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.     

NCAM-dependent neurite outgrowth is inhibited in neurons from Fyn-minus mice

Src-related nonreceptor protein tyrosine kinases in nerve growth cones (p59fyn, pp60c-src, and pp62c-yes) are potential intracellular signaling molecules for cell adhesion molecule-directed axonal growth. To determine whether src-related tyrosine kinases mediate NCAM- dependent neurite outgrowth, cultures of cerebellar and sensory neurons from fyn-, src-, and yes- minus mice were analyzed for neurite outgrowth on monolayers of NCAM140-transfected L fibroblasts. NCAM- dependent neurite outgrowth was selectively inhibited in cultures of cerebellar and dorsal root ganglion neurons from fyn-, but not src- or yes- mice. Neurite outgrowth by fyn-, src-, or yes- neurons on untransfected fibroblast monolayers was unaffected, indicating that these kinases do not contribute significantly to axon growth on at least some integrins or other adhesive substrates present on fibroblasts. This study demonstrates that p59fyn is an essential component of the NCAM signaling pathway leading to axonal growth.     

Stimulation by melanocortins of neurite outgrowth from spinal and sensory neurons in vitro

Using ELISAs for B-50/GAP43 and neurofilament (NF), we tested ACTH(1–24), -MSH, ACTH(4–10), and an ACTH(4–9) analogue (ORG2766) for their ability to induce sprouting and neuritogenesis from spinal and sensory neurons. Dissociated fetal rat spinal cord neurons or neonatal rat dorsal root ganglion (DRG) cells were cultured with peptide and assayed after 24, 48, or 96 h. In spinal neurons, -MSH and ACTH(1–24) induced the expression of B-50 dose dependently. After 24 h -MSH had a stimulatory effect (from 10 nM onwards), with a maximum at 100 μM (36% increase). After 96 h the maximal effect of 100 μM -MSH on B-50/GAP43 was lower (19%). ACTH(1–24) (100 μM) stimulated B-50/GAP43 by 19%. Neurofilament levels (96 h) were elevated maximally by 64% at 100 μM -MSH. In DRG neurons a bell-shaped dose-response curve was found for -MSH, the maximal effect being observed after 48 h at 100 nM: 54% for B-50/GAP43 and 22% for NF. In both culture systems neither ACTH(4–10) nor ORG2766 was effective. We conclude that -MSH stimulates the expression of B-50/GAP43 (sprouting) and the formation of NF (neurite elongation) and may therefore be considered a neurotrophic factor.     

Expression of NCAM containing VASE in neurons can account for a developmental loss in their neurite outgrowth response to NCAM in a cellular substratum

Binding of the neural cell adhesion molecule (NCAM) in neurons to NCAM on non-neuronal cells can stimulate axonal growth. A developmentally regulated loss of this response is associated with the insertion of 10 amino acids (called VASE) into the fourth Ig domain in up to 50% of the NCAM receptors in neurons. In the present study we have transfected PC12 cells with the major neuronal isoforms of human NCAM and tested cells expressing these isoforms for their ability to respond to NCAM in a cellular substratum. Whereas both the 140- and 180-kD isoforms of NCAM can act as functional receptors for neurite outgrowth, the presence of the VASE sequence in a minority of the receptors specifically inhibited this response. A synthetic peptide containing the VASE sequence inhibits neurite outgrowth from PC12 cells and primary neurons stimulated by NCAM. The same peptide has no effect on integrin dependent neurite outgrowth or neurite outgrowth stimulated by N-cadherin or L1. We discuss the possibility that the VASE peptide inhibits the NCAM response by preventing NCAM from binding to the FGF receptor in the plasma membrane.     

Amitriptyline inhibits neurite outgrowth in chick cerebral neurons: A possible mechanism

Previous studies showed that amitriptyline (AMI), a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants and inhibited adenylyl cyclase activity in cerebral membrane preparations. In the present study, we have investigated the possibility that AMI may have additional effects on cellular metabolism and signal transduction that underlie AMI-mediated inhibition of neurite outgrowth. In vitro AMI inhibited phospholipase C in a dose- and GTP-dependent manner in membranes from 8-day-old chick forebrain. Brain homogenates from 8-day-old chick embryos, treated in vivo for 6 days with AMI (20 μg/g/day), showed significant reductions in (1) phosphorylation of two polypeptides (49 and 105 kD), and (2) levels of three polypeptides (43, 53, and 92 kD). Western blots showed that the 43- and 53-kD polypeptides corresponded to actin and tubulin, respectively. Diolein and dilinolein, potent activators of protein kinase C, stimulated neurite outgrowth and reversed the inhibitory effects of AMI. Sphingosine, a protein kinase C inhibitor, significantly inhibited neurite outgrowth and eliminated the stimulatory effects of diolein and dilinolein on neurite outgrowth. These data suggest that AMI-mediated inhibition of neurite outgrowth involves multiple effects on cellular metabolism and signal transduction. A hypothesis consistent with our data is that AMI interferes in some manner with the action of G proteins in the signal transduction cascade. © 1993 John Wiley & Sons, Inc.     

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against β-tubulin III and S-100 β, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 μm on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.     

Isolation and some characteristics of an adhesive factor of brain that enhances neurite outgrowth in central neurons

Fractionation of octyl glucoside-solubilized proteins from young rat brain was monitored using rat brain neurons, which were cultured in microwells coated with various protein fractions to be studied. An adhesive protein that promotes neurite outgrowth in rat brain neurons was isolated by chromatography on heparin-Sepharose followed by Affi-Gel blue. The apparent molecular mass of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was about 30 kilodaltons (p30). Under nonreducing conditions a closely spaced doublet band was observed corresponding to 27-28-kilodalton size. Gel filtration in the presence of 4 M urea indicated the molecular size of 58 kilodaltons suggesting a dimeric structure. Western blotting experiments using affinity-purified rabbit antibodies detected p30 as an immunochemically distinct protein in brain and in N18 neuroblastoma cells. The p30 protein was also detected in the N18 cells by lactoperoxidase-catalyzed cell surface iodination. Western blotting of heparin-binding proteins solubilized from brains of rats of various age groups indicated that p30 is clearly more abundant in perinatal brain as compared to adult tissue. The neuron-binding and neurite outgrowth-promoting properties of p30 as well as the developmental regulation of its content in brain tissue suggest a role in neuronal growth.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Myosin II activity regulates neurite outgrowth and guidance in response to chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.     

Power metabolism from neurons and a glia to the whole brain: norm, pathology and correction

The review outlines current state of the thepretical, methodological and applies aspects of brain's energy homeostasis. Authors suggest reconsidering the exclusive role of glucose as an energy substrate (ES) at both neuronal and systemic levels discussing recent research data on qualitative composition of ES pool in the brain. The role of ES alternative to glucose, e.g., lactate and ketone bodies, is examined. The hypotheses of intracellular and astrocyte-neuron lactate shuttles are discussed along with the hypotheses of astrocyte-neuron shuttle of ketone bodies, the selfish brain theory and suppositions on homeostatic versus non-homeostatic ES supply chains. In conclusion, authors argue that exogenous native ES may be used for prevention and treatment of neurodegenerative diseases.     

Distinct effect of neurotrophins delivered simultaneously by an adenoviral vector on neurite outgrowth of neural precursor cells from different regions of the brain

For many years, it has been demonstrated that neurotrophins regulate the adult nervous system, implicating their potential as therapeutic agents for the treatment of neurodegenerative diseases. We generated adenoviral vectors encoding brain-derived neutotrophin factor (BDNF) and neurotrophin-3 (NT3) and tested either separately or together for the ability to induce differentiation of neuronal precursor cells with two different origins. Separate transduction of adenovirus delivering BDNF (BDNF-Ad) or NT3 (NT3-Ad) induced the neuronal differentiation in hippocampal and cortical precursor cells. NT3-Ad infected cells extended short neurites, whereas BDNFAd infected cells had longer neurites. In the early differentiation of hippocampal precursor cells, simultaneous infection of BDNFAd and NT3-Ad promoted further differentiation and neurite elongation compared with the separate infection of each virus. In contrast, simultaneous infection did not show the synergistic effect in the cortical precursor cells, suggesting that the neurotrophins play distinct roles in different regions of the brain. However, the numbers of neurites and spines per differentiated cells were markedly increased in cortical as well as hippocampal precursor cells, indicating the promotion of efficient neurite elongation and formation of dendritic spine, when BDNF-Ad and NT3-Ad were co-infected. These results suggest more studies in the effect of a combinatorial use of neurotrophins on different sites of brain need to be carried out to develop gene therapy protocols for neurodegenerative diseases.     

Arterial cells and CNS sheath cells from Aplysia californica produce factors that enhance neurite outgrowth in co-cultured neurons

Substrate-bound and soluble factors regulate neurite outgrowth and synapse formation during development, regeneration, and learning and memory. We report that sheath cells from CNS connectives and arterial cells from the anterior aorta of the sea slug, Aplysia californica, enhance neurite outgrowth from co-cultured Aplysia neurons. Sheath and arterial cell cultures contain several cell types, including fibrocytes, myocytes, and amoebocytes. When compared to controls (neurons with defined growth medium alone), the percentage of neurons with growth and the average neurite lengths are significantly enhanced by sheath and arterial cells at 48 h after plating of the neurons; these parameters are comparable to those of neurons cultured in medium containing hemolymph. Our results indicate that sheath cells produce substrate-bound factor(s) and arterial cells produce diffusible factor(s) that promote growth. These growth factors likely promote neuron survival and neurite outgrowth during neural plasticity exhibited in the adult CNS. Electronic Publication     

Astroglia demonstrate regional differences in their ability to maintain primary dendritic outgrowth from mouse cortical neurons in vitro

To determine whether glia from different regions of the central nervous system (CNS) initiate or maintain primary dendritic growth, embryonic day 18 mouse cortical neurons were co-cultured with rat (postnatal day 4) astroglial cells derived from retina, spinal cord, mesencephalon, striatum, olfactory bulb, retina, and cortex. Axon and dendrite outgrowth from isolated neurons was quantified using morphological and immunohistochemical techniques at 18 h and 1, 3, and 5 days in vitro. Neurons initially extend the same number of neurites, regardless of the source of glial monolayer; however, glial cells differ in their ability to maintain primary dendrites. Homotypic cortical astrocytes maintain the greatest number of primary dendrites. Glia derived from the olfactory bulb and retina maintained intermediate numbers of dendrites, whereas only a small number of primary dendrites were maintained by glia derived from striatum, spinal cord, or mesencephalon. Longer axons were initially observed from neurons grown on glia that did not maintain dendrite number. Axonal length, however, was similar on the various monolayers after 5 days in vitro. Neurons that were grown in media conditioned by either mesencephalic or cortical glia for the first 24 h followed by culture media from glia of the alternate source for 4 days in vitro confirmed that glia maintained, rather than initiated, the outgrowth of the primary dendritic arbor. These results indicate that glial cells derived from various CNS regions differ in their ability to maintain the primary dendritic arbor from mouse cortical neurons in vitro. © 1995 John Wiley & Sons, Inc.     

Electrically induced changes in Ca2+ in Helisoma neurons: Regional and neuron-specific differences and implications for neurite outgrowth

The present experiments addressed the questions of how electrical stimulation influenced the magnitude, time course, and regional levels of free intracellular calcium of different identified neurons. The calcium concentration in the growth cones, neurites and cell bodies of Helisoma buccal neurons B4 and B19 was measured while somata were electrically stimulated via an intracellular electrode. The findings showed that calcium levels in B4 and B19 increased monotonically with increasing stimulation frequency. However, the range of calcium levels evoked by electrical stimulation differed significantly for each type of neuron. The greater increase in calcium concentration in B4 was correlated with its longer duration action potential compared to B19. The increase in calcium concentration was much smaller in the cell bodies than in the growth cones and neurites. Extending the duration of the B19 action potential produced a sixfold increase in the change in calcium concentration at 2 Hz stimulation. Under conditions where the electrical stimulation produced a calcium concentration of < 160 nM, the elevated level of free intracellular calcium remained constant. When calcium concentration increased above 200 nM in both identified neurons, an initial peak concentration was followed by a decline to a lower concentration suggesting increased calcium buffering occurring above 200 nM. By correlating the calcium concentration data herein with growth data from a previous study, we suggest that specific calcium levels that influence neurite outgrowth may differ widely between neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 150–162, 1997.