Dynamics and plasticity of peptidergic control centres in the retino-brain-pituitary system of Xenopus laevis.

This review deals particularly with the recent literature on the structural and functional aspects of the retino-brain-pituitary system that controls the physiological process of background adaptation in the aquatic toad Xenopus laevis. Taking together the large amount of multidisciplinary data, a consistent picture emerges of a highly plastic system that efficiently responds to changes in the environmental light condition by releasing POMC-derived peptides, such as the peptide alpha-melanophore-stimulating hormone (alpha-MSH), into the circulation. This plasticity is exhibited by both the central nervous system and the pituitary pars intermedia, at the level of molecules, subcellular structures, synapses, and cells. Signal transduction in the pars intermedia of the pituitary gland of Xenopus laevis appears to be a complex event, involving various environmental factors (e.g., light and temperature) that act via distinct brain centres and neuronal messengers converging on the melanotrope cells. In the melanotropes, these messages are translated by specific receptors and second messenger systems, in particular via Ca(2+) oscillations, controlling main secretory events such as gene transcription, POMC-precursor translation and processing, posttranslational peptide modifications, and release of a bouquet of POMC-derived peptides. In conclusion, the Xenopus hypothalamo-hypophyseal system involved in background adaptation reveals how neuronal plasticity at the molecular, cellular and organismal levels, enable an organism to respond adequately to the continuously changing environmental factors demanding physiological adaptation.

Transient prenatal expression of NPY-Y1 receptor in trigeminal axons innervating the mystacial vibrissae.

Using immunohistochemistry in combination with confocal laser scanning microscopy, we studied the ontogeny of neuropeptide Y-Y1 receptor (Y1-R) expression in the trigeminal system of the rat. The study was limited to the nerve fibers innervating the mystacial pad and the trigeminal ganglia. In the trigeminal ganglia, Y1-R-immunoreactive (IR) neurons were first observed at E16.5. At this same stage some nerve fibers in the trigeminal ganglia also exhibited Y1-R-like immunoreactivity (LI). Strongly Y1-R-IR nerve fibers innervating the follicles of the mystacial vibrissae were first observed at E18. After double labeling, the Y1-R-LI was found to be colocalized with the neuronal marker protein gene product 9.5. At P1 only weak labeling for the Y1-R was found around the vibrissae follicles, whereas the neurons in the trigeminal ganglia were intensely labeled. The same was true for the adult rat, but at this stage no Y1-R labeling at all was observed in nerve fibers around the vibrissal follicles. These results strongly support an axonal localization of the Y1-R at this developmental stage. The transient expression of the Y1-R during prenatal mystacial pad development suggests a role for the Y1-R in the functional development of the vibrissae.

Excitatory amino acids, monoamine, and nitric oxide synthase systems in organotypic cultures: biochemical and immunohistochemical analysis.

The nigrostriatal and mesolimbic systems of the rat have been re-constructed using the organotypic culture model, whereby neonatal brain tissue is grown in vitro for approximately one month. The nigrostriatal cultures consisted of tissue from the substantia nigra, dorsal striatum and frontoparietal cortex; while the mesolimbic cultures included the ventral tegmental area, ventral striatum and cingulate cortex. The cultures were grown at 35 degrees C in normal atmosphere, using a tuberoller device placed in a cell incubator and changing the medium every 3-4 days. The in vitro development was evaluated with an inverted microscope equipped with a variable relief contrast function. Samples were taken directly from the medium in the culture tube and analysed for several amino acids with HPLC. After a month the cultures were fixed and processed for immunohistochemistry. High levels of glutamate and aspartate were observed every time the medium was changed, but the levels rapidly decreased reaching a steady state after approximately 24h. A decrease in the levels was also observed along development, reaching stable values (approximately 2 microM and approximately 0.12 microM for glutamate and aspartate, respectively) at approximately two weeks, but only when the cultures showed an apparently healthy development. The levels were approximately 10 times higher in deteriorating or apparently damaged cultures. Glutamine levels were in the mM range and remained stable along the entire experiment. No differences were observed among nigrostriatal and mesolimbic cultures. Immunohistochemistry confirmed the impressions obtained from microscopic and biochemical analysis along the in vitro development, revealing apparently healthy neuronal systems with characteristics similar to those observed in vivo, when tyrosine hydroxylase and nitric oxide synthase, markers for dopamine and nitric oxide containing neurons, respectively, were analysed. In the substantia nigra, nitric oxide synthase-positive networks surrounded tyrosine hydroxylase-positive neurons, while in the striatum nitric oxide synthase dendrites were surrounded by tyrosine hydroxylase-positive nerve terminals, suggesting a reciprocal interaction among dopamine and nitric oxide containing neurons. Thus, the organotypic model appears to capture many of the neurochemical and morphological features seen in vivo, providing a valuable model for studying in detail the neurocircuitries of the brain.

Neuropeptide Y expression in Schwann cell precursors.

Some years ago we showed that in the adult rat and mouse neuropeptide Y (NPY) is expressed by olfactory ensheathing cells, a special type of glial cell involved in guiding of the continuously renewing olfactory axons. In the present study, using immunohistochemistry combined with confocal laser scanning microscopy, and mRNA in situ hybridization, we have analyzed whether NPY is also expressed in other axon-related glial cell systems during prenatal development. NPY was found to be expressed in the dorsal and ventral rootlets along the spinal cord from E13 and onward and in the rootlets of the cranial nerves and in the sensory ganglia from E14 and onward. In some cases, NPY-immunoreactivity (IR) was also found along peripheral nerves. NPY-IR was expressed in a dot-like fashion, similar to the NPY expression observed in olfactory ensheathing cells. At E18 the NPY-immunoreactive dots had disappeared from almost all ganglia and rootlets, and only in the most central part of the rootlets some weak dot-like NPY-IR was observed. At E20 it had disappeared completely from all rootlets and nerves, except the olfactory nerve. Most of the dot-like NPY-IR did not co-localize with the neuronal marker PGP 9.5. Based on its spatiotemporal expression, it is concluded that NPY is expressed by Schwann cell precursors. NPY expressed by Schwann cell precursors might have a role in axonal growth or axonal guidance, or both.

Expression of neuropeptide Y in olfactory ensheathing cells during prenatal development.

Neuropeptide Y (NPY) is expressed in a special type of glial cell, the olfactory ensheathing cells, that surround the axons of olfactory sensory neurons on their way from the olfactory epithelium to the glomeruli in the olfactory bulb. The expression of NPY in ensheathing cells was examined during prenatal development of the olfactory system by using immunohistochemistry and in situ hybridization. NPY expression was compared with the expression of growth associated protein-43, olfactory marker protein, the low-affinity nerve growth factor receptor (p75) and S-100, factors expressed in the olfactory system at known stages of development. NPY-like immunoreactivity (NPY-LI) and NPY mRNA expression was first detected in the olfactory nerve layer of the olfactory bulb at embryonic day 15. From embryonic day 16 and onward, a clear segregation could be observed in the intensity of both NPY-LI and NPY mRNA expression within the olfactory nerve layer. NPY expression was most intense in the inner part of the olfactory nerve layer. In the outer olfactory nerve layer, a clear decrease in NPY expression was observed. The inner olfactory nerve layer, showing high NPY expression, did not stain for S-100 or p75. However, NPY-LI was found to coexist with S-100-LI from the outer olfactory nerve layer until the olfactory epithelium and with p75-LI in cells surrounding the olfactory nerve. These results show that NPY is expressed in ensheathing cells before olfactory sensory neurons mature and the formation of the glomerular layer starts. NPY might be involved in the guidance, growth, or both, of olfactory sensory axons toward their target glomeruli in the olfactory bulb or have a function in the maturation of the olfactory sensory neurons.

Neuropeptides--an overview.

The present article provides a brief overview of various aspects on neuropeptides, emphasizing their multitude and their wide distribution in both the peripheral and central nervous system. Interestingly, neuropeptides are also expressed in various types of glial cells under normal and experimental conditions. The recent identification of, often multiple, receptor subtypes for each peptide, as well as the development of peptide antagonists, have provided an experimental framework to explore functional roles of neuropeptides. A characteristic of neuropeptides is the plasticity in their expression, reflecting the fact that release has to be compensated by de novo synthesis at the cell body level. In several systems peptides can be expressed at very low levels normally but are upregulated in response to, for example, nerve injury. The fact that neuropeptides virtually always coexist with one or more classic transmitters suggests that they are involved in modulatory processes and probably in many other types of functions, for example exerting trophic effects. Recent studies employing transgene technology have provided some information on their functional role, although compensatory mechanisms in all probability could disguise even a well defined action. It has been recognized that both 'old' and newly discovered peptides may be involved in the regulation of food intake. Recently the first disease-related mutation in a peptidergic system has been identified, and clinical efficacy of a substance P antagonist for treatment of depression has been reported. Taken together it seems that peptides may play a role particularly when the nervous system is stressed, challenged or afflicted by disease, and that peptidergic systems may, therefore, be targets for novel therapeutic strategies.

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

Neurocircuitries of the basal ganglia studied in organotypic cultures: focus on tyrosine hydroxylase, nitric oxide synthase and neuropeptide immunocytochemistry.

The nigrostriatal and mesolimbic systems of the rat were reconstructed using an organotypic culture model, whereby neonatal brain tissue was grown in vitro for approximately one month. The nigrostriatal system comprised of tissue from the substantia nigra, the dorsal striatum and the frontoparietal cortex, while the mesolimbic system included the ventral tegmental area, ventral striatum (including the fundus striati, accumbens nucleus, olfactory tubercle, lateral septum, ventral pallidum and piriform cortex) and cingulate cortex. These regions were also cultured alone or in pairs. The cultures were monitored in vitro, and after one month fixed in a formalin-picric acid solution, and processed for immunohistochemistry using antibodies raised against tyrosine hydroxylase, nitric oxide synthase, preprocholecystokinin, glutamate decarboxylase, neuropeptide Y, dopamine- and cyclic AMP-regulated phosphoprotein-32 and glial fibrillary acidic protein. The tissue survived in single, double or triple cultures, although differences were found depending upon the source and combination of cultured region. Neurons had localization and shape as in vivo. Local networks were especially prominent in the mesencephalon, where both tyrosine hydroxylase-positive axons spread from the "substantia nigra" to the rest of the tissue, and where nitric oxide synthase-positive networks also surrounded tyrosine hydroxylase-positive neurons. Glutamate decarboxylase-positive nerve terminals formed dense networks around tyrosine hydroxylase-positive neurons. In the striatum, nitric oxide synthase and dopamine- and cyclic AMP-regulated phosphoprotein-32 neurons were surrounded by tyrosine hydroxylase-positive nerve terminals. The nigral and ventral tegmental area dopamine neurons projected to striatal and cortical structures, but the projection from the ventral tegmental area to the cingulate cortex was more prominent. With regard to co-existence, preprochole-cystokinin-like immunoreactivities was found in many tyrosine hydroxylase-positive neurons and neuropeptide Y- and nitric oxide synthase-like immunoreactivity co-existed in striatal and cortical tissues. In general terms, the chemical neuroanatomy in the cultures was similar to that described earlier in vivo. Nitric oxide synthase staining was particularly intense. Taken together, the organotypic model captures many of the morphological and neurochemical features seen in vivo, providing a valuable model for studying neurocircuitries of the brain in detail, where 'normal' and 'pathological' conditions can be simulated.

Identification of suprachiasmatic melanotrope-inhibiting neurons in Xenopus laevis: a confocal laser-scanning microscopy study.

The amphibian Xenopus laevis is able to adjust its skin color to the light intensity of the environment. Paling of the skin is achieved by inhibiting the release of alpha-melanophore-stimulating hormone (alpha-MSH) from the melanotrope cells in the pars intermedia of the pituitary gland. The release of alpha-MSH is inhibited by gamma-aminobutyric acid (GABA), neuropeptide Y (NPY), and dopamine (DA). To locate and identify neurons that might be responsible for the inhibitory input, double and triple immunocytochemistry, retrograde tracing from the pars intermedia with the carbocyanine membrane probe 1,1'dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzene-sulfonate (Fast DiI), and confocal laser-scanning microscopy were combined. Glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and NPY were found to coexist in an axonal network innervating the pars intermedia. The suprachiasmatic nucleus (SC) contained different populations of neurons that were single, double, or triple labelled for GAD, NPY, and TH. In the lateral SC, NPY+ neurons were observed. TH-immunoreactive (TH-IR) neurons occurred in the medial, dorsolateral, lateral, and ventrolateral SC. Neurons that were double labelled for NPY and TH and triple labelled for Fast DiI, NPY, and TH were present in the ventrolateral SC. This same area contained neurons that were triple labelled for GAD, NPY, and TH. It is concluded that the triple-labelled and probably the double-labelled ventrolateral SC neurons (suprachiasmatic melanotrope-inhibiting neurons) innervate the pituitary pars intermedia and are responsible for the NPY-, DA-, and GABA-mediated inhibition of melanotrope cell activity in Xenopus laevis.

Forebrain differentiation and axonogenesis in amphibians: I. Differentiation of the suprachiasmatic nucleus in relation to background adaptation behavior.

Functional forebrain development is the result of a complex series of early developmental processes which include cell division, cellular rearrangements, tissue-tissue interactions, cellular determinative and differentiation events, and axonogenesis. In these studies, Xenopus laevis embryos were examined for early forebrain neuronal determination, differentiation and axonogenesis with special emphasis on the hypothalamic area known to be involved in regulating pars intermedia function. Whole brain acetylcholine esterase (AChE) histochemistry was used to follow the early pattern of forebrain neuronal differentiation, and whole brain acetylated-tubulin immunocytochemistry was done to follow early forebrain axonogenesis. AChE histochemistry indicated that the source of the tract of the postoptic commissure (stpoc) was the first forebrain area to begin differentiation (stage 22). Whole brain immunocytochemistry for acetylated-tubulin indicated that the tpoc is also the first forebrain tract to develop (at stage 25/26). The main forebrain tracts have developed and become interconnected by stage 35/36. The forebrain undergoes a pronounced extension, with much cellular mixing and rearrangement during stages 37/38 to 43/44. This results in bending and contortions in the already developed tracts. Whole brain immunocytochemistry for tyrosine hydroxylase and extirpation of the stage 14 presumptive suprachiasmatic (SC) area indicated that the dopaminergic cells of the SC are determined by stage 14 and initially undergo differentiation between stages 37/38 and 40. Tadpoles with stage 14 presumptive SC extirpated lacked TH-positive tracts to the pars intermedia, lacked most midline TH-positive forebrain cells, and also failed to background adapt to white background. Thus, the SC tracts to the pars intermedia that inhibit melanotrope secretion probably form during the extension stages of 37/38 and contact the pars intermedia by stage 40 when animals are first capable of background adaptation.

Distribution of pro-opiomelanocortin and its peptide end products in the brain and hypophysis of the aquatic toad, Xenopus laevis.

Using in situ hybridization with a pro-opiomelanocortin (POMC)-mRNA probe and immunocytochemistry with antisera to POMC and to various POMC-derived peptides, it is shown that melanotrope cells in the pars intermedia of the hypophysis of the South African aquatic toad Xenopus laevis contain POMC, alpha-melanophore-stimulating hormone (alpha-MSH), gamma-MSH, acetylated and non-acetylated endorphins and adrenocorticotropic hormone (ACTH). With the exception of gamma-MSH, these peptides are also found in the corticotrope cells in the rostral pars distalis. In the Xenopus brain, neuronal cell bodies in the ventral hypothalamic nucleus express POMC, alpha-MSH, gamma-MSH, non-acetylated endorphins and ACTH, neurones in the anterior preoptic area reveal POMC, alpha-MSH, gamma-MSH and non-acetylated endorphin, neurones in the suprachiasmatic nucleus contain alpha-MSH, non-acetylated endorphin and ACTH and neurones in the posterior tubercle show alpha-MSH, non-acetylated endorphin and ACTH immunoreactivities. In the locus coeruleus POMC and ACTH coexist, whereas alpha-MSH and non-acetylated endorphin occur together in the nucleus accumbens, the striatum and the nucleus of the paraventricular organ. Finally, alpha-MSH alone is present in the olfactory bulb, the medial septum, the medial and lateral parts of the amygdala, the ventromedial and posterior thalamic nuclei, the optic tectum and the anteroventral tegmental nucleus, and non-acetylated endorphin alone appears in the epiphysis. It is suggested that neurones that form POMC-derived peptides may play a direct or indirect role in the control of POMC-producing hypophyseal cells and/or in the physiological processes these endocrine cells regulate. This idea is supported by the fact that the suprachiasmatic nucleus and the locus coeruleus, both involved in melanotrope cell control, show POMC and POMC-peptide expression. A possible involvement in melanotrope and/or corticotrope control of the anterior preoptic and ventral hypothalamic nuclei, which both express POMC and various POMC-derived peptides, deserves future attention.

Physiologically induced Fos expression in the hypothalamo-hypophyseal system of Xenopus laevis.

The amphibian Xenopus laevis adjusts the color of its skin to the degree of background illumination. The neuroendocrine mechanism responsible for this adaptation behavior involves various brain centers that control the synthesis and release of alpha-melanophore-stimulating hormone (alpha-MSH) by the pituitary melanotrope cells. The aim of the present study was to investigate the possible use of Fos as a tool to determine the activity of known and novel components of this mechanism. For this purpose, a quantitative Fos-immunocytochemical method (ABC) was successfully introduced for Xenopus, and the degree of specificity of background illumination as a regulator of Fos expression was tested by comparing this stimulus with two other stimuli, viz. a strong stressor (saline immersion) and a mild stressor ('handling'). Without stimulation basal Fos-like immunoreactivity (Fos-LI) was found in the telencephalon, the lateral pallium, the anterior, central and lateral thalamic nuclei, the suprachiasmatic nucleus, the ventral hypothalamic nucleus and the torus semicircularis. Handling had no effect on this basal pattern of Fos-LI. Saline immersion induced Fos-LI only in the magnocellular preoptic nucleus, the corticotrope cells and, less strongly, in the melanotrope cells. Melanotropes, and no other cells, expressed Fos-LI very strongly when Xenopus was transferred from a white to a black background. This Fos-LI expression continued to increase up to 7 days of stimulation. When such toads were returned to a white background it took the same time before Fos-LI expression significantly dropped. It is concluded that the ABC-Fos immunocytochemistry can be successfully applied to assess the occurrence and degree of expression of Fos-LI in the Xenopus brain and pituitary gland. The prolonged expression of Fos-LI in the pars intermedia under black background stimulation and the presence of an AP-1 binding site on the Xenopus proopiomelanocortin gene suggest an important role for c-Fos and/or Fos-related antigens in the control of the biosynthesis and secretion of alpha-MSH by the Xenopus melanotrope cell.

Nitric oxide synthase and background adaptation in Xenopus laevis.

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by alpha-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). alpha-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain. In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background. Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3',5' guanosine monophosphate (cGMP)-accumulating fibers in the PI. The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.

Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy.

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [125I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I-IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae I-II. [125I]PYY binding was most strongly attenuated by NPY13-36, a Y2 agonist, and partially inhibited by [Leu31,Pro34]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY13-36 completely abolished PYY binding, which was also reduced by [Leu31,Pro34] NPY. However, the increase in PYY binding seen in laminae I-IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu31,Pro34] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I-IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous immunoenzymatic staining of catecholamines, catecholamine-biosynthesizing enzymes, and bromodeoxyuridine in adrenal medullary cells of the rat.

The rat adrenal medulla consists mainly of low proliferating, highly differentiated parenchymal cells. By immunocytochemical techniques, two types of parenchymal cells can be identified, norepinephrine (NE)- and epinephrine (E)-storing cells. Bromodeoxyuridine (BrdU), a thymidine analogue often used to identify proliferating cells, can also be detected by immunocytochemical techniques. We developed double- and triple-labeling procedure(s) for simultaneous visualization of NE, E, dopamine beta-hydroxylase (DBH), phenylethanolamine-N methyltransferase (PNMT), and BrdU in rat adrenal medulla. BrdU was administered to 7-week-old Wistar rats by mini-osmotic pumps. Tissues were fixed by perfusion with 4% paraformaldehyde and embedded in paraffin. By immunocytochemistry, first NE, E, DBH, and/or PNMT was detected by an indirect immunoalkaline phosphatase technique with Fast Red or Fast Blue as substrate. Next, incorporation of BrdU was detected with an indirect immunoperoxidase procedure using diaminobenzidine (DAB). Both NE- and E-storing cells, as well as endothelial cells, can incorporate BrdU, i.e., are able to divide. Occasionally, we also found BrdU-stained mitotic figures in E, PNMT and DBH immunoreactive cells. No BrdU incorporation was found in the post-ganglionic neurons of the adrenal medulla. The procedures described enable a detailed cell kinetic study of the NE- and E-storing cells in the adrenal medulla, particularly in the rat, which can lead to a better understanding of cell renewal in the adrenal medullary tissue under normal and pathological conditions.

Immunologically induced sympathectomy of preganglionic nerves by antibodies against acetylcholinesterase: increased levels of peptides and their messenger RNAs in rat adrenal chromaffin cells.

Systemic administration of murine monoclonal acetylcholinesterase antibodies to rats has been shown to cause selective degeneration of sympathetic preganglionic neurons. In the present study rats were subjected to a single i.v. injection of these acetylcholinesterase antibodies, or to normal IgG or saline for control. Exophthalmos, piloerection and eyelid-drooping (ptosis) were observed within 1 h after administration of the antibodies. Rats were killed at different time-points after antibody administration, and the adrenal glands were analysed by means of indirect immunohistochemistry and in situ hybridization histochemistry. As soon as 3 h after the antibody treatment, a marked increase in the number of chromaffin cells expressing mRNA encoding, respectively, enkephalin, calcitonin gene-related peptide, galanin, neurotensin and substance P was seen. At 12 h the peptide mRNA levels were still elevated and there was a concomitant increase in the number of peptide-immunoreactive cells. All peptide levels remained high for at least 48 h; however, 77 days after the antibody treatment only enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-positive fibers was already seen 3 h after the antibody treatment, and after 24 h no fibers were encountered. In contrast, up until 48 h there was no apparent change in the number or intensity of immunofluorescent fibers expressing calcitonin gene-related peptide, galanin, neurotensin or substance P. However, 77 days after the antibody treatment the number of calcitonin gene-related peptide- and substance P-immunoreactive fibers was increased as compared to controls. In addition, reappearance of acetylcholinesterase- and enkephalin-immunoreactive fibers was seen 77 days after antibody administration, although their number was still low as compared to controls. Double-labeling immunohistochemistry revealed that the chromaffin cells expressing peptides after the antibody treatment preferentially were adrenaline storing cells (noradrenaline-negative). The majority of these cells expressed only one peptide. Both surgical transection of the splanchnic nerve as well as treatment with acetylcholine receptor antagonists mimicked the effects seen after the acetylcholinesterase-antibody treatment, although changes were less pronounced. The present results show that interruption of splanchnic transmission induces fast, marked, and selective increases in peptide expression in rat adrenal chromaffin cells.

Neuropeptide tyrosine is expressed in ensheathing cells around the olfactory nerves in the rat olfactory bulb.

The olfactory bulbs of young and adult normal rats and of colchicine-treated rats and of some other species were analysed for the presence of neuropeptide Y and neuropeptide Y messenger RNA, using immunohistochemistry at the light- and electron-microscopic levels and with in situ hybridization. In the rat and mouse, but not in monkey and guinea-pig, neuropeptide Y-like immunoreactivity and neuropeptide Y messenger RNA were observed in ensheathing cells in the olfactory nerve layer of the olfactory bulb and within nerve bundles in the olfactory mucosa. Double staining experiments revealed that neuropeptide Y-like immunoreactivity was often present in a restricted compartment, mainly the Golgi apparatus, of S-100 protein-positive ensheathing cells. After colchicine treatment a different distribution of neuropeptide Y-like immunoreactivity and neuropeptide Y messenger RNA was observed. Thus, in the outer olfactory nerve layer both neuropeptide Y-like immunoreactivity and neuropeptide Y messenger RNA disappeared, whereas in the inner part messenger RNA levels remained high and neuropeptide Y-like immunoreactivity was observed in many granule-like structures distributed diffusely in the cytoplasm. The present findings suggest that neuropeptide Y may be involved in the control of regeneration, growth and/or guiding of the axons of the olfactory sensory neurons, the only mammalian neurons known to have a continuous renewal and growth during adult life.