Olle Johansson

Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain

With the help of immunohistochemical studies using antibodies against bovine adrenal phenylethanolamine-N-methyltransferase (PNMT), the enzyme converting noradrenaline (NA) to adrenaline (A), the cellular localization of PNMT in the rat central nervous system has been demonstrated. The brains were fixed by perfusion with ice cold 4% formalin and sectioned on a cryostat, after which the sections were stained using the indirect immunofluorescence technique. Preimmune serum and PNMT antiserum adsorbed with PNMT served as control sera. Specific immunofluorescence was localized in two groups of reticular nerve cell bodies in the medulla oblongata and in nerve terminals in special nuclei of the brain stem and the spinal cord. A PNMT positive axon bundle was also observed in the reticular formation of the pons-medulla oblongata. The distribution and morphology of these PNMT containing neurons was such that they probably are identical with some catecholamine nerve terminals and cell bodies previously demonstrated with the Falck-Hillarp technique (see Dahlstro¨m and Fuxe18). The hypothesis is therefore given that the PNMT containing neurons represent A containing neurons, and that A may act as a neurotransmitter in the rat brain. The reticular A neurons appear to have similar morphological characteristics as the NA neurons with long ascending and descending fibers to the brain stem and spinal cord. The A terminals are mainly found in certain visceral afferent and efferent nuclei of the lower brain stem, in the locus coeruleus, in certain nuclei or the hypothalamus and in the periventricular grey. The well defined distribution of the A networks to these nuclei underline the view that the A neuron may participate in the control of oxytocin secretion, food and water intake, body temperature, gonadotrophin secretion, blood pressure and respiration and sleep and wakefulness.

Immunohistochemical studies using antibodies to leucine-enkephalin: Initial observations on the nervous system of the rat

Enkephalins are peptides which have pharmacological properties similar to those of morphine. Guinea pigs were immunized with a leucine-enkephalin/thyroglobulin conjugate. Immunofluorescence histochemistry with antiserum revealed a widely distributed system of axons and their terminals in the nervous system of the rat. Prominent networks of enkephalin-like immunoreactivity were found in some brainstem nuclei and in portions of the limbic forebrain. The myenteric plexus in the gastrointestinal tract also contained fluorescent fibers. The distribution of the positive immunofluorescence parallels the occurrence of enkephalin as revealed by biochemical techniques. Some areas known to have a high opiate receptor density were also shown to contain striking networks of enkephalin-like immunoreactivity. Such findings provide morphological support for the hypothesis that enkephalins are contained in nerve terminals close to opiate receptors.

Immunohistochemical support for three putative transmitters in one neuron: Coexistence of 5-hydroxytryptamine, substance p- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord

Abstract Using indirect immunofluorescence histochemistry, in part combined with the elution and restaining technique of Tramu , Pillez & Leonardelli (1978) , the distribution of 5-hydroxytryptamine (5-HT), thyrotropin releasing hormone (TRH) and substance P immunoreactive neurons has been studied in the medulla oblongata and spinal cord of normal and colchicine-treated rats. Evidence was obtained that at least some cell bodies in the medullary raphe nuclei and adjacent areas contained all three compounds, 5-HT, TRH and substance P. Other cell bodies in the same areas may contain two or only one of these three putative transmitters. Alternatively, the intraneuronal levels of one or two of the substances may be too low to be detected with the present technique, in spite of the fact that colchicine treatment was used to elevate peptide levels in the cell somata. In a quantitative evaluation the proportion of 5-HT, TRH and substance P neurons was calculated at different levels and in different nuclei of the medulla oblongata. Out of all immunoreactive neurons, there were approximately twice as many 5-HT (56%) as TRH (23%) and substance P (21%) cells respectively, and this relation was also found in several major subnuclei, such as the nucleus raphe magnus and nucleus raphe obscurus. In the ‘arcuate’ region very high proportions of 5-HT cells (about 60–80%) were observed with only few substance P cells (2–12%). The ‘parapyramidal’ and ‘paraolivar’ regions, which include the nucleus interfascicularis hypoglossi, had more substance P (26–36%) than TRH (15–17%) cells. The most ‘even’ distribution was observed in the nucleus raphe pallidus (5-HT: 43%; TRH: 32%; substance P: 25%). The evaluation also indicated how the respective cell type (5-HT, TRH and substance P cells) distributed between the different subnuclei. Thus, at rostral levels the ‘suprapyramidal’ region contained a large proportion (about 30%) of the total numbers of counted 5-HT, TRH and substance P cells, respectively. Furthermore, the nucleus raphe magnus contained a large part (about 30%) of the TRH and substance P cells, but a smaller fraction (about 20%) of the 5-HT cells. Analysis of adjacent sections at regular intervals confirmed the overall quantitative evaluation. Generally, the distribution of 5-HT, TRH and substance P cells were roughly parallel. An exception was the midportion of the rostral medulla oblongata, where 5-HT cells were very numerous. Of particular interest was the fact that, especially in the nucleus raphe pallidus, there were in several series almost the same number of 5-HT, TRH and substance P cells, supporting the view that many cells in this nucleus contained all these compounds. In the spinal cord overlapping networks of 5-HT, TRH and substance P immunoreactive fibres were observed in the ventral horn. The number of 5-HT immunoreactive fibres seemed higher than the TRH and substance P immunoreactive ones. After treatment with the neurotoxins 5,6- or 5,7-dihydroxytryptamine there was an almost complete disappearance of all three types of fibres in the ventral horn, further supporting the occurrence of the two peptides in 5-HT neurons, either both of them together in the same 5-HT neuron or each of them in separate 5-HT neurons. It is, however, important to note that there are, in all probability, 5-HT neurons in the lower medulla oblongata which contain neither TRH nor substance P. Furthermore, in other brain regions there is no certain correlation between the distribution patterns of 5-HT, TRH and substance P immunoreactive cells. The results are consistent with the coexistence of 5-HT, TRH and substance P in neurons of the medulla oblongata that project to the spinal cord. Some neurons may contain detectable levels only of 5-HT and substance P, others only of 5-HT and TRH, while others contain all three substances. It can, however, not be excluded that some neurons contain only one of these compounds or that other combinations exist.

The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system ☆

With the indirect immunofluorescence technique the distribution of methionine-enkephalin-immunoreactive cell bodies was studied in the central nervous system of rats pretreated with colchicine. The antiserum used did cross-react to 10% with leucine-enkephalin but to less than 0.1% with alpha-, beta-, and gamma-endorphine. Cell bodies with a specific immunofluorescence were observed in the tel-, di-, mes- and rhombencephalon and in the spinal cord.

European federation of neurological societies/peripheral nerve society guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. report of a joint task force of the european fe-deration of neurological societies and the peripheral nerve society

Background:  Revision of the guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy, published in 2005, has become appropriate owing to publication of more relevant articles. Most of the new studies focused on small fiber neuropathy (SFN), a subtype of neuropathy for which the diagnosis was first developed through skin biopsy examination. This revision focuses on the use of this technique to diagnose SFN.

EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy

Skin biopsy has become a widely used tool to investigate small calibre sensory nerves including somatic unmyelinated intraepidermal nerve fibres (IENF), dermal myelinated nerve fibres, and autonomic nerve fibres in peripheral neuropathies and other conditions. Different techniques for tissue processing and nerve fibre evaluation have been used. In March 2004, a Task Force was set up under the auspices of the European Federation of Neurological Societies (EFNS) with the aim of developing guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathies. We searched the Medline database from 1989, the year of the first publication describing the innervation of human skin using immunostaining with anti‐protein‐gene‐product 9.5 (PGP 9.5) antibodies, to 31 March 2005. All pertinent papers were rated according to the EFNS guidance. The final version of the guidelines was elaborated after consensus amongst members of the Task Force was reached. For diagnostic purposes in peripheral neuropathies, we recommend performing a 3‐mm punch skin biopsy at the distal leg and quantifying the linear density of IENF in at least three 50‐μm thick sections per biopsy, fixed in 2% PLP or Zambonis solution, by bright‐field immunohistochemistry or immunofluorescence with anti‐PGP 9.5 antibodies (level A recommendation). Quantification of IENF density closely correlated with warm and heat‐pain threshold, and appeared more sensitive than sensory nerve conduction study and sural nerve biopsy in diagnosing small‐fibre sensory neuropathy. Diagnostic efficiency and predictive values of this technique were very high (level A recommendation). Confocal microscopy may be particularly useful to investigate myelinated nerve fibres, dermal receptors and dermal annex innervation. In future, the diagnostic yield of dermal myelinated nerve fibre quantification and of sweat gland innervation should be addressed. Longitudinal studies of IENF density and regeneration rate are warranted to correlate neuropathological changes with progression of neuropathy and to assess the potential usefulness of skin biopsy as an outcome measure in peripheral neuropathy trials (level B recommendation). In conclusion, punch skin biopsy is a safe and reliable technique (level A recommendation). Training in an established cutaneous nerve laboratory is recommended before using skin biopsy as a diagnostic tool in peripheral neuropathies. Quality control at all levels is mandatory.

Dopamine Nerve Terminals in the Rat Limbic Cortex: Aspects of the Dopamine Hypothesis of Schizophrenia

The existence of cortical dopamine nerve terminals is demonstrated with a highly sensitive modification of the Falck-Hillarp fluorescence technique. This confirms previous biochemical reports of high dopamine levels in the cortex. The histochemistry reveals that the distribution is regional and confined to the limbic cortex.

Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord

With the indirect immunofluorescence technique the localization of thyrotropin releasing hormone (TRH) has been studied in the lower brain stem and spinal cord. Networks of TRH-positive nerve terminals were observed in many cranial nerve cell nuclei and around the motoneurons in the spinal cord.

Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin.

SummarySections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.

Serotoninergic and melatoninergic systems are fully expressed in human skin

We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N‐acetyltransferase (AANAT), and hydroxyindole‐Omethyltransferase (HIOMT). The products of AANAT activity were identified by RP‐HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N‐acetylserotonin (NAS) was detected by RP‐HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways.