorescence microscopy, is much better controllable (Rho and Sidman, 1986; Schwerdtfeger and Buhl, 1986; Buhl and Schlote, 1987; Wouterlood et al., 1990; Buhl, 1993; Belichenko and Dahlstrom, 1995), and has been applied successfully in human brain slices by a number of researchers (Buhl and Schlote, 1987; Belichenko, 1992; Einstein et al., 1993). A second advantage of intracellular injection is, that it is relatively easy to combine with various immunocytochemical techniques (Wouterlood et al., 1990). Since fluorescence tends to fade quickly during prolonged microscopical inspection, the fluorescent dye must be stabilised following the intracellular injection. In human post-mortem material, this is seldomly attempted, since this typically non-perfused and immersion-fixed tissue contains peroxidase-laden erythrocytes in blood vessels and capillaries that produce a dense background staining which interferes with AChE staining.

In the present report, we describe a procedure by which we expose thick sections of post-mortem rat and human brain to ultraviolet (UV) irradiation. Neurones in these sections are subsequently intracellularly injected with LY. The sections are then cut into thinner subsections which are subjected to either AChE staining or to stabilisation of the intracellularly injected dye to delineate the boundaries of the striosomal compartments containing the injected neurones.