Addition Of Nerve Growth Factor Research Articles

Bacopa monnieri protects neuronal cell line and Caenorhabditis elegans models of Alzheimer’s disease through sigma-1 receptor antagonist sensitive and antioxidant pathways

BACKGROUND: Due to better health care and improved nutritional status of the world’s population, many people live into old age. This has resulted in more diseases related to aging, such as neurodegenerative diseases. Bacopa monnieri (BM) is a medicinal herb found in Southeast Asia and is a popular memory-enhancing supplement. OBJECTIVE: This study aimed to investigate how BM may provide protection in neurodegenerative disease, and whether the sigma-1 receptor is involved. METHODS: PC-12 cells were differentiated with the addition of nerve growth factor. The potentiation by BM of PC-12 neurite growth was measured by counting the number of differentiated cells and by measuring their length. Differentiated PC-12 cells were also subjected to amyloid-β (Aβ) toxicity in the presence and absence of BM. The cell survival (MTT and cell counting) and neurite lengths were then measured as indicators of cellular health. Total protein was extracted from control and treated cells and expression of various signalling pathway molecules was assessed via western blotting. We also assessed the effects of BM on the lifespans of various mutant strains plus wild-type C. elegans. RESULTS: We show that BM can protect against Aβ toxicity in PC-12 cells. Furthermore, BM can potentiate neurite outgrowth in PC-12, in a sigma-1 receptor antagonist sensitive fashion, and Neuro2A cell lines. BM induced a reduction in pAKT expression and upregulated BDNF expression in PC-12 cells. BM was also able to increase the lifespan and health-span of Aβ expressing C. elegans mutants via the DAF-16 mediated pathway. BM reduced oxidative stress in wild-type C. elegans exposed to UV-A with pre-exposure and post-exposure treatments. CONCLUSIONS: This all further identifies BM as a potential agent to treat neurodegenerative diseases, by modulating different pathways.

How Microfluidics Can Help to Understand and Promote Nerve Healing after Injury: A Neurobiological Microfluidic Device with Electrophysiological Functionality

We present a microfluidic device that has been designed to study the neuroregeneration of nerve tissue after injury. Reconnection between neurons of the peripheral nervous system after injury or dissection is essential for regaining the full motoric and sensory capabilities. The neuroregeneration of damaged axons and the functional reconnection to a target cell is a complex processes, where (1) chemical guidance and (2) topographic guidance of the neurite growth cone play a crucial role. To identify new chemical guidance substances and to understand how geometry guides neurite growth, we have developed a microfluidic in-vitro system for studying neuronal cell cultures. The microchannels act as artificial guidance conduits for the growth of nerve processes (dendrites, axons), also named neurites in the following text. The microstructured system consists of a microelectrode array with sub-10 µm microelectrodes for recording neuronal signals plus a microfluidic attachement. This microfluidic attachment has 2 macrochannels with a width above 100 µm that can accommodate entire cells. Both macrochannels are connected by up to 60 microchannels which are 100 to 500 µm long but only 10 µm wide and 2 µm high, so that cell somata cannot enter the microchannels while cell processes (axons, dendrites) are small enough to enter the channel. A neuronal culture of mouse superior cervical ganglion (SCG) is seeded in the source macrochannel, while the target macrochannel stays cell-free and is filled cell medium with neurotrophic factors added. These soluble neurotrophic factors act as chemical guidance cues for the neurites and have been under debate as enhancers of axon regeneration. In our microfluidic system the neurites can reach the target macrochannel by growing through the small microchannels that are too small for entire cell somata. This topographic guidance provided by the microchannels mimics’ topographic guidance of the endoneurial channel formed by Schwann cells for a reconnecting neuron. We will present the design considerations as well as the actual fabrication of this microfluidic system on the microelectrode system. Simulations on the diffusion of chemical entities through the microchannels will be presented. Finally, images of the neurites grown through the microchannels will be shown and prove the research concept. Neurotrophic factors are also under debate for accelerating the functional recovery, i.e. the recovery of the neuron to propagate action potentials along the neurite. With our microfluidic setup the microelectrodes of the microelectrode array have been aligned such that every microchannel contains at least one (up to four) microelectrodes. By implementing microelectrodes inside of the microfluidic channels also the electrophysiological activity of the reconnecting neurites can be monitored. This electrical activity of neurons is a measure for the health status of a neuron. With the microelectrodes providing electrochemical sensor functionality to the microfluidic channels, this important question could be answered. We will present the effect, that various gradients of neurotrophic factors have on the growth and electrical activity of growing neurons. Finally, a recommendation will be given on the optimum concentration for the addition of nerve growth factor in order to promote a faster growth of electrophysiologicallly active neurites. The potential to use this microfluidic platform for identifying new nerve growth promotors and for quantifying the effects will be discussed. Concepts for advancing this microfluidic system further as cell culture reactors as well as for other interdisciplinary areas including neurobiological and medical applications will be presented. Figure 1

Biodegradable Nanotopography Combined with Neurotrophic Signals Enhances Contact Guidance and Neuronal Differentiation of Human Neural Stem Cells.

Biophysical cues provided by nanotopographical surfaces have been used as stimuli to guide neurite extension and regulate neural stem cell (NSC) differentiation. Here, we fabricated biodegradable polymer substrates with nanoscale topography for enhancing human NSC (hNSC) differentiation and guided neurite outgrowth. The substrate was constructed from biodegradable poly(lactic-co-glycolic acid) (PLGA) using solvent-assisted capillary force lithography. We found that precoating with 3,4-dihydroxy-l-phenylalanine (DOPA) facilitated the immobilization of poly-l-lysine and fibronectin on PLGA substrates via bio-inspired catechol chemistry. The DOPA-coated nanopatterned substrates directed cellular alignment along the patterned grooves by contact guidance, leading to enhanced focal adhesion, skeletal protein reorganization, and neuronal differentiation of hNSCs as indicated by highly extended neurites from cell bodies and increased expression of neuronal markers (Tuj1 and MAP2). The addition of nerve growth factor further enhanced neuronal differentiation of hNSCs, indicating a synergistic effect of biophysical and biochemical cues on NSC differentiation. These bio-inspired PLGA nanopatterned substrates could potentially be used as implantable biomaterials for improving the efficacy of hNSCs in treating neurodegenerative diseases.

Protocol and cell responses in three-dimensional conductive collagen gel scaffolds with conductive polymer nanofibres for tissue regeneration.

It has been established that nerves and skeletal muscles respond and communicate via electrical signals. In regenerative medicine, there is current emphasis on using conductive nanomaterials to enhance electrical conduction through tissue-engineered scaffolds to increase cell differentiation and tissue regeneration. We investigated the role of chemically synthesized polyaniline (PANI) and poly(3,4-ethylenedioxythiophene) (PEDOT) conductive polymer nanofibres for conductive gels. To mimic a naturally derived extracellular matrix for cell growth, type I collagen gels were reconstituted with conductive polymer nanofibres and cells. Cell viability and proliferation of PC-12 cells and human skeletal muscle cells on these three-dimensional conductive collagen gels were evaluated in vitro. PANI and PEDOT nanofibres were found to be cytocompatible with both cell types and the best results (i.e. cell growth and gel electrical conductivity) were obtained with a low concentration (0.5 wt%) of PANI. After 7 days of culture in the conductive gels, the densities of both cell types were similar and comparable to collagen positive controls. Moreover, PC-12 cells were found to differentiate in the conductive hydrogels without the addition of nerve growth factor or electrical stimulation better than collagen control. Importantly, electrical conductivity of the three-dimensional gel scaffolds increased by more than 400% compared with control. The increased conductivity and injectability of the cell-laden collagen gels to injury sites in order to create an electrically conductive extracellular matrix makes these biomaterials very conducive for the regeneration of tissues.

Nerve growth factor with fibrin glue in end-to-side nerve repair in rats

To determine the effects of end-to-side nerve repair performed only with fibrin glue containing nerve growth in rats. Seventy two Wistar rats were divided into six equal groups: group A was not submitted to nerve section; group B was submitted to nerve fibular section only. The others groups had the nerve fibular sectioned and then repaired in the lateral surface of an intact tibial nerve, with different procedures: group C: ETS with sutures; group D: ETS with sutures and NGF; group E: ETS with FG only; group F: ETS with FG containing NGF. The motor function was accompanied and the tibial muscle mass, the number and diameter of muscular fibers and regenerated axons were measured. All the analyzed variables did not show any differences among the four operated groups (p>0.05), which were statistically superior to group B (p<0.05), but inferior to group A (p>0.05). The end-to-side nerve repair presented the same recovery pattern, independent from the repair used, showing that the addition of nerve growth factor in fibrin glue was not enough for the results potentiating.

Bio-electrospinning of poly(l-lactic acid) hollow fibrous membrane

We present a bio-electrospinning technique for preparing cell-containing, oriented tubular scaffold intended for use as a nerve guide conduit (NGC). With a coaxial spinneret, solutions of PC-12 cells and poly(l-lactic acid) (PLLA) were delivered to the core and shell parts of the spinneret, respectively. When electrostatic field was applied, core/shell fiber was electrospun and collected. The electrospun fibers’ morphology was characterized via scanning electron microscopy (SEM) and optical microscopy. Two structural characteristics were found: (1) the larger, hollow fibers had diameters in tens of micrometers and wall thicknesses around few micrometers, and (2) improved orientation in a preferred direction with the aid of a rotating collection device. Finally, fluorescent PC12 cells were found not only to exist and survive inside these hollow fibers, but also, upon addition of nerve growth factor, to undergo differentiation with neurite extended along the hollow fibers in the desired direction. All of these results demonstrate its potential application for NGC.

Neuroprotective effect of bellis perennis and hypericum perforatum on PC12 cells

The rat pheochromocytoma (PC12) cell line is widely applied as a model system to study a variety of neuronal functions. Upon addition of nerve growth factor (NGF), these cells undergo differentiation characterized by an increase in acetylcholinesterase (AChE) activity, and extension of neurite-like processes. Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and the purpose of this study was to evaluate the effect of Bellis perennis (Be) and Hypericum perforatum (Hy) (6C and 30C) on healthy neuronal cells. Both homeopathic medicines have been studied at three different concentrations of 2 μl/ml, 4 μl/ml and 8 μl/ml for 96 h in PC12 cells differentiated with NGF. The cell viability was tested by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and NRU (Neutral Red Uptake). To observe the oxidative damage and evaluate the antioxidative status after exposure to homeopathic medicines, the level of thiobarbituric acid reactive species (TBARS), glutathione (GSH) level, activities of glutathione peroxidase (GPx), glutathione reductase (GR), AcetylCholine Esterase (AChE), Na+K+ATPase and Monoamine Oxidase (MAO) were assayed. These results were compared with positive control (90% alcohol). The content of LPO was significantly decreased in drug treated groups as compared to positive control while the level of GSH was significantly increased. The activities of all other enzymes were significantly restored in drug treated groups as compared to positive control. In conclusion, these medicines have preventive role on differentiated PC 12 cells.

Homodimerization of Nemo-like kinase is essential for activation and nuclear localization

Nemo-like kinase (NLK) is an evolutionarily conserved protein kinase that phosphorylates several transcription factors. However, the molecular mechanisms that regulate NLK activity have been poorly understood. Here we show that homodimerization of NLK is required for its activation and nuclear localization. Biochemical analysis revealed that NLK is activated through intermolecular autophosphorylation of NLK dimers at Thr-286. Mutation of NLK at Cys-425, which corresponds to the defect in the Caenorhabditis elegans NLK homologue lit-1, prevented NLK dimerization, rendering NLK defective in both nuclear localization and kinase activity. By contrast, the external addition of nerve growth factor, which has been previously identified as an NLK activator, induced dimerization and Thr-286 autophosphorylation of endogenous NLK proteins. In addition, both dimerization and Thr-286 phosphorylation of NLK were found to be essential for induction of neurite-like cellular processes by NLK. The present findings suggest that dimerization is an initial key event required for the functional activation of NLK.

Pancreatic Islet Transplantation Using Vascularised Chambers Containing Nerve Growth Factor Ameliorates Hyperglycaemia in Diabetic Mice

Intraportal islet transplantation has shown initial promise for the treatment of type 1 diabetes. However, the portal vein site is associated with complications such as thrombosis and hepatic steatosis, leading to transplant failure. The aims of this study were to (1) test the feasibility of an alternative islet transplantation method that utilises a FDA-approved gelatin sponge as a novel islet carrier and (2) assess if exogenous addition of nerve growth factor (NGF) has any additional beneficial effects on graft performance in diabetic mice. Mice were rendered diabetic by a single intraperitoneal injection of streptozotocin. Five hundred syngeneic islets were seeded onto a Gelitaspon® disc in the presence or absence of NGF, and placed into a silicone chamber surrounding the femoral neurovascular pedicle. Islet function was assessed by weekly monitoring of blood glucose levels and an intraperitoneal glucose tolerance test performed at the end of the study. Chambers were harvested for further histological analysis. Four of five mice transplanted with islets seeded onto Gelitaspon with NGF showed a significant reduction in blood glucose levels by 4 weeks after transplantation, and demonstrated a response similar to non-diabetic mice when tested with an intraperitoneal glucose tolerance test. Chamber tissue from this group contained islets with insulin-producing β cells adjacent to the vascular pedicle. Islets seeded onto Gelitaspon with NGF and sited on femoral vessels using a tissue-engineering chamber offer an alternative method for islet transplantation in diabetic mice. This may have potential as a method for clinical islet transplantation.

A PC-12 cell expressing GFP-peripherin in the region of a newly emerging growth cone observed a short time following the addition of nerve growth factor: Movie 31_3.

A PC-12 cell expressing GFP-peripherin in the region of a newly emerging growth cone observed a short time following the addition of nerve growth factor: Movie 31_3.

Neurotrophin Receptors TrkA and TrkB in Retinoblastoma Are Differentially Expressed Depending on Cellular Differentiation

Retinoblastoma is the most common primary intraocular malignancy in children. With the progression of retinoblastoma, retinoblastoma cells lose their ability to differentiate. Regardless of many attempts to identify prognostic factors in retinoblastoma, further investigation for prognostic factors of retinoblastoma progression is still required because of the lack of sensitivity and specificity of these prognostic factors in predicting disease progression. We demonstrated that the differential expression of the neurotrophin receptors TrkA and TrkB is closely related to the differentiation of retinoblastoma cells. While retinoblastoma cells expressed TrkA as well as TrkB, their growth rates were not influenced by the addition of nerve growth factor to the culture medium. In experimental animal models of retinoblastoma, TrkA expression was primarily detected in more differentiated areas with high nm23 immunoreactivity whereas TrkB expression was apparent in more proliferative areas with high Ki67 immunoreactivity. With retinoic-acid-induced differentiation of retinoblastoma cells, TrkA expression significantly increased whereas TrkB significantly decreased. The differential expression of TrkA and TrkB with differentiation of retinoblastoma cells was mediated by extracellular-signal-regulated kinase 1/2 activation, which was confirmed by immunocytochemistry of TrkA. Therefore, our results suggest that the differential expression of TrkA and TrkB could be valuable as a therapeutic target, for instance using specific inhibitors.

Peptide Microarray Analysis of Substrate Specificity of the Transmembrane Ser/Thr Kinase KPI-2 Reveals Reactivity with Cystic Fibrosis Transmembrane Conductance Regulator and Phosphorylase

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Human Intervertebral Disc Cells Promote Nerve Growth Over Substrata of Human Intervertebral Disc Aggrecan

Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

The role of p-c-Jun in survival and outgrowth of developing sensory neurons

c-Jun activation has been implicated not only in neuronal apoptosis, but also in survival and regeneration. This Janus facet of c-Jun activation could be related to neuronal cell type or to the developmental stage of the neuron. We investigated c-Jun activation in E18 sensory neurons. Cultures of rat dorsal root ganglia neurons were maintained with or without the addition of nerve growth factor or the c-Jun N-terminal kinase inhibitor, (D)-JNKI1. Few dorsal root ganglia neurons survived nerve growth factor deprivation, whereas neurons supplied with nerve growth factor survived and exhibited extensive axonal outgrowth. Activated c-Jun was present in the nuclei of neurons with regenerating axons, but not in apoptotic neurons. c-Jun N-terminal kinase inhibition reduced the number of p-c-Jun immunoreactive and regenerating neurons, and increased cell death. Thus, activation of c-Jun seems to be required for survival and regeneration of developing sensory neurons.

Sema4D stimulates axonal outgrowth of embryonic DRG sensory neurones.

Several semaphorins are thought to function as potent inhibitors of axonal growth. We have found that Sema4D stimulates axonal outgrowth of embryonic dorsal root ganglion (DRG) neurones in stead of retraction. Neutralizing antibodies to Sema4D inhibit this action. This action appears to differ slightly from that on PC12 cells, because DRG neurones respond to Sema4D without addition of nerve growth factor (NGF), while PC12 cells do not. On the other hand, it is blocked by deprivation of endogenous NGF with antibodies to NGF and also by Trk-inhibitor K252a, suggesting that endogenously produced-NGF and the activation of Trk receptor are required for Sema4D-action on DRG neurones. These indicate that neurite-outgrowth promoting actions of Sema4D are similar between DRG neurones and PC12 cells, since NGF-Trk signalling are required for these actions. Since Schwann cells can produce NGF, the contamination of these cells in our DRG culture might explain this action. In addition to plexin-B1 that is known as a Sema4D receptor, binding experiments indicate plexin-B2 as another receptor candidate for Sema4D. These plexins and Sema4D are expressed in embryonic DRGs. We suggest a new function of Sema4D as a neurite-outgrowth stimulating, autocrine/paracrine factor in embryonic sensory neurones.

Induction of nerve growth factor by butanol fraction of Liriope platyphylla in C6 and primary astrocyte cells.

Liriope platyphylla (LP) has been used as a tonic, antitussive, and expectorant in Korea for many years. In this study, we found that the buthanol fraction of Liriope platyphylla (BLP)-conditioned media of C6 and primary astrocyte induced the neurite outgrowth of PC12 cells, and that the effect was reversed by addition of nerve growth factor (NGF)-antibody and GF109203X, an inhibitor of protein kinase (PKC). Furthermore, we demonstrated that BLP increased the expression and secretion of NGF. GF109203X also decreased NGF expression in C6 cells. Taken together, our results suggest that astroglial NGF enhanced by BLP in a PKC-dependent pathway contributed to the induction of neurite outgrowth of PC12 cells.

Xenon prevents cellular damage in differentiated PC-12 cells exposed to hypoxia

BackgroundThe neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon.ResultsPheochromocytoma cells differentiated by addition of nerve growth factor were placed in a N2-saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N2-treated cells. Replacement of N2 by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering of Ca2+ using the Ca-chelator 1, 2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the essential participation of intracellular Ca2+-ions in the process of xenon-induced neuroprotection.ConclusionsThe results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation.

In vitro development of a tissue-engineered model of peripheral nerve regeneration to study neurite growth.

A unique tissue-engineered model of peripheral nerve regeneration was developed in vitro to study neurite outgrowth. Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human endothelial cells and/or human fibroblasts. Addition of nerve growth factor (NGF; 10 ng/ml) was not required for sensory neurons survival but was necessary to promote neurite outgrowth, as assessed by immunostaining of the 150 kDa neurofilament. A vigorous neurite elongation was detected inside the reconstructed tissue after 14 and 31 days of neurons culture, reaching up to 770 microm from day 14. Axons were often observed closely associated with the capillary-like tubes reconstructed in the model, in a similar pattern as in the human dermis. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. Besides the addition of NGF, axonal growth did not necessitate B27 supplement or glial cell coculture to be promoted and stabilized for long-term culture. Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on neurite outgrowth in vitro.

Involvement of Gas7 in nerve growth factor-independent and dependent cell processes in PC12 cells.

Gas7, a growth arrest-specific gene originally isolated from serum-starved mouse fibroblast cells, is expressed in vivo predominantly in the brain and is required for neurite formation in cultured mouse cerebellar neurons (Ju et al. [1998] Proc. Natl. Acad. Sci. USA 95: 11423-11428). Here we report that Gas7 plays a key role in the morphological differentiation of PC12 preneuronal rat pheochromocytoma cells (PC12 cells). We found that overexpression of murine Gas7 in PC12 cells leads to an expanded cell morphology and promotes spike-like cell processes that resemble the early stages of neurite formation. These processes undergo elongation upon addition of nerve growth factor (NGF). We also found that the addition of NGF induces the production of endogenous rat-Gas7 (rGas7), which is transiently elevated prior to the appearance of NGF-promoted neurite outgrowths. Furthermore, inhibition of endogenous rGas7 production by antisense nucleotides complimentary to the translation initiation region of a rGas7 cDNA (AJ131902) reduces the NGF-promoted neurite outgrowths. Our results demonstrate that Gas7 by itself influences early cell morphological development and likely functions as an early-stage intermediary in NGF-induced neuronal differentiation of PC12 culture cells.

No overlap of sensitivity to capsaicin and expression of galanin in rat dorsal root ganglion neurons after axotomy.

The neuropeptide galanin is known to have an antinociceptive effect under neuropathic conditions. After axotomy, galanin is upregulated in sensory neurons, presumably in the capsaicin-sensitive ones. Here, the sensitivity to capsaicin and the expression of galanin were simultaneously examined by double-staining in individual, dissociated rat dorsal root ganglion neurons (1) after axotomy of the sciatic nerve for up to 14 days and (2) in culture for up to 4 days without prior nerve injury. Ten days after axotomy, the proportion of capsaicin-sensitive neurons had decreased by 36 percentage points (from 63% to 27%), whereas the proportion of galaninergic neurons had increased by 33 percentage points (from 3% to 36%). These changes were also observed in neurons kept in culture, where the regulation was attenuated by the addition of nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) to the medium. After axotomy, galaninergic neurons had a soma size-distribution profile similar to the capsaicin-sensitive neurons, but there was no colocalization of capsaicin sensitivity and galanin expression in individual neurons. In culture, some neurons showed colocalization after 30 h and 48 h, but not after 6 h or 96 h. We conclude that the upregulation of galanin in an individual neuron is preceded by downregulation of its capsaicin sensitivity both in NGF-dependent peptidergic and in GDNF-dependent non-peptidergic neurons, indicating a change in phenotype.