ca(v)2.1 channels  (Mochida Pharmaceutical)

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: a Representative confocal image of a nerve terminal arborization. Singly, dually, and innervated by three or more axons NMJs from YFP muscles and also images of the morphologic maturation (S1, the most inmature, and S4, almost fully differentiated, stages) of the postsynaptic clusters from P9 mice. The bar indicates 10 μm. b Confocal immunofluorescence location of α 1D L-, N-, and P/Q-type voltage-dependent calcium channels (VDCCs) at the NMJ. Triple labeling of VDCCs (green fluorescence) with syntaxin (blue fluorescence) and nAChR-α-bungarotoxin (red fluorescence) in merge images. Figure shows the presence of α 1D L-, N-, and P/Q-type-VDCC (in green) in the nerve terminal of P9 Levator auris longus (LAL) muscle endplates. The bar indicates 10 μm

Article Snippet: Muscles were incubated overnight at 4 °C with anti-Ca V 1.3 (CACNA1D) antibody voltage-dependent L-type calcium channel subunit α 1D (1/100; ACC-005, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.1 (CACNA1A) antibody voltage-dependent P/Q-type calcium channel subunit α 1A (1/100; ACC-001, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.2 (CACNA1B) antibody voltage-dependent N-type calcium channel subunit α 1B (1/100; ACC1-002, Alomone Labs, Jerusalem, Israel), and anti-mouse syntaxin (1/1000, S066, Sigma, St Louis, MO, USA).

Techniques: Immunofluorescence, Labeling, Fluorescence

Journal: Frontiers in Neuroanatomy

Article Title: Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images

doi: 10.3389/fnana.2022.855218

Figure Lengend Snippet: Application of Gold Rippler and Gold Star for synaptic protein analysis. (A) Ca V 2.1 and Munc13-1 colocalize at intramembrane protein-rich regions of the calyx of Held membrane which presumably represent the presynaptic active zone. Only a small portion of the larger membrane face (ROI) is shown. (B) Overall gold particle density across the calyx of Held membrane for two different SDS-digestion conditions. Gray data points represent individual ROIs and gray lines connect Ca V 2.1 and Munc13-1 densities coming from the same ROI. Solid black dots represent mean densities. Ca V 2.1 and Munc13-1 densities from the ROI partially shown in (A) are indicated by light blue circles. (C) Zoomed view of the gray box in (A) showing a conceptual illustration of Gold Rippler for visual clarity. (D) LCPI curve for real and random particle distributions and two SDS-digestion conditions, where Munc13-1 particles are set as landmarks. (E) Zoomed view of the gray box in (A) showing an illustration of Gold Star. When Munc13-1 is set as the landmark, NNDs (blue lines) are measured between each Ca V 2.1 and the closest Munc13-1 particle (blue dots). (F) Cumulative frequency distribution of real and random particle-particle NNDs pooled across the 6 ROIs for each SDS-digestion condition. (G) Grand mean particle-particle NND for each SDS-digestion condition. Error bars represent S.E.M. n = 6 ROIs for each condition. Scale bars: 100 nm.

Article Snippet: The replicas were then incubated at room temperature for overnight with following antibodies per each experiment: (1) cerebellum replicas—a guinea pig anti Ca V 2.1+ channel P/Q-type alpha-1A antibody (Synaptic Systems, Göttingen, Germany; 152–205 at 0.7 μg/ml), (2) brain stem replicas used for the large terminal image analysis—a guinea pig anti Ca V 2.1+ channel P/Q-type alpha-1A antibody (Synaptic Systems, Göttingen, Germany; 152–205 at 0.7 μg/ml) and rabbit anti GFP antibody (Abcam, Cambridge, United Kingdom; ab6556 at 1μg/ml), 3) brain stem replicas used for Ca V 2.1 and Munc13-1 localization analysis—the same guinea pig anti Ca V 2.1+ channel P/Q-type alpha-1A antibody and a rabbit anti Munc13-1 antibody (Synaptic Systems, Göttingen, Germany; 126–103 at 1 μg/ml).

Techniques: