Kjell Fuxe

Immunohistochemical evidence of vasoactive intestinal polypeptide-containing neurons and nerve fibers in sympathetic ganglia

Abstract Neurons and nerve fiber networks showing immunoreactivity for vasoactive intestinal polypeptide have been observed in some sympathetic ganglia with the help of the indirect immunofluorescence technique. Antisera to tyrosine hydroxylase and dopamine β-hydroxylase were used to identify catecholamine cells on consecutive sections. In the coeliac-superior mesenteric ganglion complex and the inferior mesenteric ganglion of the guinea-pig very dense networks of vasoactive intestinal polypeptide-positive varicose nerve fibers were observed around the principal ganglion cells. Only very few immunofluorescent fibers were seen in relation to the small intensely fluorescent cells. Occasionally some immunoreactive principal ganglion cells were found. In the same ganglia of the rat a considerably less dense network of vasoactive intestinal polypeptide-positive nerve fibers was present. The superior cervical ganglia of both species contained only a few vasoactive intestinal polypeptide-immunofluorescent fibers and no positively reacting cells were found.

Occurrence of neuropeptide Y (NPY)-like immunoreactivity in catecholamine neurons in the human medulla oblongata.

Abstract We report here the coexistence of a neuropeptide and catecholamines in neurons of the human brain. Using indirect immunofluorescence histochemistry, combined with elution and restaining experiments, neurons in the medulla oblongata of man were demonstrated to contain both a neuropeptide Y-like peptide and the catecholamine synthesizing enzyme tyrosine hydroxylase.

Fluorescence Microscopy in Neuroanatomy

The ever-increasing use of fluorescence methods in bio-medical research clearly bespeaks a steadily increasing demand for more sensitive methods allowing the identification of biologically active substances present in tissues in low — and often extremely low — concentration. Beside improvements in chemical methodology, the development of more adequate and commercially available equipment has played a major role in promoting the use of fluorescence methods.

Localization and characterization of phenylethanolamine N-methyl transferase in the brain of various mammalian species

The distribution of PNMT activity in various regions and nuclei of the rat, monkey and human brain was investigated. The distribution of PNMT activity in the rat brain correlates with the distribution of PNMT immunofluorescence. The PNMT activity in the primate brain is more widely distributed than in the rat brain. High and intermediate enzyme activity values were dound in the same regions of the primate brain as in the rat brain. Intermediate or low levels were also found in various other regions of the primate brain, e.g., basal ganglia, amygdala, septum, habenula. The brain PNMT has the same substrate specificity and similar kinetic properties as the adrenal enzyme. Immunotitration studies revealed cross-reactivity between the homologous adrenal and brain PNMT.

Dopaminergic Systems in the Brain and Pituitary

It is now 20 years since Swedish scientists described the existence of the nigrostriatal, mesolimbic, and tuberoinfundibular dopaminergic (DA) neurons in the rat brain [4, 8, 13, 17, 24, 50]. Since then new types of DA neuronal systems in the brain have been mapped out and the existence of peptide comodulators in certain subpopulations of DA neuronal systems has been described [27–29]. Of considerable importance in the mapping of new types of DA systems (Tables 1, 2, and 3) has been the development of new sensitive fluorescence methods for the demonstration of DA, based on the same histochemical principles as the classical formaldehyde method, and the biochemical purification of tyrosine hydroxylase (TH) [41] has made it possible to use TH immunocytochemistry in the mapping of the central DA neuronal systems [23, 25, 27, 33, 34, 38]. It is important to emphasize that although the various DA neuronal systems have been described mainly in the rat brain, they also exist in the primate and human brains, although the details of their anatomy remain to be completely worked out [43].

Applications of immunohistochemistry to studies on monoamine cell systems with special reference to nervous tissues.

The principles for the histochemical demonstration of substances on the basis of immunologic reactions were worked out by Coons et al. many years ago and have since been used in a wide variety of research disciplines for identification and tracing purposes. Only comparatively recently have the potentials of this technique been utilized by neurobiologists, but it may be anticipated that immunohistochemistry will play a fundamental role in the neuroanatomic and histochemical analysis of the central nervous system. In fact, the complexity of this tissue has for many years posed a giant problem in neurobiology, and although the synaptic connections are well known, a t least in a few brain regions, such as the cerebellum (see Reference 2), the chemical identification of neurons is still incomplete. The successful application of immunohistochemistry t o neurotransmitterrelated problems was largely based on the fact that certain enzymes present in catecholamine (CA) neurons are also found in the glandular cells of the adrenal medulla. Thus, a rich source of tyrosine hydroxylase (TH), dopa decarboxylase (DDC), dopamine 0-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT), four enzymes involved in CA synthesis (see FIGURE l ) , is available from which the enzymes can be purified. The first important discovery in this field was the finding of Gibb et al.3 that DBH possesses antigenic activity. With this background, Geffen et aL4 purified DBH, raised antibodies t o this enzyme, and later conducted immunofluorescence studies on the adrenal gland and the peripheral autonomic nervous system with both the direct and indirect techniques. In addition, they also used antibodies to chromogranin A, a protein component of the matrix of CA storage vesicles. Similar studies on DBH5 and on DDC and PNMT6-8 were performed by other groups. In all of these studies, only cell bodies could be demonstrated, but by addition of Triton@ X-100 to the antisera, as introduced by Hartman et a L 9 DBH could also be visualized in axons and nerve terminals; that is, the entire neuron could be stained.

Cellular Localization of Dopamine-β-hydroxylase and Phenylethanolamine-N-methyl Transferase as Revealed by Immunohistochemistry

Publisher Summary This chapter discusses the results of immunohistochemical studies on dopamine-β-hydroxylase (DBH) and phenylethanolamine-N-methyl transferase (PNMT). The studies with the histochemical fluorescence method for the demonstration of catecholamines (CA) and 5-hydroxytryptamine (5-HT) on the central nervous system have clearly indicated that the mapping out of new specific neuron systems in the brain will result in markedly increased knowledge of the function of the brain. This progress was made on the basis of the localization of the transmitters themselves, dopamine (DA), noradrenaline (NA) and 5-HT respectively. Findings discussed in the chapter show that antibodies toward adrenal bovine DBH can react with rat DBH of both adrenal and nervous origin. It was possible to localize DBH at the cellular level in both the peripheral and central nervous system. The findings in the central nervous system have greatly contributed to the mapping out of central NA neurons.

Central regulation of the endocrine system

Opening address.- Session I. The Peptidergic Neuron.- Neuroendocrine regulation: The peptidergic neuron Introduction and historical background.- The biochemistry of peptidergic neurons.- Hypothalamic hormones regulating pituitary (and other) functions: Their physiology and biochemistry as well as recent studies with their synthetic analogues.- Presynaptic mechanisms in peptidergic neurons.- Neurotransmitters and neuropeptides: Distribution patterns and cellular localization as revealed by immuno-cytochemistry.- Activation of release and mechanism of release of neurohypophyseal hormones.- Studies on the release and degradation of hypothalamic releasing hormones by the hypothalamus and other CNS areas in vitro.- Postsynaptic mechanisms in peptidergic transmission.- The role of GTP in the coupling of hormone receptors and adenylate cyclase.- Interactions between hypothalamic and peripheral hormones at the anterior pituitary level.- Peptide and neurotransmitter receptors in the brain: Regulation by ions and guanyl nucleotides.- Neurophysiology of hypothalamic peptidergic neurons.- The opioid receptors and their ligands.- Session II. Transmitter and Neuropeptide Synaptic Mechanisms.- Regulation of the ?-adrenergic receptor in the pineal gland and red cell membranes.- Cyclic nucleotide and protein phosphorylation mechanisms in the central nervous system.- Contrasting principles of synaptic physiology: Peptidergic and non-peptidergic neurons.- Regulation of neuropeptide release in rat brain.- Studies on interactions of epinephrine neuronal systems with other neuronal systems.- Effect of peptides on brain monoamines and on gross behaviour.- Session III. Hormonal Control of Peptidergic Neurons.- Current approaches to steroid hormone-cell interactions.- Steroid hormone receptors in brain and pituitary.- Feedback effects on central mechanisms controlling neuroendocrine functions.- Pituitary neuropeptides and behavior.- Sexual differentiating actions of steroids on the hypothalamopituitary-liver axis.- Session IV. Interactions Between Hormones and Neurotransmitters in the Control of Peptidergic Neurons.- Neurotransmitters in the control of anterior pituitary function.- Neurotransmitter mechanisms in the control of the secretion of hormones from the anterior pituitary.- Session V. Controls of Peptidergic Neurons in Humans.- Control of peptidergic neurons in humans. An introduction.- Studies of the role of dopamine in the control of prolactin and gonadotropin secretion in humans.- Neurotransmitter control of growth hormone and prolactin secretion.- Clinical neuroendocrine relationships in normal and disordered prolactin secretion.- Session VI. Reporters Overviews.- Role and regulation of neuropeptide neurons.- Some principles of neuronal regulation at the postsynaptic level.- Reporters remarks.- Overview of Session IV: Interactions between hormones and neurotransmitters in the control of peptidergic neurons.- Discussion of clinical neuroendocrinology section.- Participants.

Preferential vulnerability of A8 dopamine neurons in the primate to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Immunohistochemical examination of the midbrain of vervet monkeys treated with MPTP revealed a marked loss of dopaminergic neurons in the lateral, but not medial, region of the A8 dopamine (DA) cell group. In the same animals, the number of DA neurons in the substantia nigra was only slightly decreased. Biochemical assessment revealed a marked (greater than 85%) depletion of DA in the striatum. However, in the DA cell body regions significant decreases in DA and homovanillic acid were observed only in the lateral A8 region, and not in the medial A8 region or substantia nigra. These data suggest that those A8 DA neurons which project to the striatum are preferentially vulnerable to MPTP.

Quantitative Neuroanatomy in Transmitter Research

The usefulness of computer-assisted image analysis with particular emphasis on fluorescence microscopy was evaluated and exemplified. Problems associated with image pick-up and transfer between microscope and computer are discussed. The importance of fully supported software programs adjusted to the needs of histology are emphasized. One such system, the !BAS (Kontron/Zeiss, West Germany) has been found suitable for use also by people with little or no background computer knowledge. Three examples where image analysis clearly adds a unique quantitative dimension to the evaluation of the results have been presented. (1) Nerve density measurements: A semi-automatic interactive program was used to evaluate the potentially neurotoxic effects of hexachlorophene and chlorhexidine, two disinfectant agents, using an intraocular screening model in which the density of the sympathetic autonomic ground plexus of the iris is studied by Falck-Hillarp fluorescence histochemistry applied to iris whole mounts. Pronounced neurotoxic effects were described. Dopaminergic nerve density measurements in striatum following neurotoxic drug treatments correlate well with other measurements of degree of denervation. (2) Transmitter release and diffusion: Experimentally induced unilateral parkinsonism in rats can be counteracted by intrastriatal implants of chromaffine tissue. These grafts work by releasing large quantities of catecholamines which diffuse through host neuropil. Image analysis was used to characterize in detail the diffusion of catecholamines using Falck-Hillarp fluorescence histochemistry. Linear scans of fluorescence intensity and imaging fluorescence gradients using false color look-up tables enables fast visual quantitative interpretation of the results. (3) Morphometry of smeared and sectioned astrocytes: A program was used that calculated area and perimeter of smeared astrocytes stained with an antiserum against glial fibrillary acidic protein, GFA. A study of astrocyte growth from adolescence to senescence revealed continuous growth of astrocytes throughout life. In this case the extreme complexity of astrocyte morphology necessitated