Injection of dye into neurones in rat and human post-mortem brain, in combination with acetylcholinesterase histochemistry: permanent preparations
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.
Section snippets
Collection of tissue
Ten female Wistar rats (Harlan-Wistar, body weight 180–200 g) were sacrificed by decapitation. The heads were stored for 6 h at 4°C, after which the brains were carefully dissected from the skull and cut with a sharp razor blade in the frontal plane into slabs of approximately 5 mm thick. Slabs containing the caudate-putamen were immediately fixed by immersion in fixative (see below).
Of two normally aged human brains (brain 1, male, age 71 years, post-mortem delay 7 h, obtained from the
Results
The present procedure resulted in stable preparations of rat and human brain tissue in which the fluorescent dye, LY, injected intracellularly into striatal neurons, labels somata and dendritic trees. The simultaneous AChE staining of selected subsections enabled the determination of the boundaries of the striosomal compartments in which the injected neurones are located. To illustrate this, Fig. 2A and 2B represent photographs of two 40 μm thick subsections, taken with one subsection in
Discussion
In this paper we describe a method for stabilising preparations of intracellularly injected neurones in immersion fixed tissue in combination with AChE histochemistry. Thus, it is possible to obtain in human brain tissue, (a) information about the dendritic geometry of neurones, and simultaneously, (b) information about the location of the injected cells in striatal compartments as visualised by the histochemical staining for AChE. Protocols for the preservation of LY in preparations of
Acknowledgements
We would like to thank Dr Thomas Ruigrok (Erasmus University, Rotterdam, The Netherlands) for donating the antibody against Lucifer Yellow. Annaatje Pattiselanno is thanked for assisting in the intracellular injection technique, and Dirk de Jong is gratefully acknowledged for photographical work. This study was supported by funds of L.D.L.
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