Central Adrenaline Neurons (eBook)

Basic Aspects and Their Role in Cardiovascular Functions

Kjell Fuxe, Menek Goldstein, Bernt Hokfelt (Herausgeber)

eBook Download: EPUB

2013 | 1. Auflage
362 Seiten
Elsevier Science (Verlag)
978-1-4831-5469-5 (ISBN)

Infos

Central Adrenaline Neurons: Basic Aspects and their Role in Cardiovascular Functions contains the proceedings of an international symposium held at The Wenner-Oren Center, Stockholm on August 27-28, 1979. The purpose of the meeting is to summarize the knowledge of central adrenaline neurons and their role in cardiovascular functions. Organized into four sections, this book begins with a discussion on the morphology, biochemistry, and pharmacology of central adrenaline neurons. Subsequent sections detail the cardiovascular functions of central catecholamine neurons and the effects of centrally acting drugs on sympathetic function in normotensive and hypertensive patients. An overview lecture of the concept of a- and ß-adrenergic receptors is also shown.

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Central Adrenaline Neurons: Basic Aspects and their Role in Cardiovascular Functions contains the proceedings of an international symposium held at The Wenner-Oren Center, Stockholm on August 27-28, 1979. The purpose of the meeting is to summarize the knowledge of central adrenaline neurons and their role in cardiovascular functions. Organized into four sections, this book begins with a discussion on the morphology, biochemistry, and pharmacology of central adrenaline neurons. Subsequent sections detail the cardiovascular functions of central catecholamine neurons and the effects of centrally acting drugs on sympathetic function in normotensive and hypertensive patients. An overview lecture of the concept of a- and -adrenergic receptors is also shown.

Textauszug

HISTOCHEMICAL IDENTIFICATION OF ADRENALINE CONTAINING CELLS WITH SPECIAL REFERENCE TO NEURONS

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.

Fig. 1 Excitation and emission spectra for dopamine, noradrenaline, adrenaline and 5-hydroxytryptamine (from ref. 39).

Fig. 2 Schematic illustration of the synthesis of adrenaline from tyrosine (from ref. 64).

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

Aldehyde induced fluorescence

The formaldehyde-induced fluorescence method allows demonstration of secondary amines such as adrenaline, although the fluorescence yield is lower than for the corresponding primary amines and although stronger reaction conditions (higher temperature and/or longer reaction) are required for maximum fluorescence (see 1, 58–60). Thus, adrenaline can easily be visualized in the gland cells of adrenal medulla, if the tissue has been freeze-dried and reacted with formaldehyde vapours. On the other hand, if liquid formaline is used, the adrenaline will diffuse and only the islands of noradrenaline containing cells remain (61–63). Whether or not the central adrenaline neurons in fact were observed in the original studies of Dahlström and Fuxe (4), i.e. whether or not adrenaline is visualized routinely with the formaldehyde-fluorescence method, is not clear. The description of the localization of cell bodies by Dahlström and Fuxe (4) indicates that primarily noradrenaline cells were observed and only few, if any adrenaline cells could be seen. With regard to nerve terminals, which have much higher amine concentrations and thus should be more easy to visualize, they mostly seem to be intermingled with noradrenaline nerve endings and a distinction between the two types has so far not been achieved. Finally, it may be added that with the glyoxylic acid method the fluorescence induced from adrenaline is of a very low intensity and, due to a difference in the excitation peak, this catecholamine will “appear essentially non-fluorescent in the routine fluorescence microscope” (60).

Immunohistochemistry – enzyme antisera

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, since mostly fixation is needed to obtain a good retention and since fixation decreases antigenicity. In a methodological paper (64) we have systematically investigated the influence of various fixatives and sectioning and incubation procedures on the possibility to obtain acceptable immunohistochemistry with antisera to catecholamine synthesizing enzymes. That study confirmed that the membrane-bound enzyme DBH could be visualized after short fixation with alcohol, as originally used by Geffen et al. (16) and subsequently by other groups (18–22). For the other enzymes, DDC and PNMT (Fig. 3) (and TH), which all are cytoplasmic, stronger fixatives seemed necessary to avoid diffusion. For these enzymes perfusion with formalin turned...

Produkt-Details

• Erscheint lt. Verlag: 22.10.2013
• Sprache: englisch
• Themenwelt: Sachbuch/Ratgeber ► Natur / Technik ► Naturführer
• Themenwelt: Medizin / Pharmazie
• Themenwelt: Naturwissenschaften ► Biologie ► Zoologie
• ISBN-10: 1-4831-5469-6 / 1483154696
• ISBN-13: 978-1-4831-5469-5 / 9781483154695