Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

The ovine sexually dimorphic nucleus,aromatase, and sexual partner preferences in sheep

We are using the domestic ram as an experimental model to examine the role of aromatase in the development of sexual partner preferences. This interest has arisen because of the observation that as many as 8% of domestic rams are sexually attracted to other rams (male-oriented) in contrast to the majority of rams that are attracted to estrous ewes (female-oriented). Our findings demonstrate that aromatase expression is enriched in a cluster of neurons in the medial preoptic nucleus called the ovine sexually dimorphic nucleus (oSDN). The size of the oSDN is associated with a ram's sexual partner preference, such that the nucleus is 2–3 times larger in rams that are attracted to females (female-oriented) than in rams that are attracted to other rams (male-oriented). Moreover, the volume of the oSDN in male-oriented rams is similar to the volume in ewes. These volume differences are not influenced by adult concentrations of serum testosterone. Instead, we found that the oSDN is already present in late gestation lamb fetuses (～day 135 of gestation) when it is ～2-fold greater in males than in females. Exposure of genetic female fetuses to exogenous testosterone during the critical period for sexual differentiation masculinizes oSDN volume and aromatase expression when examined subsequently on day 135. The demonstration that the oSDN is organized prenatally by testosterone exposure suggests that the brain of the male-oriented ram may be under-androgenized during development.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

Directional control of neurite outgrowth from cultured hippocampal neurons is modulated by the lectin concanavalin A.

Cell surface carbohydrates play an important role in the regulation of neurite outgrowth during neuronal development. We have investigated the actions of the plant lectin concanavalin A (Con A), a carbohydrate-binding protein, on neurite outgrowth from hippocampal pyramidal neurons in primary cell culture. Neurons plated in culture medium containing nanomolar concentrations of Con A have a larger number of primary neurites arising directly from the cell soma than do neurons plated in culture medium alone. Furthermore, Con A causes counterclockwise turning of neurites in over 70% of the cultured neurons. Both of these effects of Con A are blocked by the hapten sugar alpha-methyl-D-mannopyranoside, suggesting that they result from the interaction of Con A with a cell surface carbohydrate. Another lectin with a different sugar specificity, wheat germ agglutinin, does not modulate neurite outgrowth. Analysis of neurite outgrowth using video-enhanced microscopy reveals that the counterclockwise turning is accompanied by directionally biased extension of filopodia from the growth cones of growing neurites. Treatment of the neurons with cytochalasin, which disrupts actin polymerization, eliminates the neurite turning induced by Con A, suggesting that actin microfilaments are involved in directional control of neurite outgrowth.     

Directional control of neurite outgrowth from cultured hippocampal neurons is modulated by the lectin concanavalin A

Cell surface carbohydrates play an important role in the regulation of neurite outgrowth during neuronal development. We have investigated the actions of the plant lectin concanavalin A (Con A), a carbohydrate-binding protein, on neurite outgrowth from hippocampal pyramidal neurons in primary cell culture. Neurons plated in culture medium containing nanomolar concentrations of Con A have a larger number of primary neurites arising directly from the cell soma than do neurons plated in culture medium alone. Furthermore, Con A causes counterclock-wise turning of neurites in over 70% of the cultured neurons. Both of these effects of Con A are blocked by the hapten sugar α-methyl-d-mannopyranoside, suggesting that they result from the interaction of Con A with a cell surface carbohydrate. Another lectin with a different sugar specificity, wheat germ agglutinin, does not modulate neurite outgrowth. Analysis of neurite outgrowth using video-enhanced microscopy reveals that the counter-clockwise turning is accompanied by directionally biased extension of filopodia from the growth cones of growing neurites. Treatment of the neurons with cytochalasin, which disrupts actin polymerization, eliminates the neurite turning induced by Con A, suggesting that actin microfilaments are involved in directional control of neurite outgrowth. © 1992 John Wiley & Sons, Inc.     

Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.     

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.     

Neurite outgrowth from progeny of epidermal growth factor-responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: effect of brain-derived neurotrophic factor

Epidermal growth factor (EGF)-responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self-renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF-responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF-generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain-derived neurotrophic factor (BDNF) (5 ng in 0.5 microL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF-responsive stem cell-derived neurons possess limited intrinsic capability for long-distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF-responsive stem cell-derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF.     

Effects of Brain-Derived Neurotrophic Factor on Neurite Growth of Deutocerebral Neurons in Brain of the Silk Moth Bombyx mori

This study was conducted to investigate effects of brain‐derived neurotrophic factor (BDNF) on the neurite growth of deutocerebral neurons in vitro, and production of BDNF‐like neuropeptide from brain of the silk moth, Bombyx mori. In primary culture of antennal lobe (AL) neurons with BDNF, it promoted a significant neurite extension of putative AL projection neurons and an outgrowth of branches from principal neurites of putative AL interneurons. Results from immunolabeling of brain and retrocerebral complex showed that BDNF ‐like neuropeptide labeled in brain was synthesized by median and lateral neurosecretory cells, then transported to corpora allata for storage.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

Modulation of growth of Aplysia neurons by an endogenous lectin.

We have purified and characterized a galactose-binding lectin from the gonads of the mollusk Aplysia californica that modulates neurite outgrowth from cultured Aplysia neurons. Agglutination of sheep red blood cells (RBC) by this lectin, termed Aplysia gonad lectin (AGL), is inhibited strongly by galactose and to a lesser extent by fucose. On SDS-PAGE, AGL appears as a single species with a molecular weight of 34 kD under reducing conditions, and 65 kD under nonreducing conditions. This suggests that AGL is a disulfide-linked dimer in its native state. Amino terminal sequence analysis of purified AGL indicates a similarity to another galactose-binding lectin, phytohemagglutinin-E (E-PHA), found in red kidney beans. By using polyclonal antibodies prepared against AGL, we have found that the lectin is present in the gonads and eggs but not in other tissues of adult Aplysia californica. We have examined biological actions of AGL on Aplysia neurons growing in primary cell culture. AGL affects several properties of these neurons. The addition of 100 nM AGL to cultured neurons enhances neurite outgrowth from the cell soma, resulting in a greater number of primary processes. In addition, AGL acts as a neurotrophic agent, increasing neurite viability in vitro. This trophic effect is not seen with concanavalin A (con A), another lectin known to affect several properties of cultured Aplysia neurons. The results are consistent with the suggestion that AGL may play a role in neuronal differentiation and/or maintenance of viability.     

Developmental and spatial expression pattern of syntaxin 13 in the mouse central nervous system

Vesicular transport involves SNARE (soluble- N-ethylmaleimide-sensitive-factor-attachment-protein-receptor) proteins on transport vesicles and on target membranes. Syntaxin 13 is a SNARE enriched in brain, associated with recycling endosomes; its overexpression in PC12 cells promotes neurite outgrowth. This suggests an important role for receptor recycling during neuronal differentiation. Here we describe the spatiotemporal pattern of syntaxin 13 expression during mouse brain development. During early embryogenesis (E12-E15), it was found in the forebrain ventricular zone and in primary motor and sensory neurons in the brainstem, spinal cord and sensory ganglia. In the forebrain at E15, syntaxin 13 was not detected in neuroblasts in the intermediate zone of the embryonic hemispheric wall, while there was labeling in cortical neurons in deeper layers starting at E15-18, and progressively in later-generated neurons up to layer II around P6. Syntaxin 13 reached maximal expression in all brain divisions at about P7, followed by a decrease, with heterogeneous neuron populations displaying various staining intensities in adult brain. While usually restricted to the soma of neurons, we transiently detected syntaxin 13 in dendrites of pyramidal neurons during the first postnatal week. In conclusion, the developmentally regulated syntaxin 13 expression in various neuronal populations is consistent with its involvement in endocytic trafficking and neurite outgrowth.     

Reduction in nerve growth factor availability leads to a conditioning lesion‐like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target‐derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF‐treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Actin in emerging neurites is recruited from a monomer pool

Does actin in the emerging axons of regenerating neurons arise from the assembled or unassembled actin pool in the cell soma? We investigated this question by loading neurons with one of two fluorescently labeled molecules: rhodamine actin (r-actin) and rhodamine phalloidin (r-phalloidin). The assembly behavior of r-actin in vitro was identical to unlabeled actin. R-phalloidin binds tightly only to the filamentous form of actin (F-actin) and stabilizes filaments against disassembly. Hence, r-phalloidin-tagged filaments should be less likely to disassemble than r-actin-tagged filaments. Neurons of 10-d-old chick embryos were loaded with r-actin or r-phalloidin by triturating trypsinized dorsal root ganglia in isotonic sucrose containing the fluorescently tagged molecule. Isolated neurons were plated on glass coverslips in modified L15 medium containing nerve growth factor. Video images of the live cells on a thermoregulated stage were acquired with a computer imaging system. After 24 h in culture, the fluorescence distribution of r-phalloidin and r-actin was examined in live neurons of comparable morphology, neurite outgrowth, and intensity of somal fluorescence. Greater than 90% of the neurons labeled with r-actin (n=81) contained detectable levels of fluorescence in emerging neurite fibers, often extending to the tip of the growing process. Less than 10% of the neurons labeled with r-phalloidin (n=53) contained any fluorescence in the neurite fibers. In those that did contain fluorescence, the r-phalloidin usually was confined to the proximal segment of the neurite, and in no case was it found at the growing tip. Confocal microscopy and cooled CCD imaging of fixed neurons showed that all structures that incorporated r-actin or r-phalloidin also stained with bodipy phallacidin. This colocalization confirms the association of rhodamine-tagged species with F-actin. Our data support a model in which actin, needed in early stages of neurite outgrowth, arises from a pool in the soma that is capable of disassembly.     

Early development of voltage-dependent sodium currents in cultured mouse spinal cord neurons

Spinal cord neurons were dissociated from 13-day embryonic mice and grown in culture for 1-28 days. Sodium currents of neurons in culture for 1-2 days were compared with those in culture for 2-4 weeks, using the whole-cell voltage clamp method. Rapid neurite outgrowth created space clamp limitations so that unclamped neuritic sodium action potentials prevented accurate analysis of sodium current properties. Therefore neurons were bathed in sodium-free solution and brief puffs of sodium were delivered to the cell soma so that only somatic sodium currents were recorded. Sodium currents of neurons at 1-2 days in culture had voltage-dependent activation and inactivation characteristic of these channels, both in mature cultured spinal neurons and in other preparations. However, the estimated channel density on the soma of neurons 1-2 days in culture was less than two channels per micron2. Since the available sodium conductance (as measured by action potential rise rates) increases during development of spinal cord neurons in culture (Westbrook and Brenneman, 1984), we suggest that changes in channel density and/or distribution, rather than in channel kinetics, may underlie the increase in sodium conductance.     

Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron.

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiation from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22 degrees-24 degrees C when the injury was located 800 microns from the soma. Elevating the temperature to 32 degrees C or moving the lesion site to 400 or 1500 microns from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 x 10(-5) M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10(-5) M) did. The calcium channel blocker lanthanum (10(-6)-10(-4) M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10(-3)-10(-5) M), and the calcium ionophore A23187 (10(-5) M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH4Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10(-5) M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10(-5) M) had no effect. Inhibition of protein synthesis with anisomycin (2 x 10(-4) to 2 x 10(-6) M) did not significantly suppress sprouting 24 h after axotomy. Both D and L isomers of glutamate (300 microM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.     

Neuron survival in vitro is more influenced by the developmental age of the cells than by glucose condition

The objective of this study was to determine whether the sensitivity to varying glucose conditions differs for the peripheral and central nervous system neurons at different developmental stages. Ventral horn neurons (VHN) and dorsal root ganglion neurons (DRG) from rats of different postnatal ages were exposed to glucose-free or glucose-rich culture conditions. Following 24 h at those conditions, the number of protein gene product 9.5 positive (PGP⁺) DRG neurons and choline acetyltransferase positive (ChAT⁺) VHN were counted and their neurite lengths and soma diameters were measured. For both DRG and VHN, the highest number of cells with and without neurite outgrowth was seen when cells from postnatal day 4 donors were cultured, while the lowest cell numbers were when neurons were from donors early after birth and grown under glucose-free conditions. The length of the neurites and the soma diameter for VHN were not affected by either glucose level or age. DRG neurons, however, exhibited the shortest neurites and smallest soma diameter when neurons were obtained and cultured early after birth. Our results indicate that survival of neurons in vitro is more influenced by the developmental stage than by glucose concentrations.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Effect of HDAC Inhibitors on Neuroprotection and Neurite Outgrowth in Primary Rat Cortical Neurons Following Ischemic Insult

Histone deacetylase inhibitors (HDACi)—valproic acid (VPA) and trichostatin A (TSA) promote neurogenesis, neurite outgrowth, synaptic plasticity and neuroprotection. In this study, we investigated whether VPA and TSA promote post-ischemic neuroprotection and neuronal restoration in rat primary cortical neurons. On 6 days in vitro (DIV), cortical neurons were exposed to oxygen-glucose deprivation for 90 min. Cells were returned to normoxic conditions and cultured for 1, 3, or 7 days with or without VPA and TSA. Control cells were cultured in normoxic conditions only. On 7, 9, and 13 DIV, cells were measured neurite outgrowth using the Axiovision program and stained with Tunel staining kit. Microtubule associated protein-2 immunostaining and tunel staining showed significant recovery of neurite outgrowth and post-ischemic neuronal death by VPA or TSA treatment. We also determined levels of acetylated histone H3, PSD95, GAP 43 and synaptophysin. Significant increases in all three synaptic markers and acetylated histone H3 were observed relative to non-treated cells. Post-ischemic HDACi treatment also significantly raised levels of brain derived neurotrophic factor (BDNF) expression and secreted BDNF. Enhanced BDNF expression by HDACi treatment might have been involved in the post-ischemic neuroprotection and neuronal restorative effects. Our findings suggest that both VPA and TSA treatment during reoxygenation after ischemia may help post-ischemic neuroprotection and neuronal regeneration via increased BDNF expression and activation.