Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the nerves of human axillary sweat glands.

The cholinergic innervation of the human axillary sweat glands of hyperhidrotic patients was demonstrated by using the specific Karnovsky-Roots thiocholine method. The cholinergic innervation pattern was compared with the immunohistochemically demonstrated vasoactive intestinal polypeptide (VIP)-like activity at light and electron microscopic levels. The innervation patterns were identical in the light microscopic serial sections. In the electron microscope sections, VIP-like immunoreactivity was localized to the nerve terminals containing large, dense-cored vesicles 100-140 nm in size. No synapses were found, however positively stained nerve terminals were located immediately outside the basement membrane but close to the glandular secretory and myoepithelial cells, blood vessels, and occasionally the mast cells. Our results suggest the coexistence of the two neurotransmitters, acetylcholine and VIP, in the same nerves innervating both eccrine and apocrine sweat glands in human axillae.

Expression of diazepam-binding inhibitor peptide in human skin: an immunohistochemical and ultrastructural study.

Human diazepam binding inhibitor (DBI) was originally isolated from the brain and subsequently found to be present in several peripheral tissues. The various physiologic effects recently attributed to DBI include acting as an endogenous ligand for the central and peripheral (mitochondrial) benzodiazepine receptors. The present work provides, for the first time, evidence of DBI immunoreactivity in skin. DBI immunoreactivity was found in the epidermis, in the eccrine sweat and in sebaceous glands. Ultrastructurally, DBI was distributed throughout the cytoplasm. Although the physiologic role of DBI in skin is unknown, our results indicate that DBI may serve as an endogenous ligand for mitochondrial benzodiazepine receptors. Its activity could be related to the regulation of lipid and cholesterol synthesis in keratinocytes and sebaceous glands and to the secretion of sweat in sweat glands.

Scanning electron microscopy and X-ray microprobe analysis in detection of acetylcholinesterase in cultured embryonal carcinoma cells.

Mouse F9 cells, induced by retinoic acid and dibutyryl cyclic adenosine monophosphate (cAMP) to differentiate into neural-type cells, were incubated for localization of specific acetylcholinesterase (AChE) activity according to the Karnovsky-Roots method where the final enzymatic reaction product is crystalline cupric ferrocyanide and cuprous thiocholine iodide. By scanning electron microscopy (SEM) neural-type cells with long processes were seen. Most of these cells exhibited crystalline precipitates on their surface that in microprobe analysis contained copper, iron, and sulfur. These elements were also detected in some of the neural-type cells that had no visible surface precipitates. Thus, the X-ray analysis also revealed intracellular enzymatic activity. Undifferentiated rounded cells, devoid of AChE activity at the light microscope level, did not show any surface precipitates by SEM and lacked copper, iron, and sulfur emission peaks in the elementary analysis. These results demonstrate that elementary analysis of cytochemical enzymatic reaction products by SEM can be used in identifying cells.

Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe.

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.

Rod-like intramitochondrial inclusions after hypothermic chemical cardioplegia during cardiac operations.

Rod-like intramitochondrial inclusions in the myocardial cells were observed after hypothermic chemical cardioplegia in three out of 20 patients who underwent coronary bypass operations. They were not seen in another group of 20 patients who underwent an aortic valve replacement operation in whom only topical cooling was used for myocardial protection. The occurrence of rod-like intramitochondrial inclusions could not be correlated with other signs of ischemic myocardial injury. X-ray microanalysis did not reveal any inorganic substance in the intramitochondrial inclusions. Therefore, we believe that their occurrence was not related to the calcium paradox phenomenon, a feared complication of cardiac operations.

Cerium ions in the histochemical demonstration of second-messenger enzymes.

Second-messenger systems are involved in the regulation of numerous cellular processes. Adenylate cyclase (AC) and guanylate cyclase (GC) enzymes are in key positions in the regulation of these systems. The cerium method has been successfully applied to demonstrate amine- and neuropeptide-stimulated AC in rat nervous and adipose tissues and human sweat glands at the electron microscopic level. AC was also localized in cultured neurons. Nitric oxide compounds stimulated GC were demonstrated in rat hippocampal areas. Enzyme reactions were located in neurons pre- and postsynaptically in synapses; in addition, GC activity was seen intraneuronally and in glial cells. Adipocytes and eccrine glandular cells exhibited reaction products in their plasmalemmas. Optimal histochemical conditions are described, combined with control experiments. Some handicaps, related to the sensitivity of the enzymes to the fixatives, penetration problems of cerium salts, and especially the specificity of the method in phosphatase enzyme histochemistry in general are discussed.

Fos and jun in rat central amygdaloid nucleus and paraventricular nucleus after stress.

The present paper describes the effect of capsaicin-induced stressful stimulus on the expression of immediate early genes (IEGs) c-fos, c-jun, junB and junD in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (ACe) using in situ hybridization. Stress caused an intense expression of c-fos, c-jun and junB especially in the PVN and ACe and also a clear induction of junD was observed in the PVN. This suggests that the PVN and the ACe are two major targets of stress in the brain. The intense expression of the IEGs in the ACe and PVN suggests that stress may affect neurotransmitter gene expression through Fos and Jun proteins in both these nuclei.

Serotonin-like immunoreactivity in rat carotid body.

Serotonin-like immunofluorescence was demonstrated in the glomus cells of the rat carotid body. Similar immunoreactivity was noted in mast cells in the organ, while no immunoreactive nerve fibers were seen. It is suggested that glomus cell serotonin could participate in the modulation of chemoreceptor activity.

Distribution of acidic fibroblast growth factor-like immunoreactivity in rat skeletal muscle fibers.

Acidic fibroblast growth factor (aFGF) is a mitogenic, angiogenic and neurotrophic growth factor which promotes proliferation, but delays differentiation of cultured myoblasts. Its mRNA is expressed in the skeletal muscle, however, the distribution of aFGF in the postnatal skeletal muscle is poorly characterized. In the present study, the distribution of aFGF-like immunoreactivity (LI) was examined in developing and adult rat skeletal muscle fibers. In addition, the effect of the transection of the sciatic nerve on aFGF-LI in calf muscle fibers was examined. From the first postnatal day on, aFGF-immunoreactive (IR) muscle fibers were observed in different calf muscles. From the 7th postnatal day on a large number of muscle fibers exhibited aFGF-LI in the soleus muscle, some in plantaris and only few in gastrocnemius and extraocular muscles. Double-labelling with fast-myosin antibody showed that aFGF-LI was restricted to the slow oxidative muscle fibers. aFGF-IR intrafusal muscle fibers were seen in developing and mature muscle spindles. In addition, aFGF-IR nerve fibers and myoneural junctions were observed in different muscles. Transection of the sciatic nerve did not noticeably alter the expression pattern of aFGF-LI in calf muscles during two-week period. The present study demonstrates aFGF-LI in the rat slow oxidative muscle fibers where it may have fiber-type specific functions in addition to its known trophic effects.

Calbindin D-28k-immunoreactivity in rat muscle spindle; a light and electron microscopic study.

The localization of the vitamin D-dependent calcium-binding protein, calbindin D-28k (CaBP), was studied immunocytochemically in rat striated muscle. CaBP-like immunoreactivity was found in some of the intrafusal fibres in muscle spindles. The spindle capsule and the perineurial sheath of the nerve bundles were occasionally immunoreactive to CaBP. In electron microscope the labelling for CaBP was found diffusely in sarcoplasm, in Z-bands and inside the terminal cisternae of intrafusal muscle fibres. The present findings suggest that CaBP may have a role in maintaining the appropriate microenvironment in the intracapsular space of muscle spindle and that CaBP may be involved in the function of intrafusal muscle fibres.

Co-localization of peptide-like immunoreactivities with glucocorticoid receptor- and Fos-like immunoreactivities in the rat parabrachial nucleus.

The parabrachial nucleus (PB) is a brainstem nucleus, which mediates autonomic information from the viscera to various forebrain nuclei, e.g. to the central nucleus of the amygdala (ACe) and to the medial preoptic area (MPOA). The neurons of the PB contain several neuropeptides, of which calcitonin-gene related peptide-immunoreactive (CGRP-IR) and neurotensin (NT)-IR neurons provide input to the ACe, whereas corticotropin-releasing factor-IR (CRF) neurons project to the MPOA. The aim of the present paper was to study whether the neurons containing CGRP-, NT- and CRF-like immunoreactivities (LIs) in the PB also contain glucocorticoid receptor (GR)- and/or Fos-LIs after stress. No co-localization was observed with the GR-LI and peptide-LIs, suggesting that plasma glucocorticoids do not have direct effects on these neurons of the PB. After stress, the vast majority of the peptide-IR perikarya exhibited Fos-LI, suggesting that the peptidergic pathways from the PB to ACe and MPOA are activated in stress. The ACe and MPOA have been connected in various stress related responses, e.g. inhibiting the hypothalamo-pituitary-gonadal axis, raising the blood pressure and pulse, and increasing the secretion of glucocorticoids. Therefore, the activation of the peptidergic pathways between the PB and the ACe and MPOA suggests that some of these responses may be elicited by the peptidergic input from the PB. Furthermore, since Fos acts as a transcription factor, stress may affect the expression of the neuropeptides studied.

Ornithine decarboxylase-like immunoreactivity in rat spinal motoneurons and motoric nerves.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the formation of the polyamines putrescine, spermidine and spermine. In the present study ornithine decarboxylase-like immunoreactivity (ODC-LI) was localized immunocytochemically in rat spinal motoneurons, motoric nerves and myoneural junctions in several muscles. In the spinal cord ODC-LI was expressed in most of the large multipolar neurons located in the ventral horn at cervical and lumbar levels. ODC-LI was localized in the cytoplasm, dendrites and axons of the labelled neurons. The nuclei of motoneurons were unlabelled; however, the nuclear membranes and the surrounding cytoplasm were strongly stained. ODC-immunoreactive (IR) axons could be traced through the white matter entering the ventral roots. The myelinated axons in the ventral roots and in the nerve bundles among the muscles were intensely stained with ODC antiserum. The myoneural junctions apposing individual muscle fibers showed ODC-LI with slightly less intensity. Some ODC-IR nerve fibers were seen in the muscle spindles. The present results show that motoneurons in adult rat spinal cored express ODC-LI and that OCD-LI is transported to motoric nerves and myoneural junctions. This suggests that polyamines can be synthesized both in the motoneuron somata and in their peripheral projections. Polyamines may thus regulate cellular functions in all parts of motoneurons. In addition, polyamines may be secreted from their distal projections and have tropic effects on Schwann cells and/or muscular tissue.

Protein kinase C beta-subtype-immunoreactive motor and sensory nerves in rat muscle spindle.

The localization of protein kinase C beta-subtype-like immunoreactivity (PKC-beta-LI) was studied in the muscle spindles of rat neck muscles. In the equatorial regions of muscle spindles PKC-beta-LI was detected in spiral sensory nerve endings surrounding intrafusal muscle fibers. In polar regions single PKC-beta-immunoreactive (IR) nerve fibers were found between intrafusal muscle fibers and some PKC-beta-IR motor nerve endings were seen on the surface of intrafusal fibers. These results suggest that PKC-beta may be involved in regulation of muscle contraction and tonus by modulating the sensitivity of afferent and efferent innervation of muscle spindles.

Simultaneous localization of calcitonin gene-related peptide and neurotensin in rat central amygdaloid nucleus.

The distribution of calcitonin gene-related peptide (CGRP) and neurotensin (NT)-like immunoreactivities (LI) was studied in rat central amygdaloid nucleus (ACe) with immunocytochemical double staining. A dense network of CGRP- and NT-immunoreactive (IR) nerve fibers and some NT-positive neurons were found in the lateral and lateral capsular subnuclei. Light microscopically CGRP-immunoreactive nerve endings were in close contact to most of the NT-immunoreactive neurons. Under the electron microscope CGRP-positive terminals formed symmetric axo-somatic synapses with part of the NT-IR neurons. These results indicate that the NT- and CGRP-containing neuronal systems are in contact with each other in the ACe. Both peptides have marked effects on the circulatory system when administered intracerebrally. Thus the NT-IR neuronal system receiving synaptic input from CGRP-IR nerve terminals may mediate the cardiovascular effects of these two peptides.

Immunohistochemical evidence for the presence of protein kinase C beta-subtype in rat motoneurons and myoneural junctions.

The localization of protein kinase C beta-subtype-like immunoreactivity (PKC-beta-LI) was studied in the spinal cord and in different striated muscles of rat. In the spinal cord, large motoneurons in the ventral horn were PKC-beta-immunoreactive (IR). Strong immunoreactivity to PKC-beta was found in large nerve bundles between muscles, and in smaller nerves among muscle fibers. Myoneural junctions, which showed weaker immunoreactivity to PKC-beta, were also demonstrated in all muscles studied; the external ocular muscles, the diaphragma and the triceps surae muscle. Muscle cells were not labelled.

The expression of ornithine decarboxylase antizyme mRNA and protein in rat motoneurons.

The distribution of ornithine decarboxylase antizyme messenger ribonucleic acid (AZ mRNA) and AZ-like immunoreactivity (LI) was studied in the brainstem and spinal cord motoneurons and in the extraocular and triceps surae muscles of rat. In situ hybridization showed AZ mRNA in the gray matter of the spinal cord at different levels of spinal cord with highest AZ mRNA levels in the ventral horn of the spinal cord. No apparent changes in AZ mRNA contents were seen after unilateral transection of the sciatic nerve in lumbar motoneurons. AZ-immunoreactive (IR) motoneurons were observed in the nucleus of the VI cranial nerve and in the ventral horn of the spinal cord. These motoneurons also showed ornithine decarboxylase (ODC)-LI. Subcellularly, AZ-LI was observed both in the nuclei and cytoplasm of labeled motoneurons. Heavily stained AZ-IR nerve fibers and myoneural junctions were observed among muscle fibers in different muscles. In addition, the nuclei of muscle fibers showed AZ-LI.

Localization of neurocalcin-like immunoreactivity in rat cranial motoneurons and spinal cord interneurons.

Neurocalcin (NC) is a calcium-binding protein with at least three putative calcium-binding domains called EF-hands. In this study, the distribution of neurocalcin-like immunoreactivity (LI) was examined in the rat motor system. Motoneurons in the III, IV and VI cranial nerve nuclei were NC-immunoreactive (IR) and strong labelling was seen in the nerve bundles and in the myoneural junctions in all extraocular muscles. In the ventral horn of the spinal cord, interneurons were NC-IR, whereas motoneurons, identified by Fluorogold tracing, were unlabelled. A large number of NC-IR neurons was present in the dorsal horn. NC-IR nerve fibers were seen in the ventral roots and, more abundantly, in the dorsal roots. The present results demonstrate NC-LI in the supraspinal motoneurons and spinal cord interneurons, both of which are fast-firing neurons. Provided neurocalcin regulates the concentration of free intracellular Ca2+, it may participate in several cellular functions in the fast-firing neurons.

Simultaneous demonstration of cholinesterases and glyoxylic acid-induced fluorescence of catecholamines in stretch preparations.

A combined simultaneous method to demonstrate adrenergic nerves using glyoxylic acid-induced fluorescence and nerves showing cholinesterase activity using the thiocholine technique is described in whole-mount preparations. The subcutaneous fascia and the right atrium of the heart of the rat and guinea-pig were used as tissue specimens, and the innervation patterns of adrenergic and cholinergic nerves were demonstrated in UV and transmitted light. Technical points and the limitations of the method are discussed.

Second messenger enzymes in glial cells: a cytochemical point of view.

Knowledge about second messenger metabolizing enzymes in neuroglia is still rather fragmentary. Therefore, the aim of the present investigation was to localize adenylate cyclase, guanylate cyclase, cyclic nucleotide phosphodiesterase and protein kinase A in glial cells of the rat hippocampus and cerebellum. Enzyme histochemical and immunohistochemical methods were used to detect the enzymes at the light and electron microscopic level. Astroglial cells were found to contain all 4 enzymes. Especially the microvascular glial cell processes were reactive. Oligodendroglial cells were only stained for adenylate cyclase acticity. Intracellularly, microtubules and intracellular membranes were frequently stained. The results point to the regulation of glial cell metabolism and of transport processes by cyclic nucleotides.

Reinnervation of human skin grafts: a histochemical study.

The reinnervation of nine human skin grafts was investigated using histochemical thiocholine methods for the demonstration of cholinesterases. The regenerated cutaneous nerves showed both specific acetylcholinesterase and nonspecific cholinesterase reactions. In the youngest specimens, taken 3 weeks after the grafting, such regenerated nerves were seen both at the subdermal level under the graft and at the margins of the graft. These nerves seemed to orient toward the denervated graft area. The growing nerves were generally distributed in a random fashion. The reinnervation of some hair follicles, erector pili muscles, and sweat glands were observed in well-innervated full-thickness and thick partial-thickness skin grafts. It is suggested that this target-organ control of regenerating nerves occurs as a result of the action of chemotactic factors. A well-innervated graft bed seems to be important for optimum reinnervation of skin grafts. Fibrosis and scarring seem to hamper nerve regeneration.