Knockdown of microglial Cav2.2 N-type voltage-dependent Ca2+ channel ameliorates behavioral deficits in a mouse model of Parkinson's disease.

Cav2.2 N-type voltage-dependent Ca2+ channel (VDCC) expressed in neurons is known to be essential for neurotransmitter release. We have shown previously that this channel is also expressed in nonexcitable microglia and plays pivotal roles in microglial functions. Here, we have examined the effects of microglia-specific knockdown (KD) of Cav2.2 channel in a mouse model of Parkinson's disease (PD). We found that the KD of Cav2.2 channel reduces the accumulation of microglia in the substantia nigra and ameliorates the behavioral deficits in PD model mice. These results are in marked contrast with those found in microglia-specific KD of Cav1.2 L-type channel, where exacerbated symptoms are observed. Our results suggest that blockade of microglial Cav2.2 N-type VDCC is beneficial for the treatment of PD.

Neuroanatomy of pain-deficiency and cross-modal activation in calcium channel subunit (CACN) α2δ3 knockout mice.

The phenotype of calcium channel subunit (CACN) α2δ3 knockout (KO) mice includes sensory cross-activation and deficient pain perception. Sensory cross-activation defines the activation of a sensory cortical region by input from another modality due to reorganization in the brain such as after sensory loss. To obtain mechanistic insight into both phenomena, we employed a comprehensive battery of neuroanatomical techniques. While CACNα2δ3 was ubiquitously expressed in wild-type mice, it was absent in α2δ3 KO animals. Immunostaining of α1A, α1B, and α1E revealed upregulation of N-type and R-type, but not P/Q-type Cav2 channels in cortical neurons of CACNα2δ3 KO mice. Compared to wild-type mice, axonal processes in somatosensory cortex were enhanced, and dendritic processes reduced, in CACNα2δ3 KO mice. Immunohistochemical and MRI analyses, investigating morphology, thalamocortical and intra-/intercortical trajectories, revealed a disparity between projection and commissural fibers with reduction of the number of spatial specificity of thalamocortical projections. L1cam staining revealed wide-ranging projections of thalamocortical fibers reaching both somatosensory/motor and visual cortical areas. Activation (c-fos+) of excitatory and inhibitory neurons suggested that deficient pain perception in α2δ3 KO mice is unlikely to result from cortical disinhibition. Collectively, our data demonstrate that knock out of CACN α2δ3 results in some structural abnormalities whose functional implications converge to dedifferentiation of sensory activation.

Intracellular calcium mobilization of the aganglionic intestine in the endothelin B receptor gene-deficient rat.

BACKGROUND AND AIM: Up to now, numerous reports have analyzed the pathogenesis of Hirschsprung's disease (HD) by means of physiologic, pathologic, or molecular biologic methods. However, very little is still known about the smooth muscle cell itself. The endothelin B receptor gene-deficient (EDNRB(-/-)) rat, which is suitable for research of HD, has an aganglionic segment of the total colon. Our purpose is to investigate the myogenic mechanisms using simultaneous measurements of the intracellular Ca2+ concentration ([Ca2+]i) and tension and reverse transcriptase polymerase chain reaction for L-type Ca2+ channel (L-VOC) expression. METHODS: The muscle strips of the rat distal colon were loaded with a Ca2+ indicator dye, fura-PE3/AM, for 3 to 4 hours. The changes in the fluorescence intensity of Ca2+-fura-PE3 complex of the strips were monitored with a front surface fluorometer (CAM-230). The fluorescence intensities at 340- and 380-nm excitation and their ratio (F340/F380) were recorded as the level of [Ca2+]i. The comparison of L-VOC alpha1c subunit messenger RNA (mRNA) expression in both wild and homozygous rat was performed by reverse transcriptase polymerase chain reaction. RESULTS: The peak levels of force development induced by carbachol were 139.1% +/- 5.0% in EDNRB(-/-) rat, whereas the peak levels were 242.1% +/- 27.7% in EDNRB(+/+) rat. The changes in the [Ca2+]i elevation induced by carbachol were 101.7% +/- 12.2% in the homozygous rat, whereas these were 143.8% +/- 8.9% in the wild-type rat. Both results in the homozygous rat significantly decreased in comparison with those of the wild rat (P < .05). The expression of the L-VOC channel mRNA also decreased in the homozygous rat. CONCLUSIONS: This is the first report to show the [Ca2+]i mobilization in the smooth muscles of the rat model of HD. The decrease in both [Ca2+]i and force development was thus considered to be due to the decrease in the Ca2+ channel expression.

Altered inactivation of Ca2+ current and Ca2+ release in mouse muscle fibers deficient in the DHP receptor gamma1 subunit.

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit gamma1 have previously been studied using coexpression with the cardiac alpha1C polypeptide in nonmuscle cells and primary-cultured myotubes of gamma1-deficient mice. Data from single adult muscle fibers of gamma-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from gamma+/+ and gamma-/- mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of -80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in gamma-/- compared with gamma+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the gamma1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.