T. HÖKFELT, M. GOLDSTEIN, K. FUXE, O. JOHANSSON, A. VERHOFSTAD, H. STEINBUSCH, B. PENKE and J. VARGAS,     Department of Histology, Karolinska Institutet, Stockholm, Sweden; Department of Psychiatry, Neurochemistry Laboratories, New York University Medical Center, New York, New York, USA; Department of Anatomy and Embryology, University of Nijmegen, The Netherlands; Department of Medical Chemistry, University of Szeged, Szeged, Hungary

Publisher Summary

This chapter describes a formaldehyde-induced fluorescence method that allows demonstration of secondary amines such as adrenaline although the fluorescence yield is lower for the corresponding primary amines. On the other hand, if liquid formaline is used, the adrenaline will diffuse and only the islands of noradrenaline containing cells remain. The visualization of enzymes and other substances with immunohistochemical methods is dependent upon (1) the retention of the compound in question at the proper site in the tissue and (2) the preservation of immunoreactivity. These two requirements are in principle difficult to reconcile as fixation is mostly needed to obtain a good retention and as fixation decreases antigenicity. The specificity of the immunoreaction is dependent of several factors. Firstly, the enzyme preparation used for immunization has to be pure. Secondly, it is difficult to exclude the possibility that cross-reacting enzymes and other transferases may react with phenylethanolamine-N-methyltransferase (PNMT) antisera. The catecholamine synthesizing enzymes are protein molecules and possess antigenic characteristics.

INTRODUCTION

In 1962 Falck, Hillarp and collaborators (1, 2) introduced a specific and sensitive formaldehyde-induced fluorescence method for the demonstration of certain biogenic amines in histological sections. It became possible to localize dopamine, noradrenaline and to a certain extent 5-hydroxytryptamine in neurons and to map the distribution of these systems (3–8). This mapping work has continued since then, adding more detailed information on the systems as well as revealing the existence of new systems. Information on dopamine, noradrenaline and 5-hydroxytryptamine systems can be obtained in several review articles (9–15). More recently the formaldehyde-induced fluorescence histochemistry has been supplemented by studies based on immunohistochemical techniques. In 1969 Geffen, Livett and Rush (16) were able to visualize peripheral catecholamine neurons and adrenal catecholamine containing gland cells using the indirect immunofluorescence technique of Coons and collaborators (see 17) and antibodies raised to dopamine-β-hydroxylase (DBH), the enzyme responsible for the conversion of dopamine to noradrenaline. Subsequently this approach has extensively been used for identifying not only catecholamine neurons (20-34d)but also other types of enzymes, for example glutamate decarboxylase (see 35) and numerous peptides (see 36). For general reviews, see refs. 37, 38.

Using the routine Falck-Hillarp formaldehyde fluorescence technique, it is not possible to differentiate between the three catecholamines, since they have the same excitation and emission characteristics (Fig. 1) (39). This fact has through the years represented a problem when trying to distinguish primarily between dopamine and noradrenaline neurons, and it was therefore necessary to rely on biochemical measurements and experimental and pharmacological manipulations (see e.g. 40–46). Little attention was given to the third catecholamine adrenaline, which was early observed in brain by biochemical techniques (47, 48). Gunne (49) showed that the adrenaline levels in the brain stem were only about 5% of the total noradrenaline and adrenaline levels, and no good pharmacological manipulations have existed to demonstrate preferentially adrenaline neurons. The isolation and characterization of all four enzymes in the catecholamine synthesis (Fig. 2), tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (dopadecarboxylase, DDC) (DBH) and phenylethanolamine-N-methyltransferase (PNMT) (see 50–54) and the production of antisera to each of these enzymes, have given us possibilities to differentiate not only between dopamine and noradrenaline neurons but also to identify adrenaline neurons, i.e. neurons containing PNMT.

More recently a new exciting approach has been utilized to visualize adrenaline in tissue sections. Verhofstad and colleagues have raised antisera directly to various amines, such as 5-hydroxytryptamine (55) and dopamine, noradrenaline and adrenaline (56). Surprisingly, antisera to these small molecules can be used for the immunohistochemical demonstration of the amines in tissue sections. This has convincingly been demonstrated for 5-hydroxytryptamine (55, 57) and studies are now in progress to use this approach also for visualization of catecholamines. So far good results have been obtained on the adrenal gland and nervous tissue with noradrenaline antisera and on the former tissue with adrenaline antiserum (56), but hopefully it will be possible to study adrenaline also in nervous tissue.

In the following we would like to describe attempts to localize adrenaline cell systems, particularly by immunohistochemistry using antiserum to PNMT under the assumption that this enzyme represents a marker for adrenaline neurons and that the antiserum specifically reacts with the PNMT. We will only deal with the hlstochemical approaches in this paper, since biochemical studies will be described in the paper by Goldstein and collaborators and by others in this Volume. Furthermore, a detailed description of the distribution of central PNMT immunoreactive neurons will be given in the following article (Hökfelt et al., this Volume).

ASPECTS ON METHODOLOGY