Visual system development of the spotted unicornfish, Naso brevirostris (Acanthuridae).

Ontogenetic changes of the visual system are often correlated with shifts in habitat and feeding behaviour of animals. Coral reef fishes begin their lives in the pelagic zone and then migrate to the reef. This habitat transition frequently involves a change in diet and light environment as well as major morphological modifications. The spotted unicornfish, Naso brevirostris, is known to shift diet from zooplankton to algae and back to mainly zooplankton when transitioning from larval to juvenile and then to adult stages. Concurrently, N. brevirostris also moves from an open pelagic to a coral-associated habitat before migrating up in the water column when reaching adulthood. Using retinal mapping techniques, we discovered that the distribution and density of ganglion and photoreceptor cells in N. brevirostris changes primarily during the transition from the larval to the juvenile stage, with only minor modifications thereafter. Similarly, visual gene (opsin) expression based on RNA sequencing, although qualitatively similar between stages (all fish mainly expressed the same three cone opsins; SWS2B, RH2B, RH2A), also showed the biggest quantitative difference when transitioning from larvae to juveniles. The juvenile stage in particular seems mismatched with its reef-associated ecology, which may be due to this stage only lasting a fraction of the lifespan of these fish. Hence, the visual ontogeny found in N. brevirostris is very different from the progressive changes found in other reef fishes, calling for a thorough analysis of visual system development of the reef fish community.

Neural Ganglia Transcriptome and Peptidome Associated with Sexual Maturation in Female Pacific Abalone (Haliotis discus hannai).

Genetic information of reproduction and growth is essential for sustainable molluscan fisheries and aquaculture management. However, there is limited knowledge regarding the reproductive activity of the commercially important Pacific abalone Haliotisdiscushannai. We performed de novo transcriptome sequencing of the ganglia in sexually immature and mature female Pacific abalone to better understand the sexual maturation process and the underlying molecular mechanisms. Of the ~305 million high-quality clean reads, 76,684 transcripts were de novo-assembled with an average length of 741 bp, 28.54% of which were annotated and classified according to Gene Ontology terms. There were 256 differentially expressed genes between the immature and mature abalone. Tandem mass spectrometry analysis, as compared to the predicted-peptide database of abalone ganglia transcriptome unigenes, identified 42 neuropeptide precursors, including 29 validated by peptidomic analyses. Label-free quantification revealed differential occurrences of 18 neuropeptide families between immature and mature abalone, including achatin, FMRFamide, crustacean cardioactive peptide, and pedal peptide A and B that were significantly more frequent at the mature stage. These results represent the first significant contribution to both maturation-related transcriptomic and peptidomic resources of the Pacific abalone ganglia and provide insight into the roles of various neuropeptides in reproductive regulation in marine gastropods.

Molecular cloning and functional expression of the 5-HT7 receptor in Chinese mitten crab (Eriocheir sinensis).

Serotonin (5-HT) regulates numerous physiological functions and processes, such as light adaptation, food intake and ovarian maturation, and plays the role through 5-HT receptors. To our knowledge, this is the first study to isolate and characterize the serotonin receptor 7 (5-HT7 receptor) cDNA encoded in Eriocheir sinensis, an economically important aquaculture species in China, by performing rapid-amplification of cDNA ends. The full-length of 5-HT7 receptor gene cDNA is 2328 bp and encodes a polypeptide with 590 amino acids that are highly homologous with other crustaceans 5-HT7 receptor genes. Analysis of the deduced amino acid sequence of the 5-HT7, including 7 transmembrane domains and some common features of G protein-coupled receptors (GPCRs), indicated that 5-HT7 receptor was a member of GPCRs family. A gene expression analysis of the 5-HT7 receptor by RT-PCR revealed that the 5-HT7 receptor transcripts were widely distributed in various tissues, in which high expression levels were observed in the cranial ganglia, thoracic ganglia and intestines. Further study about the effects of photoperiods on the 5-HT7 expression in the tissues showed that a significantly increasing expression of the 5-HT7 receptor was observed in the thoracic ganglia induced by constant light. In addition, in the eyestalks, the expression levels of 5-HT7 mRNA in constant darkness and constant light were lower than control treatment. Then, the expression levels of the 5-HT7 receptor in three feeding statuses displayed that there were significantly increasing expressions in the hepatopancreas and intestines after feeding, compared with before feeding and during the feeding period. Finally, the 5-HT7 mRNA expression levels in stage III and stage IV were higher than the levels in stage I of ovarian development. Our experimental results showed that the 5-HT7 receptor structurally belongs to GPCRs, and the thoracic ganglia and eyestalks are the important tissues of the 5-HT7 receptor for light adaptation. The 5-HT7 receptor may also be involved in the physiological regulation of the hepatopancreas and intestines after ingestion in E. sinensis. In addition, the 5-HT7 receptor is involved in the process of ovarian maturation. The study provided a foundation for further research of light adaptation, digestive functions and ovarian maturation of the 5-HT7 receptor in Decapoda.

Disruption of Interneuron Neurogenesis in Premature Newborns and Reversal with Estrogen Treatment.

Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic in utero environment and supply of maternal hormones. As the production of interneurons continues until the end of pregnancy, we hypothesized that premature birth would disrupt interneuron production and that restoration of the hypoxic milieu or estrogen treatment might reverse interneuron generation. To test these hypotheses, we compared interneuronal progenitors in the medial ganglionic eminences (MGEs), lateral ganglionic eminences (LGEs), and caudal ganglionic eminences (CGEs) between preterm-born [born on embryonic day (E) 29; examined on postnatal day (D) 3 and D7] and term-born (born on E32; examined on D0 and D4) rabbits at equivalent postconceptional ages. We found that both total and cycling Nkx2.1+, Dlx2+, and Sox2+ cells were more abundant in the MGEs of preterm rabbits at D3 compared with term rabbits at D0, but not in D7 preterm relative to D4 term pups. Total Nkx2.1+ progenitors were also more numerous in the LGEs of preterm pups at D3 compared with term rabbits at D0. Dlx2+ cells in CGEs were comparable between preterm and term pups. Simulation of hypoxia by dimethyloxalylglycine treatment did not affect the number of interneuronal progenitors. However, estrogen treatment reduced the density of total and proliferating Nkx2.1+ and Dlx2+ cells in the MGEs and enhanced Ascl1 transcription factor. Estrogen treatment also reduced Ki67, c-Myc, and phosphorylation of retinoblastoma protein, suggesting inhibition of the G1-to-S phase transition. Hence, preterm birth disrupts interneuron neurogenesis in the MGE and estrogen treatment reverses interneuron neurogenesis in preterm newborns by cell-cycle inhibition and elevation of Ascl1. We speculate that estrogen replacement might partially restore neurogenesis in human premature infants.SIGNIFICANCE STATEMENT Prematurity results in developmental delays and neurobehavioral disorders, which might be ascribed to disturbances in the development of cortical interneurons. Here, we show that preterm birth disrupts interneuron neurogenesis in the medial ganglionic eminence (MGE) and, more importantly, that estrogen treatment reverses this perturbation in the population of interneuron progenitors in the MGE. The estrogen seems to restore neurogenesis by inhibiting the cell cycle and elevating Ascl1 expression. As preterm birth causes plasma estrogen level to drop 100-fold, the estrogen replacement in preterm infants is physiological. We speculate that estrogen replacement might ameliorate disruption in production of interneurons in human premature infants.

The allometry of the central nervous system during the postembryonic development of the spider Eratigena atrica.

During ontogenesis, the size of a spider body, tissues and organs increases dramatically. The aim of the study was to estimate changes in the central nervous system of postembryonic stages of Eratigena atrica and compare them with the literature data on species differing in behavioural traits. Allometric analysis involved evaluation of histological slides embedded in paraffin and stained with hematoxylin and eosin. The reduced major axis regression (RMA) was applied to find allometric relationships between the volumes of the particular parts of the body. All the measured parts of the central nervous system (CNS) were negatively allometrically related to the volume of the prosoma, showing that the increment of the CNS was lower than that of the entire body. The growth of the brain was negatively allometrically related to the growth of the CNS but the increment of the subesophageal ganglion was greater than that of the CNS, exhibiting a positive allometry. Within both these structures, the increase in neuropil volume was greater than the growth of the cortex (cell body rind). Thus, in postembryonic development, the share of the subesophageal ganglion and neuropil in the total volume of the CNS increased, whereas that of the brain and cortex decreased. The mode of the CNS development in E. atrica is similar to that observed in other arthropods, including Argiope aurantia, a spider of different ecology and behaviour.

Development of Calbindin- and Calretinin-Immunopositive Neurons in the Enteric Ganglia of Rats.

Calbindin D28 K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10 days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20 days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.

Prediction of a neuropeptidome for the eyestalk ganglia of the lobster Homarus americanus using a tissue-specific de novo assembled transcriptome.

In silico transcriptome mining is a powerful tool for crustacean peptidome prediction. Using homology-based BLAST searches and a simple bioinformatics workflow, large peptidomes have recently been predicted for a variety of crustaceans, including the lobster, Homarus americanus. Interestingly, no in silico studies have been conducted on the eyestalk ganglia (lamina ganglionaris, medulla externa, medulla interna and medulla terminalis) of the lobster, although the eyestalk is the location of a major neuroendocrine complex, i.e., the X-organ-sinus gland system. Here, an H. americanus eyestalk ganglia-specific transcriptome was produced using the de novo assembler Trinity. This transcriptome was generated from 130,973,220 Illumina reads and consists of 147,542 unique contigs. Eighty-nine neuropeptide-encoding transcripts were identified from this dataset, allowing for the deduction of 62 distinct pre/preprohormones. Two hundred sixty-two neuropeptides were predicted from this set of precursors; the peptides include members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon α, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, glycoprotein hormone ß5, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families. The predicted peptides expand the H. americanus eyestalk ganglia neuropeptidome approximately 7-fold, and include 78 peptides new to the lobster. The transcriptome and predicted neuropeptidome described here provide new resources for investigating peptidergic signaling within/from the lobster eyestalk ganglia.

Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas.

In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19-23 weeks (13 cases) and 30-34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation.

Crustacean hyperglycemic hormones of two cold water crab species, Chionoecetes opilio and C. japonicus: isolation of cDNA sequences and localization of CHH neuropeptide in eyestalk ganglia.

Crustacean hyperglycemic hormone (CHH) is primarily known for its prototypical function in hyperglycemia which is induced by the release of CHH. The CHH release takes place as an adaptive response to the energy demands of the animals experiencing stressful environmental, physiological or behavioral conditions. Although >63 decapod CHH nucleotide sequences are known (GenBank), the majority of them is garnered from the species inhabiting shallow and warm water. In order to understand the adaptive role of CHH in Chionoecetes opilio and Chionoecetes japonicus inhabiting deep water environments, we first aimed for the isolation of the full-length cDNA sequence of CHH from the eyestalk ganglia of C. opilio (ChoCHH) and C. japonicus (ChjCHH) using degenerate PCR and 5' and 3' RACE. Cho- and ChjCHH cDNA sequences are identical in 5' UTR and ORF with 100% sequence identity of the putative 138aa of preproCHHs. The length of 3' UTR ChjCHH cDNA sequence is 39 nucleotides shorter than that of ChoCHH. This is the first report in decapod crustaceans that two different species have the identical sequence of CHH. ChoCHH expression increases during embryogenesis of C. opilio and is significantly higher in adult males and females. C. japonicus males have slightly higher ChjCHH expression than C. opilio males, but no statistical difference. In both species, the immunostaining intensity of CHH is stronger in the sinus gland than that of X-organ cells. Future studies will enable us to gain better understanding of the comparative metabolic physiology and endocrinology of cold, deep water species of Chionoecetes spp.

Activation of bone marrow-derived mesenchymal stromal cells-a new mechanism of defocused low-energy shock wave in regenerative medicine.

BACKGROUND AIMS: Defocused low-energy shock wave (DLSW) therapy has shown effectiveness in regenerative medicine. The mechanism of action was mainly focused on the pathophysiological improvement at the wound tissues. In this study, the activation of stem cells treated by DLSW was first examined as an important pathway during the healing process. METHODS: Cultured rat bone marrow-derived mesenchymal stromal cells (BMSC) were treated by DLSW before each passage. The untreated BMSC served as a control. The secretions of vascular endothelial growth factor (VEGF) and CXC ligand 5 (CXCL5) were tested by means of enzyme-linked immunoassay. Flow cytometry was performed to analyze the BMSC (passage 4) surface antigen expressions (CD166, CD44 and CD34). The expressions of proliferating cell nuclear antigen and Ki67 were analyzed by means of Western blot. The healing abilities of conditioned media of shocked and unshocked BMSC were examined by Matrigel-based capillary-like tube formation assay and rat major pelvic ganglia culture test. RESULTS: The shocked BMSC secreted more VEGF and CXCL5 than did those of unshocked BMSC. The expressions of CD166, CD44 and CD34 showed no significant differences (P > 0.05) between the shocked and unshocked BMSC. The shocked BMSC demonstrated higher expressions of proliferating cell nuclear antigen (P < 0.01) and Ki67 (P < 0.01) than did those of unshocked BMSC. The shocked BMSC conditioned medium showed higher ability to enhance the growth of major pelvic ganglia neurites (P < 0.05) and Matrigel-based endothelial tube-like formation (P < 0.05). CONCLUSIONS: DLSW did not interfere with the expressions of cell surface markers. DLSW enhanced the secretion and proliferation of BMSC and promoted angiogenesis and nerve regeneration in vitro.

The homeobox gene Gsx2 controls the timing of oligodendroglial fate specification in mouse lateral ganglionic eminence progenitors.

The homeobox gene Gsx2 has previously been shown to be required for the specification of distinct neuronal subtypes derived from lateral ganglionic eminence (LGE) progenitors at specific embryonic time points. However, its role in the subsequent generation of oligodendrocytes from these progenitors remains unclear. We have utilized conditional gain-of-function and loss-of-function approaches in order to elucidate the role of Gsx2 in the switch between neurogenesis and oligodendrogenesis within the embryonic ventral telencephalon. In the absence of Gsx2 expression, an increase in oligodendrocyte precursor cells (OPCs) with a concomitant decrease in neurogenesis is observed in the subventricular zone of the LGE at mid-stages of embryogenesis (i.e. E12.5-15.5), which subsequently leads to an increased number of Gsx2-derived OPCs within the adjacent mantle regions of the cortex before birth at E18.5. Moreover, using Olig2(cre) to conditionally inactivate Gsx2 throughout the ventral telencephalon with the exception of the dorsal (d)LGE, we found that the increase in cortical OPCs in Gsx2 germline mutants are derived from dLGE progenitors. We also show that Ascl1 is required for the expansion of these dLGE-derived OPCs in the cortex of Gsx2 mutants. Complementing these results, gain-of-function experiments in which Gsx2 was expressed throughout most of the late-stage embryonic telencephalon (i.e. E15.5-18.5) result in a significant decrease in the number of cortical OPCs. These results support the notion that high levels of Gsx2 suppress OPC specification in dLGE progenitors and that its downregulation is required for the transition from neurogenesis to oligodendrogenesis.

Ex vivo neurogenesis within enteric ganglia occurs in a PTEN dependent manner.

A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU(+) cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.

Development of nervous systems to metamorphosis in feeding and non-feeding echinoid larvae, the transition from bilateral to radial symmetry.

The development of nervous system (NS) in the non-feeding vestibula larva of the sea urchin, Holopneustes purpurescens, and the feeding echinopluteus larva of Hemicentrotus pulcherrimus was examined by focusing on fate during metamorphosis. In H. purpurescens, the serotonergic NS (SerNS) appeared simultaneously and independently in larval tissue and adult rudiment, respectively, from 3-day post-fertilization. In 4-day vestibulae, an expansive aboral ganglion (450 x 100 mum) was present in the larval mid region that extended axons toward the oral ectoderm. These axons diverged near the base of the primary podia. An axonal bundle connected with the primary podia and the rim of vestopore on the oral side. Thus, the SerNS of the larva innervated the rudiment at early stage of development of the primary podia. This innervation was short-lived, and immediately before metamorphosis, it disappeared from the larval and adult tissue domains, whereas non-SerNS marked by synaptotagmin remained. The NS of 1-month post-fertilization plutei of H. pulcherrimus comprised an apical ganglion (50 x 17 mum) and axons that extended to the ciliary bands and the adult rudiment (AR). A major basal nerve of serotonergic and non-serotonergic axons and a minor non-serotonergic nerve comprised the ciliary band nerve. In 3-month plutei, axonal connection among the primary podia in the neural folds completed. The SerNS never developed in the AR. Thus, there was distinctive difference between feeding- and non-feeding larvae of the above sea urchins with respect to SerNS and the AR.

Postnatal development of the mucosal plexus in the porcine small and large intestine.

Knowledge regarding the foetal and postnatal development of the enteric nervous system is crucial for the understanding of congenital disorders. While lot of information exists regarding the myenteric and submucosal plexuses, the development of the mucosal plexus has not been previously studied. The mucosal innervation seems to play an important role in the local reflex activity of the gut. In this study, we examined the development of enteric mucosal innervation in the pig at various ages of life. Small and large bowel paraffin-embedded specimens were stained with PGP 9.5 and neurofilament protein in three piglets from six age groups (60 and 90 days gestation, newborn, 4 and 12 weeks old, and adult pigs). Small and large bowel demonstrated identical innervation patterns. Myenteric and submucosal plexuses were stained with PGP 9.5 at 60 days gestation. However, the mucosal staining was first noted clearly at the newborn period. By 4 weeks, PGP 9.5 staining was noted in small amounts within the mucosa. Inner proprial and villous fibres were seen ahead in time to the subepithelial fibres. Both inner proprial and villous staining became quiet prominent by 12 weeks of age and remained unchanged into adulthood. However, the subepithelial fibres appear to increase in adulthood. This study demonstrates for the first time that enteric mucosal innervation first appears only at birth. The immaturity of the mucosa generated reflex activity, and secretory functions may have implication in the management of functional intestinal obstruction in the premature infant.

Homology of arthropod anterior appendages revealed by Hox gene expression in a sea spider.

Arthropod head segments offer a paradigm for understanding the diversification of form during evolution, as a variety of morphologically diverse appendages have arisen from them. There has been long-running controversy, however, concerning which head appendages are homologous among arthropods, and from which ancestral arrangement they have been derived. This controversy has recently been rekindled by the proposition that the probable ancestral arrangement, with appendages on the first head segment, has not been lost in all extant arthropods as previously thought, but has been retained in the pycnogonids, or sea spiders. This proposal was based on the neuroanatomical analysis of larvae from the sea spider Anoplodactylus sp., and suggested that the most anterior pair of appendages, the chelifores, are innervated from the first part of the brain, the protocerebrum. Our examination of Hox gene expression in another sea spider, Endeis spinosa, refutes this hypothesis. The anterior boundaries of Hox gene expression domains place the chelifore appendages as clearly belonging to the second head segment, innervated from the second part of the brain, the deutocerebrum. The deutocerebrum must have been secondarily displaced towards the protocerebrum in pycnogonid ancestors. As anterior-most appendages are also deutocerebral in the other two arthropod groups, the Euchelicerata and the Mandibulata, we conclude that the protocerebral appendages have been lost in all extant arthropods.

Biotinidase reveals the morphogenetic sequence in cochlea and cochlear nucleus of mice.

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

Regional differences in nitrergic neuronal density in the developing porcine urinary bladder.

Nitric oxide (NO) is involved in normal bladder physiology by regulating local arteriolar tone and smooth muscle relaxation and modulating the production of extracellular matrix proteins in vitro. Little information is available regarding the nitrergic innervation of the bladder during development. In this study we investigated the changes in density and morphology of the intramural nitrergic neurons of the porcine urinary bladder during development using whole-mount preparation. Bladder specimens were obtained from porcine foetuses of gestational age 60 days (n=5) and 90 days (n=5) and from newborn piglets (n=5) after perfusion fixation. Bladders were divided into base, body, and dome. Whole-mount preparation using NADPH-diaphorase (NADPH-d) histochemistry was used to visualize nitrergic innervation of the urinary bladders and to measure density of NADPH-positive ganglia (including single neurons), number of NADPH-d positive neurons per ganglion, and size of individual neurons. One-way ANOVA and chi-square tests were used for statistical analysis with a p-value <0.05 considered statistically significant. NADPH-d positive ganglia were numerous in the muscular layer of all three age groups. At E60, ganglion density was significantly higher in the body (mean 880/cm(2)) than in the dome (397/cm(2)) or the base (676/cm(2)). The ganglion density significantly decreased with age. The number of NADPH-d positive neurons per ganglion increased significantly between E90 and birth (p<0.01). A marked increase in the size of individual neurons over time was also seen (p<0.001), predominantly due to an increase in cytoplasm. Our data on whole-mount preparations demonstrate that significant maturation in nitrergic neuronal density and morphology occurs in the porcine urinary bladder, at least until birth.

[Age-dependent changes of morphometric and histochemical characteristics of neurocytes in different ganglia of albino rats]. / Vozrastnye preobrazovaniia morfometricheskikh i gistokhimicheskikh kharakteristik neirotsitov razlichnykh gangliev u belykh krys.

The aim of this study was to obtain the normative data on the age-dependent transformation of morphometric and histochemical characteristics of neurocytes in different ganglia in albino rats. Cell cross-sectional area, activities of cholinesterase (demonstrated with thioacetic acid method) monoamine oxidase (demonstrated with Glenner method) were measured in neurocytes of stellate, spinal, trigeminal and gastric ganglia in rats aged 2 to 360 days. Measurements were made with the help of "Bioscan" videoanalyzer. Informational analysis was used for the evaluation of the degree of maturation of neurocyte systems. General features, age- and organ-related peculiarities of morphometric and enzyme-histochemical characteristics were established for neurocytes of different ganglia, as well as a heterochronism of their definitive state attainment. The time of stabilization for neurocytes of stellate and I thoracic spinal ganglia was the age of 60 days, for those of trigeminal ganglion and intramural gastric ganglia -90 and 120 days, respectively. By this time, neurocyte systems turned from a determined state into a probabilistic-determined one, this transformation being considered as a population stabilization.

Blocked MAP kinase activity selectively enhances neurotrophic growth responses.

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.