Deletion of the neuropeptide Y Y1 receptor affects pain sensitivity, neuropeptide transport and expression, and dorsal root ganglion neuron numbers.

Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity.

Nicotine exposure in breastfed infants.

AIM: To study exposure to nicotine in breastfed infants in relation to parental smoking habits. MATERIAL AND METHODS: Forty mother-infant pairs were studied. Twenty non-smoking mothers, 18 smoking (2-20 cigarettes per day) and two snuff-taking mothers were included. All infants were healthy, exclusively breastfed and their postnatal age was 6 wk. During a home visit, parental smoking habits were recorded, and the time of mothers' last smoke or taking of snuff and breastfeeding were recorded. Breast milk and infant urine samples were collected. Concentrations of nicotine and cotinine were analysed with gas chromatography. The amount of milk ingested during the home visit was calculated by weighing the infants. RESULTS: Two non-smoking and non-snuff-taking women had milk containing nicotine (28 and 13 microg/l, respectively). Both had smoking spouses. In the smoking and snuff-taking group, the mean (SD) milk nicotine concentration was 44 (38) microg/l (n = 36). When milk samples taken 7 h and 0.6 h after smoking were compared, the concentration of milk nicotine increased from 21 to 51 microg/l (p < 0.01). The two snuff-taking mothers exposed their children to higher milk nicotine concentrations than all but two of the smokers. The concentrations of the metabolite cotinine in infant urine correlated with the dose of nicotine ingested during the home visit (r = 0.84, p < 0.01). CONCLUSIONS: Breastfed infants with a smoking or snuff-taking mother are exposed to nicotine in breast milk. The mean intake of nicotine via milk is 7 microg/kg/d. With a shorter time between the mothers' smoking and breastfeeding, the milk nicotine concentration will increase. Both passive smoking at home and snuff-taking were associated with measurable nicotine levels in milk. Healthcare personnel promoting breastfeeding should be aware of these factors influencing exposure to nicotine in the breastfed infant.

Zinc transporter 3 and zinc ions in the rodent superior cervical ganglion neurons.

Previous studies have revealed that zinc-enriched (ZEN) terminals are present in all parts of the CNS though with great differences in intensity. The densest populations of both ZEN terminals and ZEN somata are found in telencephalic structures, but also structures like the spinal cord demonstrate impressive ZEN systems spreading terminals several segments around the respective ZEN somata. The present study evaluates whether sympathetic neurons in the superior cervical ganglia (SCG) are ZEN neurons, i.e. contain vesicles that have zinc transporter 3 (ZnT3) proteins in their membranes and contain zinc ions. ZnT3 immunoreactivity (IR) was found in the somata and processes in the postganglionic neurons of mouse SCG. Only a small fraction of neurons (less than 5%), expressed varying degrees of ZnT3. Colchicine treatment, however, increased the number of ZnT3-positive neurons three-fold, suggesting an accumulation of ZnT3 protein in the somata. A small proportion of the postganglionic axons revealed dotted accumulations of ZnT3 IR along their courses. Double labeling showed that all ZnT3-positive neurons and axons were also tyrosine hydroxylase-positive with strong immunofluorescence, while no colocalization was found between ZnT3 and the vesicular acetylcholine transporter (VAChT) or neuropeptide Y IR. VAChT-positive preganglionic neurons were found to terminate on ZnT3 neuronal somata. 6-Methoxy 8-para toluene sulfonamide quinoline fluorescence and zinc selenium autometallography (ZnSe(AMG)) revealed that a subgroup of SCG cells contained free or loosely bound zinc ions. It is therefore concluded that ZnT3 and zinc ions are present in a subpopulation of TH-positive, NPY-negative neurons in the rodent SCG, supporting the notion that vesicular zinc ions may play a special role in the peripheral sympathetic adrenergic system.

Differential localization of alpha-, beta- and gamma-synucleins in the rat CNS.

Alpha-synuclein is a presynaptic protein that normally participates in the homeostasis of synaptic vesicles. Missense mutations in its gene cause the protein to participate actively in the development of heritable forms of Parkinson's disease. Moreover, its metabolism is perturbed in all cases of Parkinson's disease where alpha-synuclein accumulates in a filamentous form in the Lewy body nerve cell lesion. Lewy bodies also develop in other common neurodegenerative disorders, like dementia with Lewy bodies and Lewy body variant of Alzheimer's disease. In the present study, we have studied the detailed distribution of alpha-, beta- and gamma-synuclein in the rat CNS. Alpha-synuclein was not observed in perikarya, but was distributed with high intensity in nerve terminals in the caudate and putamen and ventral pallidum, where beta-synuclein was much weaker and less densely distributed in the caudate and putamen. Gamma-synuclein was not found in the caudate and putamen. Alpha-synuclein was robustly distributed in the substantia nigra pars reticulata, but was very weak or virtually absent from the perikarya of the neurons in the pars compacta. In contrast, beta-synuclein was very weak or absent from the substantia nigra. gamma-Synuclein was absent from the terminals of substantia nigra pars reticulata, but sparsely distributed gamma-synuclein-containing neurons were detected in the substantia nigra pars compacta. In the brainstem, alpha-synuclein as well as gamma-synuclein were present in the locus coeruleus with high intensity, while beta-synuclein was very weak. In addition, alpha-synuclein was intense in the vagus nucleus, but weak in the oculomotor, facial, hypoglossal, accessory and ambiguous nuclei, where beta-synuclein was very intensely present. Furthermore, gamma-synuclein was localized in the terminals and in cell bodies of the Edinger-Westphal nucleus, the red nucleus, locus coeruleus, and most cranial nerve-related nuclei. In the spinal cord, alpha- and gamma-synucleins were intensely present in laminae I and II and in the preganglionic sympathetic nuclei, whereas beta-synuclein was very weak. These results indicate that alpha-synuclein is abundant in central catecholaminergic regions. Beta-synuclein is more localized in the somatic cholinergic components, while it is particularly weak or absent from catecholaminergic neurons. Gamma-synuclein appears to be present in both cholinergic and catecholaminergic regions, but very weak in the forebrain.

Distribution and intraneuronal trafficking of a novel member of the chromogranin family, NESP55, in the rat peripheral nervous system.

NESP55 (neuroendocrine secretory protein of M(r) 55000) is a novel member of the chromogranin family. In the present study, we have investigated the distribution, axonal transport and proteolytic processing of NESP55 in the peripheral nervous system. The amount of NESP55 immunoreactivity in adrenal gland was more than 240 times higher than that in the vas deferens. Double or triple immunostaining demonstrated that NESP55 immunoreactivity was highly co-localized with tyrosine hydroxylase immunoreactivity in bundles of thin axons and postganglionic sympathetic neurons; that NESP55 immunoreactivity also co-existed with vesicular acetylcholine transporter immunoreactivity in large-sized axons in sciatic nerves, and that NESP55 immunoreactivity overlapped with calcitonin gene-related peptide immunoreactivity in some large-sized axons, but NESP55 immunoreactivity was not detected in sensory neurons. Strong NESP55 immunoreactivity was found in cell bodies and axons, but it was not detectable in any terminal region by immunohistochemistry. In crush-operated sciatic nerves, NESP55 immunoreactivity could be found as early as 1 h after operation, and accumulated amounts increased substantially with time. However, NESP55 immunoreactivity was only observed in axons proximal to the crush, but none or very little distal to the crush, which was consistent with the data from radioimmunoassay. Finally, extracts of the normal and crushed sciatic nerve and vas deferens were subjected to high-performance liquid chromatography followed by radioimmunoassay. The results indicate that NESP55 is processed slowly to small peptides (GAIPIRRH) during axonal transport. NESP55 immunoreactivity was only detected in axons proximal to the crush. The data in the present study indicate that NESP55 immunoreactivity is widely distributed in adrenergic, cholinergic, and peptidergic neurons, but not in sensory neurons, and that this peptide is anterogradely, but not retrogradely, transported with fast axonal transport and slowly processed to smaller peptides during axonal transport in the peripheral nervous system.

Zinc-enriched GABAergic terminals in mouse spinal cord.

Electrophysiological experiments have shown that zinc ions modulate glutamate and GABA receptors in brain slices. All the zinc-enriched neuronal pathways in the brain analyzed up until now have been found to be glutaminergic. Many years ago, zinc-enriched terminals with flat vesicles and symmetric synapses were found to be present in rat spinal cord by Henrik Daa Schrøder, and recently these findings have been supported by immunohistochemical and electron microscopical data in lamprey, mouse and rat. In the present study we expanded these observations by revealing a colocalization of zinc ions, zinc transporter-3 (ZnT3) and glutamic acid decarboxylase (GAD) in synaptic vesicles of zinc-enriched terminals throughout the mouse spinal cord. Confocal analysis of ZnT3 and GAD immunofluorescence was used at light microscopical levels, and a combination of zinc selenium autometallography and GAD immunocytochemistry at electron microscopic levels. Zinc-enriched/GABAergic terminals were observed in all laminae of the spinal gray matter, but most densely populated were laminae I and III in the dorsal horn. In the lateral and ventral funiculi of the white matter, rows of inhibitory zinc-enriched boutons were seen radiating from the gray matter. Ultrastructurally, colocalization of zinc ions and GAD immunoreactivity was seen in a pool of presynaptic terminals in the above locations. Some zinc-enriched terminals were not GAD-positive and some GAD-positive terminals were void of zinc ions. The majority of the zinc-enriched, not GABAergic terminals could be classified as excitatory based on their morphology, i.e. round clear vesicles and symmetric synapses. We conclude that a majority of the spinal cord zinc-enriched terminals are GABAergic. The zinc-enriched terminals with excitatory morphology are most likely glutaminergic, a few have an inhibitory morphology but are not GABAergic. These are most likely glycinergic.

Presence of sst2(a) receptor immunoreactivity in rat ependyma and tanycytes.

Somatostatin sst2(a) receptor was observed, by immunofluorescence, in ependymal cells and in tanycytes of the wall of the ventricle and the hypothalamic recess of the male rat median eminence. Strong immunoreactivity for the receptor protein was observed in lateral tanycytes (alpha-type) while a moderate signal was seen in medial tanycytes (beta-type). In high magnification the immunoreactive material, of moderate intensity, had a coarse granular appearance. Only few of the alpha-tanycytes also displayed immunoreactive GFAP. The apical portion of the ependymal cells as well as of tanycytes contained immunoreactive S-100 (alphabeta). Since rather high levels of somatostatin are demonstrated to occur in the cerebrospinal fluid of the third ventricle, it is suggested that somatostatin via the sst2(a) receptor may regulate the physiology of tanycytes.

Is the postganglionic sympathetic neuron zinc-enriched? A stop-flow nerve crush study on rat sciatic nerve.

Axonal transport of endogenous zinc ions in the rat sciatic nerve was studied by a stop-flow/nerve crush technique combined with zinc selenide autometallography (ZnSeAMG) at light and electron microscopic levels. Distinct accumulations of ZnSeAMG grains were detected, in particular proximal but also distal to the crushes, 1.5 h after the operation, and the amounts of zinc ions increased further in the following 3-8 h. Ultrastructurally, ZnSeAMG grains were located predominantly in unmyelinated axons. The data suggest that a subpopulation of sciatic nerve axons contains and transports zinc ions both antero- and retrogradely, indicating that the second neuron in the sympathetic nervous system is zinc enriched (ZEN).

Synaptic vesicle proteins and neuronal plasticity in adrenergic neurons.

The neurons in the superior cervical ganglion are active in plasticity and re-modelling in order to adapt to requirements. However, so far, only a few studies dealing with synaptic vesicle related proteins during adaptive processes have been published. In the present paper, changes in content and expression of the synaptic vesicle related proteins in the neurons after decentralization (cutting the cervical sympathetic trunk) or axotomy (cutting the internal and external carotid nerves) were studied. Immunofluorescence studies were carried out using antibodies and antisera against integral membrane proteins, vesicle associated proteins, NPY, and the enzymes TH and PNMT. For colocalization studies, the sections were simultaneously double labelled. Confocal laser scanning microscopy was used for colocalization studies as well as for semi-quantification analysis, using the computer software. Westen blot analysis, in situ 3'-end DNA labelling, and in situ hybridization were also employed. After decentralization of the ganglia several of the synaptic vesicle proteins (synaptotagmin I, synaptophysin, SNAP-25, CLC and GAP-43) were increased in the iris nerve terminal network, but with different time patterns, while TH-immunoreactivity had clearly decreased. In the ganglia, these proteins had decreased at 1 day after decentralization, probably due to degeneration of the pre-ganglionic nerve fibres and terminals. At later intervals, these proteins, except SNAP-25, had increased in the nerve fibre bundles and re-appeared in nerve fibres outlining the principal neurons.

Treatment of enterotoxigenic and enteropathogenic Escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic Escherichia coli (EPEC) are important causes of diarrhoea in young children and are associated with significant mortality rates. Passive immunization with antibodies from immunized cows has previously been shown to be effective as prophylaxis against E. coli-induced diarrhoea and therapeutically against rotavirus and cryptosporidia-induced diarrhoea. METHODS: We tested the therapeutic efficacy of an oral bovine immunoglobulin milk concentrate (BIC) from cows hyperimmunized with ETEC and EPEC strains, in a randomized, placebo-controlled study in children with E. coli-induced diarrhoea. Eighty-six children between 4-24 months of age attending the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) with E. coli-induced diarrhoea (63 EPEC/ETEC and 23 with other diarrhoeagenic E. coli) were randomly assigned to receive orally administered BIC (20 g) containing anti-ETEC/EPEC antibodies or a placebo preparation daily for 4 consecutive days. Daily stool output, intake of oral rehydration solution (ORS), stool frequency, and presence of diarrhoeagenic E. coli strains in the stool were monitored for 4 days. RESULTS: Children in the treatment group tolerated the BIC with no side effects. There were no significant differences between the two groups with regard to ORS intake, stool output, frequency of diarrhoea, or clearance of pathogen. Nor was there any significant alteration in the duration of diarrhoea. CONCLUSIONS: In contrast to the prophylactic efficacy of anti-E. coli BIC and the therapeutic efficacy of a similarly prepared anti-rotavirus BIC, antibodies from hyperimmunized cows appear to have no significant therapeutic benefit in the treatment of acute diarrhoea due to EPEC/ETEC.

Cytoplasmic dynein conversion at a crush injury in rat peripheral axons.

Cytoplasmic dynein is a motor for retrograde axonal transport for movement of membranous organelles toward the neuronal cell body. However, cytoplasmic dynein is synthesized in the cell body and conveyed along the axon to nerve terminals. To characterize the axonal transport of cytoplasmic dynein in relation to synaptic vesicles and other membrane compartments, immunocytochemical and cytofluorimetric scanning analyses of crush-operated rat sciatic nerves were performed. Distal to the crush, the kinetics of dynein accumulation were consistent with its role in the retrograde transport of membranous organelles. During the initial 3 hr after crush, only small amounts of dynein-immunoreactive material accumulated proximal to the crush. This is consistent with metabolic labeling studies showing that most of the dynein moving in the anterograde direction is in the slow component of axonal transport. Thereafter, the rate of proximal accumulation of dynein increased, and by 8 hr postcrush a large amount of dynein immunoreactivity was observed. This accelerated accumulation may be due to recruitment of dynein from slow component b onto organelles proximal to the crush. Double labeling demonstrated that dynein immunoreactivity colocalized with synaptophysin, a transmembrane protein found in small, clear synaptic vesicles. In contrast, dynein immunoreactivity did not colocalize well with calcitonin gene-related peptide (CGRP), a peptide matrix marker for some large dense-cored vesicles. Finally, dynein immunoreactivity colocalized with the anterograde transport motor kinesin both proximal and distal to a crush, suggesting that kinesin may carry some dynein-containing membrane compartments during fast anterograde axonal transport.

Increased tyrosine hydroxylase immunoreactivity in bladder tissue from patients with classic and nonulcer interstitial cystitis.

PURPOSE: Interstitial cystitis is a chronic debilitating condition which mainly affects women. Accumulated evidence indicates that interstitial cystitis is a heterogeneous syndrome. The nonulcer subtype appears different than classic interstitial cystitis in regard to symptoms, and endoscopic and histological findings as well as response to various treatments. We further explore the neurogenic nature of this disease using indirect immunofluorescence to evaluate the presence and density of various autonomic and sensory nerve fibers. MATERIALS AND METHODS: Specimens from the bladder wall of 6 patients with classic interstitial cystitis, 7 with nonulcer interstitial cystitis and 6 controls were evaluated to determine the presence and density of nerve fibers containing tyrosine hydroxylase, calcitonin gene-related peptide, neuropeptide Y and substance P using specific antibodies, and the general presence of nerve fibers using a mixture of antibodies against nerve filament, neuron specific enolase and S-100 protein. RESULTS: Increased density and number of nerve fibers immunoreactive for tyrosine hydroxylase were noted in interstitial cystitis cases compared to controls. Furthermore, there was a difference between classic and nonulcer disease in the overall density of nerves using the antibody mixture. CONCLUSIONS: Our findings indicate an altered peripheral sympathetic innervation in interstitial cystitis cases, which may be an indication of primary neurogenic etiology. The difference in nerve density observed after incubation with the antibody mixture between classic and nonulcer interstitial cystitis supports the hypothesis that the 2 forms represent separate entities.

Axonal transport of synucleins is mediated by all rate components.

Synucleins are abundant nerve terminal proteins of hitherto unknown function. In diseases with Lewy bodies, human alpha-synuclein concentrates in these lesions in the cell body and mutations in alpha-synuclein lead to heritable Parkinson's disease with Lewy bodies. This indicates that changes in the normal metabolism and axonal transport of alpha-synuclein is perturbed in these diseases. To investigate the normal axonal transport of synucleins we studied the rat visual system by nerve crush operations and metabolic labelling of the retinal ganglion cells followed by immunoprecipitation of nerve segments. We found by immunofluorescence microscopy of the crush-operated nerves that synucleins are transported by fast antero- and retrograde transport and colocalize with synaptophysin and SNAP-25 around the lesion. The metabolic labelling studies demonstrated that synucleins were moved through the nerve with all the rate components, the fast component and the slow components a and b, with component b predominating. Two-dimensional gel electrophoresis revealed that both alpha- and beta-synuclein migrate through the nerve by slow component b in a ratio of 2:1.

Axonal transport and distribution of immunologically distinct kinesin heavy chains in rat neurons.

The functional significance of biochemical and immunochemical heterogeneity in neuronal kinesin remains uncertain. Confocal laser scanning microscopy, cytofluorimetric scanning, and immunoblots were used for quantitative analyses of axonal transport and cellular distribution of immunochemically distinct kinesin heavy chain isoforms (H1 and H2) in rat peripheral nerve and spinal cord. H1 and H2 immunoreactivities (IR) were observed in axons proximal to a crush as early as 1 hr after the crush operation and increased linearly with time, consistent with fast axonal transport of both. Only approximately 10% of the proximal accumulations of H1-IR and H2-IR accumulated distal to the crush, in contrast to synaptophysin-IR (approximately 70%). H2-IR was widely present in peripheral nervous system and virtually colocalized with synaptic vesicle proteins synaptophysin, synaptobrevin I, and SNAP-25 and two neuropeptides [calcitonin gene-related peptide (CGRP) and substance P (SP)], although H2-IR was weaker in spinal cord terminals. In contrast, H1-IR appeared preferentially enriched in large axons, probably motor and large sensory neurons, which contained synaptophysin-IR, synaptobrevin I-IR, SNAP-25-IR, and CGRP-IR. However, H1-IR was weak or absent from SP-containing thin and medium-sized axons. In addition, H1-IR appeared to be absent from spinal cord nerve terminals. H1- and H2-IR kinesins are both transported with fast axonal transport, and comparatively small amounts of kinesins are retrogradely transported. H2 was widely distributed in motor, sensory, and sympathetic neurons, whereas H1 was enriched in large motor and sensory neurons.

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.

[Lung function in premature infants can be improved. Surfactant therapy and CPAP reduce the need of respiratory support]. / Lungfunktionen kan förbättras hos för tidigt födda barn. Surfaktantbehandling och CPAP minskar behovet av respiratorvård.

Randomised trials have shown exogenous surfactant therapy to reduce mortality and morbidity among very low birthweight (VLBW) infants with respiratory distress syndrome (RDS). Surfactant therapy is normally given to infants on mechanical ventilation. In the Stockholm area, 12 VLBW infants born after 27-30 gestational weeks and suffering from RDS were recently treated using the INSURE (Intubation-SURfactant-Extubation) approach--i.e., surfactant therapy during brief intubation, immediately followed by extubation and continuous positive airway pressure (CPAP) treatment. The treatment was successful in all 12 cases, the mean (+/- SD) a/A ratio increasing significantly from 0.17 +/- 0.04 before the INSURE procedure to 0.46 (0.12 after (P < 0.001). Only one infant later needed mechanical ventilation for RDS.

Proteolytic processing, axonal transport and differential distribution of chromogranins A and B, and secretogranin II (secretoneurin) in rat sciatic nerve and spinal cord.

The chromogranin family comprises chromogranin A and B, and secretogranin II. The present study has focused on the axonal transport of chromogranins/secretogranin II and their detailed distribution in peripheral nerves and the spinal cord. With radioimmunoassay (RIA) and column chromatography, we first studied the processing of chromogranin B and secretogranin II during axonal transport. No larger precursors of these peptides were detected in the sciatic nerves, indicating that they are already processed to a high degree early during axonal transport. We also analysed nerve segments above and below a crush, using RIA, in order to compare these accumulation data with those obtained by the cytofluorimetric-scanning (CFS) technique. For the latter technique, the amounts of accumulation distal to the crush (presumably representing recycling and retrogradely transported peptides) were 30-40% of the amounts in the proximal accumulation for chromogranin A and secretoneurin, in contrast to chromogranin B, which showed 15% recycling. With the RIA, the corresponding values for secretoneurin and PE-11 (antibody against chromogranin B) were 42% and 14%, respectively. Therefore, the data obtained by CFS were in excellent agreement with those obtained by RIA. In crushed sciatic nerves, chromogranin A was present in large axons as well as in small- and medium-sized axons. Chromogranin B was mainly restricted to large axons, while secretoneurin was localized to bundles of small axons. This differential distribution was also found in the spinal roots and in the peripheral terminals. Chromogranin A was present in both ventral and dorsal roots, and chromogranin B was detected in ventral roots and in large sensory axons in the dorsal roots. Secretoneurin was dominant in the dorsal root. Double-labelling studies with antibodies against choline acetyltransferase/vesicular acetylcholine transporter, or against tyrosine hydroxylase, confirmed that chromogranin A was distributed in cholinergic, sensory, as well as adrenergic neurons. Chromogranin B was mainly present in cholinergic motor neurons and large sensory neurons, and secretoneurin was restricted to adrenergic and sensory neurons. The present study demonstrates that chromogranins A and B, and secretoneurin are transported with fast axonal transport in the peripheral nerves, with different amounts of recycling, and that they are differentially distributed in different types of neurons in the peripheral nervous system and the spinal cord, suggesting that each of them may play a special role in subsets of neurons.

The processing of secretogranin II in the peripheral nervous system: release of secretoneurin from porcine sympathetic nerve terminals.

The distribution of secretoneurin (SN), a peptide derived from secretogranin II (SgII), in the coeliac ganglion, the splenic nerve and the spleen was examined by immunohistochemistry. In the ganglion, SN immunoreactivity (IR) was unevenly distributed. Positive nerve terminals densely surrounded some postganglionic perikarya in which also intense SN-IR was present. In the crushed splenic nerves, intense immunoreactivities appeared proximal (but to a less extent also distal) to the crush of the nerve. Analysis by cytofluorimetric scanning (CFS) demonstrated that SN-IR and neuropeptide Y immunoreactivity (NPY-IR) were predominant in the axons proximal to the crush representing anterogradely transported components. Using radioimmunoassay (RIA) we demonstrated that upon electrical stimulation (10 Hz, 1 min) of the splenic nerve, significant amounts of SN-IR (64.2+/-2.3 fmol) were released together with NA (4. 1x106+/-0.2 fmol) and NPY (330.0+/-7.2 fmol) from the isolated perfused porcine spleen. To evaluate the processing of SgII in sympathetic neurons, boiled tissue extracts (coeliac ganglia and splenic nerve) and boiled spleen perfusate (used as a suitable source for vesicle derived peptides) were analysed by gel filtration chromatography followed by SN-RIA. In all cases immunoreactivity was present solely as SN, indicating that SgII was fully processed to the free peptide. The evidence that SN is transported to the nerve terminals and is released from the porcine spleen upon nerve stimulation, suggests that it may modulate adrenergic neurotransmission and may also play a role in the neuroimmune communication.