Elsevier

Progress in Neurobiology

Volume 90, Issue 2, 9 February 2010, Pages 101-118
Progress in Neurobiology

Looking at neurotransmitters in the microscope☆

https://doi.org/10.1016/j.pneurobio.2009.10.005Get rights and content

Abstract

This review article covers the early period of my career. I first summarize research initiated by the late Nils-Åke Hillarp, after his appointment in 1962 as professor in the Department of Histology at Karolinska Institutet. He only lived for three more years, but during this short period he started up a group of ten students who explored various aspects of the three monoamine transmitters, dopamine, noradrenaline and 5-hydroxytryptamine, using the new formaldehyde fluorescence method developed by Bengt Falck and Hillarp in Lund. This method allowed visualization of the cellular localization in the microscope of these monoamines, which introduced a new discipline in neurobiology—chemical neuroanatomy. I then deal with work aiming at localizing the monoamines at the ultrastructural level, as well as attempts to use radioactively labeled aminoacids, especially γ-aminobutyric acid (GABA), and autoradiography, to identify, in the microscope, neurons using such transmitters. Finally, our immunohistochemical work together with Kjell Fuxe and the late Menek Goldstein, using antibodies to four monoamine-synthesizing enzymes is summarized, including some aspects on the adrenaline neurons, which had escaped detection with the Falck–Hillarp technique.

Introduction

This review deals with work mainly performed during the starting period of the monoamine research at Karolinska, and may show some overlap with the preceding article by Kjell Fuxe, Annica Dahlström, Gösta Jonsson, Luigi Agnati and collaborators (this issue), but hopefully with a different perspective. I was the last one to join the Hillarp team, and my project was not based on the formaldehyde fluorescence method (FAM). I was probably the one who had the least contact with Nils-Åke Hillarp, in fact I only rarely talked directly with him.1 This in spite of the fact that I met him very early: My uncle Bernt Hökfelt and Hillarp had worked together in the 1950s (see below), and Bernt invited Nils-Åke and me for a dinner in his Stockholm home before Hillarp's move to Karolinska. I had just started as an unpaid amanuensis in the histology department and felt absolutely embarrassed and insufficient.

Section snippets

Nils-Åke Hillarp

Nils-Åke Hillarp was born in 1916 and became a scientist who changed neuroscience perhaps more than anyone in Sweden and, in fact, internationally. In 1962, Hillarp moved from Lund University in South Sweden to Karolinska Institutet (KI), having been appointed prosektor (an old title corresponding to Associate Professor, means the one who cuts and prepares the corpse for the professor) in histology. Included in his luggage was a new method, FAM, which he had developed in Lund together with

The electron microscopy of monoamines

The first project given to me by Hillarp in 1963 was to develop a fluorescence method for histamine based on orto-phtalaldehyde, but I completely failed. Hillarp then asked me to try to visualize the monoamines in the electron microscope (EM), especially in the brain, one important idea being comparing the distribution patterns described with FAM by Kjell and Annica. The reason I was assigned this task was my knowledge, albeit limited, in electron microscopy, which I had obtained under the

Histochemical neuroendocrinology and pharmacology

During the later phase of my thesis work, I started, with increasing intensity, to work together with Kjell Fuxe, one reason being that Annica Dahlström had to concentrate on her thesis dealing with fast axonal transport and then moved to Gothenburg. It seems fair to say that I, in parallel with my electron microscopic thesis work, that is before completion of my thesis, did an early, extremely productive ‘postdoc training’ with Kjell as my supervisor. In fact, it was a treat to work with one

How to identify other neurotransmitter neurons in the microscope?

In the mid and late 1960s, only the CAs and 5-HT, at least in my world, were real transmitters (of course, in addition to ACh). The dominant electrophysiologists told us elsewise—they certainly did not accept a transmitter status for monoamines which led to some controversies (see Carlsson, 2001). On a personal level, the low percentage (15%) of the DA nerve terminals in the striatum (Hökfelt and Ungerstedt, 1969, Hökfelt and Ungerstedt, 1973), and the 30–40% in the external layer of the median

Immunofluorescence thanks to Albert Coons and Menek Goldstein

In 1969 Geffen, Livett and Rush published a landmark paper which should have a major impact on neuroscience and other fields of biomedicine (Geffen et al., 1969). Thus, they purified and raised antibodies to dopamine β-hydroxylase (DBH) from the bovine adrenal medulla, the enzyme converting DA to NA. They applied the antiserum to adrenal gland and sympathetic ganglia using the indirect immunohistochemical technique developed by Coons and collaborators in the early 1940s (see Coons, 1958), that

Concluding remarks

Our laboratory has now for more than four decades been involved in characterizing neuronal systems on the basis of their messenger molecules by looking in a microscope. All this started when the late Nils-Åke Hillarp arrived at Karolinska in 1962, bringing with him the FAM for visualizing the cellular localization of DA, NA and 5-HT in a fluorescence microscope. Early on, we localized monoamines at the ultrastructural level in synaptic vesicles in the brain using the KMnO4 fixation technique

Acknowledgements

The work described here has throughout the years been supported by the Swedish MRC/Research Council (04x-2887), as well as by several Foundations (The Magnus Bergvall Foundation, The Knut and Alice Wallenberg Foundation, The Ollie and Elof Ericsson Foundation, the Margaretahemmet Foundation, the C.H. Nathorst Foundation, The Swedish Insurance Committee for Medical Research and Karolinska Institutet Research Funds). Dr. Menek Goldstein (and thus indirectly I) was supported by United States

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    ☆

    This article is dedicated to all wonderful and talented students, postdoctoral fellows, guest scientists, world-wide colleagues and, last but not least, technicians/secretaries, with whom I have collaborated over more than four decades. I have had a loose control of the work in the laboratory, so many of the ideas and projects have sprung out of their scientific minds. Also, antibodies have been the scientific oxygen in my professional life, and the vast majority of them has been generously supplied by colleagues around the world.

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