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.

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.

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.

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.

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.

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.

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.

Turning teratocarcinoma cells into neurons: rapid differentiation of NT-2 cells in floating spheres.

Cells from the human teratocarcinoma line NTera-2 can be induced to terminally differentiate into postmitotic neurons when treated with retinoic acid. However, this differentiation process is rather time consuming as it takes between 42 and 54 days. Here, we propose a modified differentiation protocol which reduces the time needed for differentiation considerably without compromising the quantity of the neurons obtained. The introduction of a proliferation step as free floating cell spheres cuts the total time needed to obtain high yields of purified NT-2 neurons to about 24-28 days. The cells obtained show neuronal morphology and migrate to form ganglion-like cell conglomerates. Differentiated cells express neuronal polarity markers such as the cytoskeleton associated proteins MAP2 and Tau. Moreover, the generation of neurons in sphere cultures induced immunoreactivity to the ELAV-like neuronal RNA-binding proteins HuC/D, which have been implicated in mechanisms of nerve cell differentiation.

The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation.

Cholinergic properties in chick sympathetic neurons are detectable early during development of paravertebral ganglia and mature after target contact. The cholinergic marker choline acetyltransferase (ChAT) is first detectable at embryonic day 6 and its expression partly overlaps with that of the noradrenergic marker tyrosine hydroxylase (TH). At late embryonic stages, when sympathetic neurons have established target contact, ganglia consist of two major neuronal populations, TH-positive noradrenergic neurons and cholinergic neurons that at this stage express vasoactive intestinal peptide (VIP) in addition to ChAT. The maturation of sympathetic neurons is paralleled by changes in their response to the neurokine ciliary neurotrophic factor (CNTF). These findings suggest that expression of neurotransmitter properties is controlled differentially before and during target innervation.

Localization of thymosin beta 4 to the neural tissues during the development of Xenopus laevis, as studied by in situ hybridization and immunohistochemistry.

Thymosin beta 4, a polypeptide of 5 kDa, is known to have capacity to regulate actin polymerization by binding to an actin monomer. Distribution of Xenopus laevis thymosin beta 4 (XT beta 4) in the developing Xenopus larva was examined by means of in situ hybridization and immunohistochemistry. Analysis with in situ hybridization revealed that XT beta 4 mRNA becomes gradually localized to the neural tissues, notochord and inner epidermis during neurula stages. Intense accumulation of XT beta 4 mRNA was observed in the ganglions of cranial nerves and in the dorsal region of the spinal cord from stage 26 and onwards. XT beta 4 immunoreactivity (XTI) was observed in larvas at all developmental stages later than stage 26, tail bud embryo. Immunoreactivity was initially distributed to the ganglion of cranial nerve V and Rohon-Beard cells. As the development progressed, the XTI appeared in other neuronal groups. By late tadpole stages (stages 42-47) the XTI was found in the pineal body, oculomotor and trochlear motoneurons of the midbrain, various neurons in the rhombencephalon, ganglions of cranial nerves V, VII/VIII and IX/X. In the spinal cord the XTI was observed in Rohon-Beard cells, dorsal root ganglion cells, motoneurons and other spinal cord neurons. Immunoreactivity was seen in both cell bodies and axons of the neurons. These findings suggest that thymosin beta 4 plays a role in the development of neurons, especially of sensory neurons.

Grafting in acute spinal cord injury: morphological and immunological aspects of transplanted adult rat enteric ganglia.

We have studied allogeneic transplants of adult rat enteric ganglia in order to evaluate their use as donor tissue for eventual autografts in rodent spinal cord injury models. Female Sprague-Dawley rats of similar weights served either as transplant donors or as recipients. A glass micropipette of 0.8 mm diameter was used to create a local penetrating injury of the lower thoracic spinal cord and the transplant material was pressure injected through the pipette within the neural parenchyma. Ganglia of the myenteric plexus adhering to the stratum longitudinal muscularis were dissected from portions of the jejunum and ileum. Following partial enzymatic digestion and mechanical disruption of the myenteric plexus and muscle tissue (labeled with adherent rhodamine conjugated microbeads), reaggregates of myenteric plexus and muscle were suspended in growth medium and cultured in vitro for one to two days prior to transplantation. Transplants were examined at three, four, six, and eight weeks after surgery. Some of the donor tissue was grown in vitro, in order to determine its cellular composition. These cultured explants were fixed after 10 days, and like myenteric plexus and muscle grafts, were stained histochemically for acetylcholinesterase and observed by fluorescence and light microscopy. At the earlier post-transplantation periods, grafts contained several clusters of enteric ganglion cells that were positive for acetylcholinesterase and exhibited ultrastructural features characteristic of the enteric nervous system. They had well-defined boundaries. Reactive astrocytes and their processes remained located within the host spinal cord adjacent to the boundary region of the grafts. Likewise, macrophages were located in areas abutting the graft. Newly formed vasculature penetrated the graft interior and appeared to be continuous with the host vessels. Grafts grown for at least eight weeks were characterized by interdigitating boundaries. Finger-like protrusions of graft tissue containing fibroblasts and collagen intermixed with adjacent gray and white matter of the host cord. Such transplants also had reactive astrocytes and ED1-positive macrophages. At this later stage, several groups of ganglion cells were identified that were intensely acetylcholinesterase-positive; however, only two of four grafts were recovered, whereas two of the transplants degenerated. We postulate that degeneration of allogeneic grafts may occur as a result of ongoing immune responses of the host which could be prevented by use of autogeneic enteric ganglia. Our studies show that fully differentiated enteric ganglia can survive transplantation to acutely injured spinal cord of adult rats.

The regulation of transmitter expression in postembryonic lineages in the moth Manduca sexta. II. Role of cell lineage and birth order.

The expression of GABA is restricted to the progeny of only six of the 24 identified postembryonic lineages in the thoracic ganglia of the tobacco hornworm, Manduca sexta (Witten and Truman, 1991). It is colocalized with a peptide similar to molluscan small cardioactive peptide B (SCPB) in some of the neurons in two of the six lineages. By combining chemical ablation of the neuroblasts at specific larval stages with birth dating of the progeny, we tested whether the expression of GABA and the SCPB-like peptide was determined strictly by cell lineage or involved cellular interactions among the members of individual clonal groups. Chemical ablation of the six specific neuroblasts that produced the GABA-positive neurons (E, K, M, N, T, and X) or of the two that produced the GABA + SCPB-like-immunoreactive neurons (K, M) prior to the generation of their lineages resulted in the loss of these immunoreactivities. These results suggest that regulation between lineages did not occur. Ablation of the K and M neuroblasts after they had produced a small portion of their lineages had no effect on the expression of GABA, but did affect the pattern of the SCPB-like immunoreactivity. Combining birth-dating techniques with transmitter immunocytochemistry revealed that it was the position in the birth order and not interactions among the clonally related neurons that influenced the peptidergic phenotype. These results suggest that cell lineage is involved in establishing the GABAergic phenotype and that both cell lineage and birth order influence the determination of the peptidergic phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen-specific critical periods for the organization of the major pelvic ganglion.

Previous studies indicate that the major pelvic ganglion (PG) is dependent on testosterone for normal development. Tyrosine hydroxylase (T-OH), DOPA decarboxylase, and choline acetyltransferase (CAT) activities are significantly reduced by postnatal castration on day 10-11, while testosterone replacement therapy reversed all developmental enzyme activity deficits (Melvin and Hamill, 1987). In the present studies castration on the day of birth combined with various testosterone-replacement paradigms produced effects demonstrating that the PG is sensitive to testosterone dosage and time of administration during early postnatal development. Gonadal hormone replacement experiments show that the androgens testosterone and dihydrotestosterone were effective in restoring T-OH and CAT activity deficits produced by neonatal castration. Estrogen therapy reversed the deficits in CAT activity, but was ineffective in reversing the alterations in T-OH activity. Treatment of pregnant dams with the anti-androgen flutamide altered the ontogeny of T-OH and CAT activities in pups despite replacement therapy on the day of birth. Thus, androgen-critical periods exist prenatally as well as postnatally. These studies suggest that the organization of PG development is critically dependent on both the time of exposure and dose of testosterone. Prenatal and postnatal critical periods appear to exist. In addition, the lack of an effect of estradiol on tyrosine hydroxylase activity suggests that androgens are specifically responsible for organizing the noradrenergic development of the PG.

Developmental changes in choline uptake and acetylcholine metabolism in the larval brain of the tobacco hornworm, Manduca sexta.

The larval brain of the tobacco hornworm, Manduca sexta, was maintained in vitro and the uptake of labelled and unlabelled choline as well as their subsequent metabolism were measured by high-voltage paper electrophoresis. Significant levels of choline lipid metabolites, phosphorylcholine and acetylcholine (ACh) were noted. Unbound choline reached equilibrium after 6-8 h of incubation, while ACh accumulation continued to increase after 24 h indicating that the rate of synthesis exceeded the rate of breakdown. An apparent Km could not be determined for these whole-organ studies; however, the Vmax for ACh accumulation for days 5 (70 pmol/brain/h) and 6 (105 pmol/brain/h) of the last larval instar did vary significantly while the level of unbound choline in the brain did not change. The level of choline uptake was dependent upon the presence of Na+ and Ca2+, while the amount of ACh accumulated was affected specifically by the presence of Mg2+, the latter ion activating acetylcholinesterase. The determination of levels of unbound choline and ACh accumulation in the developing brain during the last two larval instars demonstrated increases in acetylcholine accumulation at previously reported times of the release of the hormone that initiates the molting process, prothoracicotropic hormone. These changes in the patterns of ACh accumulation occur during 4-8 h time intervals; this is the first report of such short-range changes in neurotransmitter metabolism in whole brains. The intensity of the ACh accumulation shift is equivalent to the intensity of the hormone burst. Other fluctuations in the levels of ACh accumulation and free choline correlate with the development of the brain.

Identification of neurones containing cardioacceleratory peptides (CAPs) in the ventral nerve cord of the tobacco hawkmoth, Manduca sexta.

The abdominal ganglion neurosecretory cells responsible for the synthesis and release of two insect neurohormones, cardioacceleratory peptides 1 and 2 (CAP1 and CAP2), from the perivisceral organs (PVOs) have been identified in the tobacco hawkmoth, Manduca sexta. Previous work established the existence of two groups of abdominal ganglion cell bodies with axons projecting to the PVO: four laterally-situated pairs and five pairs lying on the midline (Taghert & Truman, 1982b). Micro-dissection and bioassay of various parts of an abdominal ganglion revealed that CAP activity was greatest in the medial portion of the ganglion, the portion containing the 10 midline neurones. Six of the 10 midline neurosecretory cells, the new midline bilateral (MB) cells, appeared to differentiate post-embryonically, commencing differentiation late in the last larval instar and reaching maturity midway through adult development. The development of the new MB cells was mirrored by the accumulation of CAP activity in the abdominal nerve cord. Not present in measurable amounts in larvae, CAP activity was first detectable a few days after pupation and reached maximal levels midway through adult development. CAP-like bioactivity was collected from the PVO in response to antidromic stimulation of the nerve containing the new MB axons. No CAP-like bioactivity was detected in those preparations in which the new MB axons were severed or in which other nerves were stimulated. Intracellular stimulation of a new MB neurone evoked the release from the PVO of measurable levels of CAP bioactivity. It was shown that this stimulation-evoked, cardioacceleratory activity was sensitive to protease treatment, and was released only from the cell that was stimulated. On the basis of these experiments, it was concluded that the CAPs are synthesized and secreted from the new MB cells.

Nerve growth factor and neural oncology.

The precise role of the nerve growth factor protein (NGF) during the growth and development of the human nervous system is not determined. Although it appears to influence a number of neural functions, its mechanism of action is poorly understood. A number of researchers have proposed that NGF may be involved in several pathological conditions including cancer. It has been shown that NGF is secreted by certain sarcoma (23), neuroblastoma (113), and glioma (7,102,136) cell lines and can bind to neuroblastoma and metastatic melanoma cell lines (42). Neuroblastoma (136,181) and pheochromocytoma (165) cells in vitro can be induced by NGF to differentiate toward a morphologically "more benign" state and appropriate NGF treatment of rats can reduce the number of chemically induced gliomas and neurinomas (174,178). NGF can also reduce the growth of intracerebrally inoculated anaplastic glioma cells (172). Anti-NGF treatment of rats (178) and mice (179) can alter the tumor distribution observed following ethylnitrosourea or benzo(a)pyrene treatment (10). In humans, it has been reported that serum levels of NGF are usually elevated in persons "at risk" for neurofibromatosis (156). The precise nature of the NGF role is not known in these instances. Further understanding of the action of NGF could be of clinical importance.

Neuritic growth and neuroma formation by an isolated molluscan ganglion.

The isolated, paired buccal ganglia of the snail Helisoma trivolvis were cultured in vivo (i.e., in host snails) for periods up to 377 days. Such ganglia typically become enveloped in a white mass of fibrous tissue which reaches a maximum plateau of size within four weeks. The scanning electron microscope revealed the newly formed tissue to be composed of fine fibers which also pervaded the neuropil. Injection of individual neurons with Lucifer Yellow CH filled many new neurites within the tissue mass. It is concluded that these masses are largely of neuronal origin and they are therefore designated as neuromas. The maximum size of neuromas may be limited by intrinsic mechanisms which limit neuronal volume.