Reduced Nicotinamide Adenine Dinucleotide Phosphate–Diaphorase Histochemistry: Light and Electron Microscopic Investigations

Publisher Summary

This chapter discusses several modifications of the procedures for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and the distribution of these NADPH-diaphorase–positive cell groups and nerve terminals in the central nervous system. It also discusses the ultrastructral localization of NADPH-diaphorase and coexistence of other neuropeptides in the positive neurons and nerve fibers. The best procedure for NADPH-diaphorase histochemistry involves washing out of blood and proper fixation. The chapter further presents several NADPH-diaphorase histochemical methods. One of these methods is a direct method using NADPH, and the other two methods are indirect methods using NADPH. Typical intensity NADPH-diaphorase positive neurons are found in the cerebral cortex, striatum, accumbens, and pedunculopontine tegmental, parabrachial, and pontine reticular nuclei in mammalian brains. Several subtypes of NADPH-diaphorase–positive neurons can be classified by their characteristic profiles. This classification is also presented in the chapter. Under the electron microscope, the NADPH-diaphorase–positive neurons show large amounts of the electron-dense reaction product formazan, which is not confined to any particular subcellular organelle.

Introduction

The accidental discovery of the Golgi impregnation staining method for neurons has allowed study of the detailed morphology of neuronal cell bodies and their associated processes in the central nervous system and contributed to establishment of the neuron theory. Use of this staining technique has allowed development of a system of precise classification of neurons in a particular nucleus on the basis of morphology without biochemical indices. The possible relation of the detailed morphological differences to neuronal function has been under discussion for a long time. In order to allow the simultaneous examination of biochemical and morphological aspects of the central nervous system, numerous histochemical methods for various neurotransmitters and neuropeptides have been developed. For example, histofluorescent methods for the transmitters have been used to visualize dopaminergic, noradrenergic, and serotonergic neuronal systems (1). Enzyme histochemistry for acetylcholinesterase, which is responsible for the metabolism of acetylcholine, was taken as a marker for the cholinergic system (2,3). Enzyme histochemical procedures for the demonstration of various dehydrogenases have also been applied to the central nervous system, although with limited success (4,5). More recently, immunohistochemical methods for enzymes, neurotransmitters, and neuropeptides have provided additional knowledge of the morphology of particular biochemically defined neuronal systems (6,7). The hybridoma technique has been widely used to obtain specific monoclonal antibodies against substances unique to particular subsets of neurons. By using these monoclonal antibodies and immunohistochemistry, morphological knowledge has been extraordinarily expanded. However, these immunohistochemical techniques remain complicated, with the results being very dependent on fixation and other procedural details. Moreover, they are often difficult to use on the autopsied human material (8).

Recently, there has been a revival of interest in stable and simple enzyme histochemical methods, especially in the central nervous system, where highly specific and selective distributions of enzymes are frequently observed and where changes in activity can indicate functional states. The most notable results have come from studies of acetylcholinesterase as a marker for cholinergic structures in mammalian brains, including human brains (8,9). Other enzymes that have been examined in the brain include 4-aminobutyrate aminotransferase (GABA transaminase), monoamine oxidase, and various dehydrogenases (10, 11, 12, 13, 14). Dehydrogenase histochemistry is based on the ability of reduced cofactors, formed in the dehydrogenases reaction, to reduce subsequently a dye, such as a tetrazolium salt, to a visible reaction product. One such dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase is found in certain neurons in the unfixed rat brain and spinal cord (15, 16, 17, 18). A novel histochemical procedure, based on this diaphorase activity, selectively stains various discrete populations of neurons in moderately fixed brain tissue, and for these neurons yields a Golgi-like picture of cell bodies, dendritic trees, and axonal networks (19). Although the function of NADPH–diaphorase in the brain remains a mystery, this simple reliable enzyme histochemical procedure provides a useful technique to obtain detailed morphological information for particular cell groups at both light and electron microscopic levels (20,21). The NADPH–diaphorase-positive neurons are not exclusively associated with any one particular neurotransmitter system, although the NADPH–diaphorase does seem to coexist with somatostatin and neuropeptide Y (NPY) in many neurons in various forebrain areas and with choline acetyltransferase in some more rostral systems (21, 22, 23, 24, 25, 26, 27).

In this article, several modifications of the procedures for NADPH–diaphorase histochemistry and the distribution of these NADPH–diaphorase-positive cell groups and nerve terminals in the central nervous system are discussed. Also discussed are the ultrastructral localization of NADPH–diaphorase and coexistence of other neuropeptides in the positive neurons and nerve fibers.