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.

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.

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.

Immunocytochemical localization of glucocorticoid receptor in rat skin.

The aim of the present study was to map immunohistochemically the distribution of the glucocorticoid receptor (GR) in rat skin. Nuclear GR-like immunoreactivity (LI) was found in both epidermis and dermis. In the epidermis, the basal cell layer showed an intense immunoreaction; the lower part of the spinous layer was also labelled. In the dermis, the fibroblasts as well as the sweat glands, sebocytes and adipocytes were GR-immunoreactive (IR). In the root sheath of the hair follicle the staining was most intensive in the outer layer. The endothelial cells comprising the smooth muscle cells of the blood vessels, as well as the arrector pili muscle, showed GR-LI. In the peripheral nerves, the immunoreaction was localized to the nuclei of the Schwann cells and in the perineurial fibroblasts. Mast cells did not show nuclear GR-LI. Based on our immunocytochemical findings that several cell types of the skin are GR-IR, the variable physiological and pharmacological effects of glucocorticoids are easier to understand.

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.

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.

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.

Calbindin D-28k-immunoreactivity in rat muscle spindles during postnatal maturation and after denervation.

Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled. The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.

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.

Colocalization of peptide and glucocorticoid receptor immunoreactivities in rat central amygdaloid nucleus.

The central amygdaloid nucleus (ACe) is part of the amygdaloid complex that participates in adrenocorticotrophin secretion, stress-related reactions and behavioral functions. The ACe contains numerous glucocorticoid receptor (GR)-immunoreactive (IR) neurons, and in addition it has been shown to contain several neuropeptide-IR somata and nerve terminals. In order to study the relationship between the GR- and neuropeptide-IR structures we mapped the distribution of GR-like immunoreactivity (LI) in amygdaloid complex and colocalized the neuropeptide- and GR-LIs in the ACe. In the amygdaloid complex the central, medial and cortical nuclei contained a high number of GR-IR neurons, whereas a moderate number of GR-IR neurons were observed in the basolateral and basomedial nuclei. Only a few GR-IR neurons were seen in the lateral nucleus. In the ACe, the majority of corticotrophin-releasing factor (CRF)-, met-enkephalin (met-ENK)-, neurotensin (NT)- and somatostatin (SOM)-IR neurons contained also GR-IR. About half of the substance P (SP)-IR neurons were seen to contain GR-IR, whereas only some of the few vasoactive intestinal polypeptide and cholecystokinin-IR neurons showed GR-LI. Nerve terminals containing calcitonin gene-related peptide and the above mentioned peptides were seen in close contact with the GR-IR neurons. These results suggest that the glucocorticoids may modulate directly the neurotransmitter synthesis of the CRF-, met-ENK, NT-, SOM- and SP-IR cells in the ACe.

Histochemistry of guanylate cyclase, phosphodiesterase, and NADPH-diaphorase (nitric oxide synthase) in rat brain vasculature.

The distribution of guanylate cyclase, phosphodiesterase, and NADPH-diaphorase [nitric oxide (NO) synthase] was studied in rat brain both at the light and electron microscopic level with special emphasis on the vascular system. We showed that the cGMP-generating enzyme is located in cells (glial cells and pericytes) surrounding cerebral vessels, but not in the endothelium. For NO synthase, a dual localization was observed. The enzyme is present in parts of the endothelium and in nerve endings apparently innervating larger brain vessels. We propose, therefore, that NO acts on guanylate cyclase both from a "synaptic" and endothelial source.

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.

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.

Neuropeptides in cutaneous neurofibromas of von Recklinghausen's disease.

The occurrence of neuropeptides was studied in neurofibromas of von Recklinghausen's disease by indirect immunofluorescence. All non-plexiform cutaneous neurofibromas contained abundant vasoactive intestinal polypeptide, peptide histidine-isoleucine and calcitonin gene-related peptide immunoreactive nerves. The nerves were small and unmyelinated. Neuropeptides might be responsible for itch that occurs especially in small cutaneous neurofibromas. Neuropeptides are also suggested to act as modulators and/or trophic factors for neurofibroma growth.

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.

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.

Immunocytochemical study of the relations of acetylcholinesterase, enkephalin-, substance P-, choline acetyltransferase- and calcitonin gene-related peptide-immunoreactive structures in the ventral horn of rat spinal cord.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity was localized immunocytochemically in the large motoneurons in the ventral horn of rat spinal cord. Using fluorescence double-labelling substance P (SP)-immunoreactive nerve fibres were found to surround both the CGRP-positive and negative motoneurons, whereas enkephalin (ENK)-immunoreactive fibres surrounded mainly CGRP-negative cells. All CGRP-like immunoreactive motoneurons were also choline acetyltransferase (ChAT)- and acetylcholinesterase (AChE)-positive. On the other hand a large population of ChAT- and AChE-positive motoneurons were devoid of CGRP-immunoreactivity. It is probable that CGRP/ChAT/AChE-positive cells surrounded by SP-positive fibres have different functions in motoric nervous system than the CGRP-negative ChAT/AChE-positive cells, which are surrounded by ENK-immunoreactive fibres.