Interaction of neurotrophin signaling with Bcl‐2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl‐2 prevents SGN death but inhibits neurite growth. Here we assessed the consequences of Bcl‐2 targeted to either the mitochondria (GFP‐Bcl‐2‐Maob) or the endoplasmic reticulum (ER, GFP‐Bcl‐2‐Cb5) on cultured SGN survival and neurite growth. Transfection of wild‐type GFP‐Bcl‐2, GFP‐Bcl‐2‐Cb5, or GFP‐Bcl‐2‐Maob increased SGN survival, with GFP‐Bcl‐2‐Cb5 providing the most robust response. Paradoxically, expression of GFP‐Bcl‐2‐Maob results in SGN death in the presence of neurotrophin‐3 (NT‐3) and brain‐derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears to be specific for neurotrophin signaling, insofar as coexpression of constitutively active mitogen‐activated kinase kinase (MEKΔEE) or phosphatidyl inositol‐3 kinase (P110), but not other prosurvival stimuli (e.g., membrane depolarization), also results in the loss of SGNs expressing GFP‐Bcl‐2‐Maob. MEKΔEE and P110 promote SGN survival, whereas P110 promotes neurite growth to a greater extent than NT‐3 or MEKΔEE. However, wild‐type GFP‐Bcl‐2, GFP‐Bcl‐2‐Cb5, and GFP‐Bcl‐2‐Maob inhibit neurite growth even in the presence of neurotrophins, MEKΔEE, or P110. Historically, Bcl‐2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl‐2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl‐2 targeted to the mitochondria results in SGN death in the presence of neurotrophins. © 2010 Wiley‐Liss, Inc.     

Effects of Kir2.1 gene transfection in cochlear hair cells and application of neurotrophic factors on survival and neurite growth of co-cultured cochlear spiral ganglion neurons

Immature inner hair cells (IHCs) produce spontaneous action potentials, which may be associated with the survival of spiral ganglion neurons (SGNs) during early development. Later, this activity ceases in part by the expression of Kir channels. In the present study, SGNs were co-cultured with organ of Corti in which a Kir2.1 channel was over-expressed in an attempt to block the spontaneous activity of IHCs. The over-expression led to a reduced survival and neurite growth accompanied by increased SGN apoptosis. The enhanced activation of apoptosis was consistent with the inhibition of the survival-promoting pathway and the disruption of [Ca²⁺]_i homeostasis. Furthermore, the effect of Kir2.1 over-expression can be reversed by exogenous neurotrophic factors (NTFs). These results are consistent with the hypothesis that the earlier-than-normal expression of Kir2.1 in HCs inhibits their spontaneous activity required for SGN survival and neurite growth.     

Chronic neurotrophin delivery promotes ectopic neurite growth from the spiral ganglion of deafened cochleae without compromising the spatial selectivity of cochlear implants

Cochlear implants restore hearing cues in the severe–profoundly deaf by electrically stimulating spiral ganglion neurons (SGNs). However, SGNs degenerate following loss of cochlear hair cells, due at least in part to a reduction in the endogenous neurotrophin (NT) supply, normally provided by hair cells and supporting cells of the organ of Corti. Delivering exogenous NTs to the cochlea can rescue SGNs from degeneration and can also promote the ectopic growth of SGN neurites. This resprouting may disrupt the cochleotopic organization upon which cochlear implants rely to impart pitch cues. Using retrograde labeling and confocal imaging of SGNs, we determined the extent of neurite growth following 28 days of exogenous NT treatment in deafened guinea pigs with and without chronic electrical stimulation (ES). On completion of this treatment, we measured the spread of neural activation to intracochlear ES by recording neural responses across the cochleotopically organized inferior colliculus using multichannel recording techniques. Although NT treatment significantly increased both the length and the lateral extent of growth of neurites along the cochlea compared with deafened controls, these anatomical changes did not affect the spread of neural activation when examined immediately after 28 days of NT treatment. NT treatment did, however, result in lower excitation thresholds compared with deafened controls. These data support the application of NTs for improved clinical outcomes for cochlear implant patients. J. Comp. Neurol. 521:2818–2832, 2013. © 2013 Wiley Periodicals, Inc.     

L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells.

The intracellular free Ca2+ concentration, [Ca2+]i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca2+]i is partly a function of Ca2+ influx through voltage-sensitive calcium channels (VSCC), Ca2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd2+, Ni2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd2+ (less than 50 microM), which have been reported to inhibit L-type channels, reduced neurite initiation, but not mean neurite length. Ni2+ (less than 100 microM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (less than 300 microM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (great than 300 microM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 microM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 microM), Cd2+ (5 microM), and streptomycin (200 microM) reduced neurite initiation, while nickel (50 microM) and neomycin (100 microM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd2+ inhibited L-type channels but not T-type channels, while Ni2+ inhibited T-type channels but not L-type channels. Streptomycin slightly inhibited L-type channels but enhanced current flow through T-type channels. Neomycin slightly inhibited both channel types. These data indicated that neurite initiation in these two cell types may be modulated by Ca2+ influx through L-type channels, but not T- or N-type channels. Neurite length was not significantly influenced by any of the agents tested, suggesting that Ca2+ influx through VSCC may not affect neurite elongation.     

Semaphorin 3A induces acute changes in membrane excitability in spiral ganglion neurons in vitro

The development and survival of spiral ganglion neurons (SGNs) are dependent on multiple trophic factors as well as membrane electrical activity. Semaphorins (Sema) constitute a family of membrane‐associated and secreted proteins that have garnered significant attention as a potential SGN “navigator” during cochlea development. Previous studies using mutant mice demonstrated that Sema3A plays a role in the SGN pathfinding. The mechanisms, however, by which Sema3A shapes SGNs firing behavior are not known. In these studies, we found that Sema3A plays a novel role in regulating SGN resting membrane potential and excitability. Using dissociated SGN from pre‐hearing (P3–P5) and post‐hearing mice (P12–P15), we recorded membrane potentials using whole‐cell patch clamp recording techniques in apical and basal SGN populations. Recombinant Sema3A was applied to examine the effects on intrinsic membrane properties and action potentials evoked by current injections. Apical and basal SGNs from newborn mice treated with recombinant Sema3A (100 ng/ml) displayed a higher resting membrane potential, higher threshold, decreased amplitude, and prolonged latency and duration of spikes. Although a similar phenomenon was observed in SGNs from post‐hearing mice, the resting membrane potential was essentially indistinguishable before and after Sema3A exposure. Sema3A‐mediated changes in membrane excitability were associated with a significant decrease in K⁺ and Ca²⁺ currents. Sema3A acts through linopirdine‐sensitive K⁺ channels in apical, but not in the basal SGNs. Therefore, Sema3A induces differential effects in SGN membrane excitability that are dependent on age and location, and constitutes an additional early and novel effect of Sema3A SGNs in vitro.     

Ca2+/calmodulin-dependent protein kinases II and IV both promote survival but differ in their effects on axon growth in spiral ganglion neurons

Spiral ganglion neuron (SGN) survival in vitro can be maintained by neurotrophins, permeant cAMP analogs, and depolarization in an additive manner, with depolarization being the most efficacious. Therefore, we used cultured SGNs to determine the mechanism by which depolarization promotes neuronal survival. Our data implicate Ca(2+)/calmodulin-dependent protein kinase (CaMK) activity by showing that it is induced by depolarization, that CaMK activity is necessary for at least part of the survival-promoting effect of depolarization, and that CaMKII or CamKIV activity suffices to support neuronal survival in the absence of other trophic stimuli. First, that depolarization of SGNs activates CaMKs is evidenced by observation of increased CaMKII phosphorylation and of CaMK-dependent CREB phosphorylation. Second, the requirement for CaMKs is shown by a reduction of SGN survival under depolarizing conditions in the presence of CaMK inhibitors. Third, transfection of COOH-terminal-truncated (lacking regulatory domain), constitutively active CaMKII or CaMKIV, but not of normal, full-length CAMKs, promotes SGN survival in the absence of other trophic stimuli, indicating that CaMK activity is sufficient to promote survival. The survival-promoting effect of truncated CaMKs is additive with that of depolarization, neurotrophins, or cyclic AMP. Although both CaMKII and CaMKIV activities converge in promoting survival, their actions on axon growth are markedly different: Transfection of truncated CaMKII, but not of truncated CaMKIV, into SGNs prevents axon outgrowth.     

CaMKII and CaMKIV mediate distinct prosurvival signaling pathways in response to depolarization in neurons

By fusing the CaMKII-inhibitory peptide AIP to GFP, we constructed a specific and effective CaMKII inhibitor, GFP-AIP. Expression of GFP-AIP and/or dominant-inhibitory CaMKIV in cultured neonatal rat spiral ganglion neurons (SGNs) shows that CaMKII and CaMKIV act additively and in parallel to mediate the prosurvival effect of depolarization. Depolarization or expression of constitutively active CaMKII functionally inactivates Bad, indicating that this is one means by which CaMKII promotes neuronal survival. CaMKIV, but not CaMKII, requires CREB to promote SGN survival, consistent with the exclusively nuclear localization of CaMKIV and indicating that the principal prosurvival function of CaMKIV is activation of CREB. Consistent with this, a constitutively active CREB construct that provides a high level of CREB activity promotes SGN survival, although low levels of CREB activity did not do so. Also, in apoptotic SGNs, activation of CREB by depolarization is disabled, presumably as part of a cellular commitment to apoptosis.     

Neuro-2a neuroblastoma cells form neurites in the presence of taxol and cytochalasin D

We have examined the role of microtubules and microfilaments in neurite outgrowth by chemically modifying their interaction in Neuro-2a neuroblastoma cells. Cells exposed to taxol (1 microM), an agent that promotes microtubule polymerization and stabilization, did not form neurites over a 24 h period. Similarly, cells exposed to cytochalasin D (4 microM), an agent which promotes microfilament depolymerization, did not develop neurites. However, cells treated simultaneously with taxol (1 microM) and cytochalasin D (4 microM) produced long (50 microns) thin, unbranched neurites. Neurites formed during this simultaneous treatment grew in a circular pattern, lacked typical growth cones, were packed densely with microtubules and were deficient in microfilaments. Untreated cells maintained in control medium for 24 h formed short (15 microns), thick, highly branched neurites containing a dense meshwork of microtubules, microfilaments and neurofilaments. These results demonstrate that taxol does not block neurite outgrowth from Neuro-2a cells maintained under microfilament-limiting conditions. They suggest further that microtubules may provide the major cytoskeletal framework for neurite elongation.     

Mechanism for increase in expression of cerebral diazepam binding inhibitor mRNA by nicotine: involvement of L-type voltage-dependent calcium channels

We investigated the mechanisms underlying the increase in diazepam binding inhibitor (DBI) and its mRNA expression induced by nicotine (0.1 microM) exposure for 24 h using mouse cerebral cortical neurons in primary culture. Nicotine-induced (0.1 microM) increases in DBI mRNA expression were abolished by hexamethonium, a nicotinic acetylcholine (nACh) receptor antagonist. Agents that stabilize the neuronal membrane, including tetrodotoxin (TTX), procainamide (a Na(+) channel inhibitor), and local anesthetics (dibucaine and lidocaine), dose-dependently inhibited the increased expression of DBI mRNA by nicotine. The nicotine-induced increase in DBI mRNA expression was inhibited by L-type voltage-dependent Ca(2+) channel (VDCC) inhibitors such as verapamil, calmodulin antagonist (W-7), and Ca(2+)/calmodulin-dependent protein kinase II (CAM II kinase) inhibitor (KN-62), whereas P/Q- and N-type VDCC inhibitors showed no effects. In addition, nicotine exposure for 24 h induced [3H]nicotine binding to the particulate fractions of the neurons with an increased B(max) value and no changes in K(d). Under these conditions, the 30 mM KCl- and nicotine-induced 45Ca(2+) influx into the nicotine-treated neurons was significantly higher than those into non-treated neurons. These results suggest that the nicotine-stimulated increase in DBI mRNA expression is mediated by CAM II kinase activation resulting from the increase in intracellular Ca(2+) through L-type VDCCs subsequent to the neuronal membrane depolarization associated with nACh receptor activation.     

Stimulation of L-type Ca2+ channel in growth cones activates two independent signaling pathways

Although growth cones respond to various modulators of neurite outgrowth, such as neurotrophins, neurotransmitters, and cell adhesion molecules, the signal-transducing mechanisms for these modulators in growth cones are unclear. Since recent studies have suggested that the signals of these modulators are mediated by Ca²⁺ influx through L-type voltage-sensitive Ca²⁺ channels (VSCCs) in the growth cone, we examined L-type VSCC-dependent signaling pathways, using isolated growth cones (IGCs) from developing rat forebrains. Binding assays revealed that L-type VSCC is enriched in growth cone membrane and gradually decreased in amount developmentally, while N-type VSCC has the opposite tendency. In intact IGCs, Bay K 8644 (BK, an L-type agonist) induced much more rapid elevation of [Ca²⁺]_i than that in adult synaptosomes. Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by protein kinase C (PKC) was enhanced in the IGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca²⁺-dependent degradation of brain spectrin (fodrin) by calpain was also enhanced by BK or GABA, consequently inducing the release of α-actinin from the membrane skeleton of the growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other, indicating that these reactions occur independently. Our results suggest that Ca²⁺ influx through L-type VSCCs activates two distinct signaling branches, probably in the different domains of the growth cone, i.e., Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein, and Ca²⁺-dependent degradation of brain spectrin and the release of α-actinin by calpain. J. Neurosci. Res. 51:682–696, 1998. © 1998 Wiley-Liss, Inc.     

Effects of sphingosine, staurosporine, and phorbol ester on neurites of rat sympathetic neurons growing in compartmented cultures

Local application of sphingosine (1-10 microM), an inhibitor of protein kinase C, to NGF-supplied, distal neurites of rat sympathetic neurons in compartmented cultures caused their retraction and/or degeneration within 24 hr. This effect was specific for distal neurites because sphingosine (even at 100 microM) applied to cell bodies and/or proximal neurites did not destroy these regions of the cells, and their distal neurites continued to elongate. However, effects of other agents suggest that the retraction/degeneration observed in distal neurites directly exposed to sphingosine is not mediated by inhibition of protein kinase C: application of staurosporine, another inhibitor of protein kinase C, to distal neurites did not cause retraction or degeneration; treatment of neurons for 24 or more hours with 2 microM phorbol 12-myristate 13-acetate (PMA), used to downregulate protein kinase C activity, slowed neurite extension about 50%, but did not cause degeneration; and neurons pretreated with PMA still displayed retraction/degeneration of neurites when they were subsequently exposed to sphingosine. Also, replacement of NGF supplied to distal neurites with anti-NGF IgG did not cause retraction/degeneration of neurites within 1 d, suggesting that the effect of sphingosine did not arise by interference with the action of NGF. The specificity of the sphingosine-induced retraction/degeneration for distal neurites suggests that this effect operates via specific mechanisms in distal neurites that can trigger their retraction/degeneration. Such mechanisms could play important roles in nerve growth inhibition, nerve fiber retraction, and degeneration that occur normally in the nervous system and in response to injury and disease. Also, the ability of neurites to grow in the presence of PMA suggests that neurite growth is not dependent upon the activity of protein kinase C. However, the reduced rate of neurite extension in the presence of PMA suggests that chronic PMA treatment may affect mechanism(s) that can modulate neurite growth.     

Developmental regulation of neuron-specific P2X3 receptor expression in the rat cochlea

ATP-gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49-56), using RT-PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthal's canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2-8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP-gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission.     

Calpain inhibitors protect auditory sensory cells from hypoxia and neurotrophin-withdrawal induced apoptosis

Inhibitors of calpain have been shown to protect nerve growth factor (NGF)-deprived ciliary ganglion neurons and hypoxic cortical neurons. Calpains have been identified in the cochlea and are active during ischemic injury. Since apoptosis can be initiated by loss of neurotrophic support, hypoxia, and ototoxins (e.g., cisplatin, CDDP), the role of calpain inhibitors under these conditions was examined in auditory hair cells and neurons. Dissociated spiral ganglion neuron (SGN) cell cultures and organ of Corti explants from P3 rats were used to test the efficacy of calpain inhibitors as otoprotective molecules. Our results indicate that calpain inhibitor I, calpain inhibitor II, and leupeptin all provided significant protection of SGNs against neurotrophin-withdrawal and hypoxia-induced apoptosis. The increase in neuronal survival ranged from 2.16 to 2.31 times greater than in untreated neurotrophin-withdrawn SGN cell cultures. BOC-Asp(Ome)-Fluoromethyl Ketone (B-D-FMK), a general caspase inhibitor, increased neuronal survival 2.16 times more. Neuronal survival rates were from 1.88 to 2.27 times greater than in untreated, hypoxic neurons and hair cell survival rates were from 1.98 to 2.03 times greater than untreated, hypoxic organ of Corti explants. However, protection of auditory hair cells and neurons from CDDP-induced damage (10 and 6 μg/ml, respectively) was limited with any of these calpain inhibitors. Apoptotic pathways initiated by neurotrophin-deprivation and ototoxic stress (e.g., CDDP) have been shown to be different. Our results agree with this finding, with neurotrophin-withdrawal and hypoxia, but not CDDP damage-induced apoptosis being calpain-dependent.     

A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements

The neurotransmitter 5-HT alters growth cone motility and neurite elongation in neuron B19, isolated from the buccal ganglion of Helisoma trivolvis (Haydon et al., 1984). The effects of 5-HT are mediated by increases in intracellular calcium levels within the growth cones (Cohan et al., 1987). 5-HT causes a receptor-mediated depolarization of the membrane, which results in the opening of voltage-sensitive calcium channels. The resulting calcium influx decreases both the elongation rate and the total outgrowth of neurites. However, the mechanism(s) mediating these calcium-dependent changes is unclear. As many of the intracellular effects of calcium in eukaryotic cells are mediated by the calcium-binding protein calmodulin, we tested the involvement of such an interaction in the regulation of neurite outgrowth. In these experiments, a new, potent calmodulin antagonist with increased selectivity, CGS 9343B (CGS; Norman et al., 1987), was used to inhibit calmodulin activity during the application of 5-HT to neuron B19. The addition of 100 microM 5-HT to the culture medium resulted in a significant decrease in the rate of neurite elongation and total neurite outgrowth. Administration of CGS to the culture medium at a concentration (1.8 microM) equivalent to its IC50 for calmodulin inhibition completely blocked the inhibitory effects of 100 microM 5-HT, on both neurite elongation and total neurite outgrowth. CGS alone caused a slight decrease in elongation rate but had no significant effect on total outgrowth. CGS did not block 5-HT-induced electrical activity, indicating that it was not acting as a 5-HT receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prosurvival and proapoptotic intracellular signaling in rat spiral ganglion neurons in vivo after the loss of hair cells

Neurons depend on afferent input for survival. Rats were given daily kanamycin injections from P8 to P16 to destroy hair cells, the sole afferent input to spiral ganglion neurons (SGNs). Most SGNs die over an approximately 14-week period after deafferentation. During this period, the SGN population is heterogeneous. At any given time, some SGNs exhibit apoptotic markers--TUNEL and cytochrome c loss--whereas others appear nonapoptotic. We asked whether differences among SGNs in intracellular signaling relevant to apoptotic regulation could account for this heterogeneity. cAMP response element binding protein (CREB) phosphorylation, which reflects neurotrophic signaling, is reduced in many SGNs at P16, P23, and P32, when SGNs begin to die. In particular, nearly all apoptotic SGNs exhibit reduced phospho-CREB, implying that apoptosis is due to insufficient neurotrophic support. However, >32% of SGNs maintain high phospho-CREB levels, implying access to neurotrophic support. By P60, when approximately 50% of the SGNs have died, phospho-CREB levels in surviving neurons are not reduced, and SGN death is no longer correlated with reduced phospho-CREB. Activity in the proapoptotic Jun N-terminal kinase (JNK)-Jun signaling pathway is elevated in SGNs during the cell death period. This too is heterogeneous: <42% of the SGNs exhibited high phospho-Jun levels, but nearly all SGNs undergoing apoptosis exhibited elevated phospho-Jun. Thus, heterogeneity among SGNs in prosurvival and proapoptotic signaling is correlated with apoptosis. SGN death following deafferentation has an early phase in which apoptosis is correlated with reduced phospho-CREB and a later phase in which it is not. Proapoptotic JNK-Jun signaling is tightly correlated with SGN apoptosis.     

Cation channel control of neurite morphology

The development of neuronal polarity and morphology is essential for a functioning nervous system. The present study was undertaken to explore whether blockade of specific channels alter neuronal morphology. Retinal ganglion cells were cultured in the presence of antagonists to NMDA, AMPA/kainate, L-, N-, P-, and Q-type voltage-dependent calcium channels (VDCCs). Five parameters were measured under these conditions: the number of neurites at the cell body, total neurite length, the length of the longest neurite, the number of branch points per neurite, and the diameter of the cell soma. Antagonists to NMDA and L-type VDCCs reduce the number of neurites at the cell body; antagonists to P- and Q-type VDCCs increase the number of neurites. Antagonists to the N-type VDCCs increase total neurite outgrowth, while antagonists to the NMDA and P-type channels reduce total neurite length. Antagonists to the NMDA and L-type channels increase the length of a single neurite, while decreasing the number of branch points; antagonists to the P- and Q-type VDCCs do essentially the opposite-increase the number of neurites, while decreasing the length of each. Blockade of one or more cation channels in developing retinal ganglion cells significantly perturbs neurite morphology. This study may help elucidate part of the role that cation channel signaling plays in neuritic development.     

Substance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons

Substance P (SP), a member of the tachykinin family of neurotransmitters and neuromodulators, has been identified on spiral ganglion neurons (SGNs) in the inner ear; however, its high affinity receptor, neurokinin-1 (NK1), has not been identified and the physiological effects of SP on SGNs are not well understood. To address these issues, immunolabeling, RT-PCR, Western blots and whole-cell patch-clamp recordings were made from SGNs in P0-P5 mouse cochlear organotypic cultures. The NK1 receptor was detected on SGNs by immunocytochemistry, the protein was detected in cochlear tissues by Western blots, and the mRNA for the NK1 receptor was also found in cochlear tissues of postnatal mice (P2) by RT-PCR. Application of SP (1 to 25 microM) significantly increased the latency of SGN action potentials (APs) (mean increase 7.8 +/- 4 ms; 25 microM of SP), prolonged the duration of the action potential and made the resting potential (RP) more positive (mean 9.0 +/- 7 mV) relative to normal values (-54 +/- 6 mV). SP (1 to 25 microM) also suppressed voltage-activated potassium currents (IK+) and calcium currents (ICa2+). Puffing 25 microM of SP onto SGNs suppressed IK+ by 43 +/- 9% (n = 7) and ICa2+ by 40.6 +/- 5.6% (n = 7); both currents recovered when SP was washed out. A SP antagonist blocked the SP-induced suppression of IK+ and ICa2+. These results indicate that SP acting through NK1 receptors can have direct neuromodulatory effects on SGNs.     

Involvement of dihydropyridine-sensitive calcium channels in nerve growth factor-dependent neurite outgrowth by sympathetic neurons

We have used a number of pharmacological manipulations of calcium influx to alter the nerve growth factor (NGF)-elicited neurite outgrowth response of SCG neurons. Our results indicate that influx of extracellular calcium is critical to sympathetic SCG neurite outgrowth. Effective blockade of this process was produced by the inorganic calcium channel blockers Cd2+ (with an IC50 of 48 microM), Co2+ (129 microM), and Ni2+ (180 microM). More specifically, there is a significant contribution from dihydropyridine-sensitive L-type calcium channels to NGF-activated neurite outgrowth, as evidenced by the significant inhibition of neurite outgrowth by diltiazem (IC50 of 17 microM) and nifedipine (3 microM). Further, increases in calcium influx can elicit an enhanced neurite outgrowth response, as shown by the calcium channel agonist Bay K 8644 which potentiated neurite outgrowth by up to 40%.     

Regulation of retinal neurite growth by alterations in MAPK/ERK kinase (MEK) activity

Activation of the extracellular-signal regulated kinase (ERK) cascade may be involved in the promotion of neurite outgrowth by a variety of stimuli. For example, we have previously shown that laminin (LN) and N-cadherin activate ERK2 in chick retinal neurons, and that pharmacological inhibition of MAPK/ERK kinase (MEK), the major upstream ERK2 activator, severely impairs neurite growth induced by these proteins. We have therefore hypothesized that ERK activation through MEK is required for optimal induction of neurite growth by these proteins. Here we show that expression of mutant MEK in transfected retinal neurons alters neuronal responses to LN in a manner consistent with this hypothesis. Neurons expressing a constitutively active MEK construct extended longer neurites on LN than controls, while neurons transfected with a dominant negative construct extended shorter neurites. Further, experiments in which transfected neurons were replated onto polylysine substrates suggest that activation of MEK is sufficient for neurite promotion on a non-inducing substrate, and neurons replated onto LN confirm the pharmacological data that inhibition of MEK activation inhibits LN-induced neurite growth. We conclude that ERK activation plays a direct role in the promotion of neurite outgrowth from retinal neurons by LN.     

Factors regulating the expression of acetylcholinesterase-containing neurons in striatal cultures: effects of chemical depolarization

The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 microM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20 microM) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCl levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2+ channels--verapamil (1 microM) and nifedipine (1 microM)--did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 microM) had a partial effect. When cultures treated with 5 microM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed.