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Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain.

ABSTRACT: The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q type voltage-gated calcium channels (VGCCs) and removal of extracellular Ca(2+) or treatment with a combination of L-type, N-type, and P/Q-type VGCC antagonists rescues SGN neurite growth under depolarizing conditions. By measuring the fluorescence intensity of SGNs loaded with the fluorogenic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (20 microM), we demonstrate that depolarization activates calpains. Calpeptin (15 microM), a calpain inhibitor, prevents calpain activation by depolarization and rescues neurite growth in depolarized SGNs suggesting that calpain activation contributes to the inhibition of neurite growth by depolarization.

SUBMITTER: Roehm PC

PROVIDER: S-EPMC2265381 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain.

Roehm Pamela C PC Xu Ningyong N Woodson Erika A EA Green Steven H SH Hansen Marlan R MR

Molecular and cellular neurosciences 20071101 2

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q ty ...[more]

PMID: 18055215

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Project description:As with other elements of the peripheral auditory system, spiral ganglion neurons display specializations that vary as a function of location along the tonotopic axis. Previous work has shown that voltage-gated K(+) channels and synaptic proteins show graded changes in their density that confers rapid responsiveness to neurons in the high frequency, basal region of the cochlea and slower, more maintained responsiveness to neurons in the low frequency, apical region of the cochlea. In order to understand how voltage-gated calcium channels (VGCCs) may contribute to these diverse phenotypes, we identified the VGCC α-subunits expressed in the ganglion, investigated aspects of Ca(2+)-dependent neuronal firing patterns, and mapped the intracellular and intercellular distributions of seven VGCC α-subunits in the spiral ganglion in vitro. Initial experiments with qRT-PCR showed that eight of the ten known VGCC α-subunits were expressed in the ganglion and electrophysiological analysis revealed firing patterns that were consistent with the presence of both LVA and HVA Ca(2+) channels. Moreover, we were able to study seven of the α-subunits with immunocytochemistry, and we found that all were present in spiral ganglion neurons, three of which were neuron-specific (Ca(V)1.3, Ca(V)2.2, and Ca(V)3.3). Further characterization of neuron-specific α-subunits showed that Ca(V)1.3 and Ca(V)3.3 were tonotopically-distributed, whereas Ca(V)2.2 was uniformly distributed in apical and basal neurons. Multiple VGCC α-subunits were also immunolocalized to Schwann cells, having distinct intracellular localizations, and, significantly, appearing to distinguish putative compact (Ca(V)2.3, Ca(V)3.1) from loose (Ca(V)1.2) myelin. Electrophysiological evaluation of spiral ganglion neurons in the presence of TEA revealed Ca(2+) plateau potentials with slopes that varied proportionately with the cochlear region from which neurons were isolated. Because afterhyperpolarizations were minimal or absent under these conditions, we hypothesize that differential density and/or kinetics of one or more of the VGCC α-subunits could account for observed tonotopic differences. These experiments have set the stage for defining the clear multiplicity of functional control in neurons and Schwann cells of the spiral ganglion.

Project description:1. In this study, intracellular Ca(2+) was measured as the Fura-2 ratio (R) of fluorescence excited at 340 and 380 nm (F(340)/F(380)) in nonpressurized rat mesenteric small arterioles ( (lumen diameter) 10-25 microm). 2. The response to depolarization using 75 mm KCl was an increase in R from a baseline of 0.96+/-0.01 ([Ca(2+)](i) approximately 74 nm) to 1.04+/-0.01 ( approximately 128 nm) (n=80). The response to 75 mm K(+) was reversibly abolished in Ca(2+)-free physiological saline solution, whereas phentolamine (10 microm) or tetrodotoxin (1 microm) had no effects. LaCl(3) (200 microm) inhibited 61+/-9% of the response. 3. A [K(+)]-response curve indicated that the Ca(2+) response was activated between 15 and 25 mm K(+). The data suggest that the Ca(2+) response was caused by the activation of voltage-dependent Ca(2+) channels. 4. Mibefradil use dependently inhibited the Ca(2+) response to 75 mm K(+) by 29+/-2% (100 nm), 73+/-7% (1 microm) or 89+/-7% (10 microm). Pimozide (500 nm) use dependently inhibited the Ca(2+) response by 85+/-1%. 5. Nifedipine (1 microm) inhibited the Ca(2+) response to 75 mm K(+) by 41+/-12%. The response was not inhibited by calciseptine (500 nm), omega-agatoxin IVA (100 nm), omega-conotoxin MVIIA (500 nm), or SNX-482 (100 nm). 6. Using reverse transcriptase-polymerase chain reaction, it was shown that neither Ca(V)2.1a (P-type) nor Ca(V)2.1b (Q-type) voltage-dependent Ca(2+) channels were expressed in mesenteric arterioles, whereas the Ca(V)3.1 (T-type) channel was expressed. Furthermore, no amplification products were detected when using specific primers for the beta(1b), beta(2), or beta(3) auxiliary subunits of high-voltage-activated Ca(2+) channels. 7. The results suggest that the voltage-dependent Ca(2+) channel activated by sustained depolarization in mesenteric arterioles does not classify as any of the high-voltage-activated channels (L-, P/Q-, N-, or R-type), but is likely to be a T-type channel. The possibility that the sustained Ca(2+) influx observed was the result of a T-type window current is discussed.

Project description:Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded spontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na+ channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Cav1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Cav1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Cav1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Cav1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies.SIGNIFICANCE STATEMENT Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca2+ indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca2+ signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.

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Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain.

Publications

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain.

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