The role of voltage-gated calcium channels in neurotransmitter phenotype specification: Coexpression and functional analysis in Xenopus laevis.
Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture.Cells expressing Cav 2.1 and Cav 2.2 displayed increased calcium spiking compared with cells not expressing this marker.The VGCC antagonist diltiazem and agonist (-)BayK 8644 were used to manipulate calcium activity.
Affiliation: Department of Biology, College of William and Mary, Williamsburg, Virginia, 23185.
- Calcium Channels/metabolism*
- Neurons/drug effects/physiology*
- Neurotransmitter Agents/metabolism*
- Xenopus Proteins/metabolism*
- Action Potentials/drug effects/physiology
- Calcium Channels, N-Type/metabolism
- Cells, Cultured
- Cranial Nerves/physiology
- GABAergic Neurons/drug effects/physiology
- Glutamic Acid/metabolism
- In Situ Hybridization, Fluorescence
- Spinal Cord/physiology
- Vesicular Glutamate Transport Protein 1/metabolism
- Vesicular Inhibitory Amino Acid Transport Proteins/metabolism
- Xenopus laevis
- gamma-Aminobutyric Acid/metabolism
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fig03: Coexpression patterns of xVIAAT and VGCC α1 subunits in the central nervous system of Xenopus laevis swimming tadpole embryos. VGCC subunit expression is labeled with fluorescein (green) and xVIAAT expression is labeled with Cy3 (red). Coexpression is indicated by the yellow overlap of both channels. A–D: Cav1.2 coexpression with xVIAAT in the (A) forebrain, (B) midbrain, (C) hindbrain, (D) anterior spinal cord, and (E) posterior spinal cord. F–J: Cav1.3 coexpression in the (F) forebrain, (G) midbrain, (H) hindbrain, (I) anterior spinal cord, and (J) posterior spinal cord. K–O: Cav2.1 coexpression in the (K) forebrain, (L) midbrain, (M) hindbrain, (N) anterior spinal cord, and (O) posterior spinal cord. P–S: Cav2.2 coexpression in the (P) forebrain, (Q) midbrain, (R) hindbrain, and (S) spinal cord. Cav3.1 coexpression in the (T) forebrain, (U) midbrain, (V) hindbrain, and (W) spinal cord. X–AA: Cav3.2 coexpression in the (X) forebrain, (Y) midbrain, (Z) hindbrain, and (AA) spinal cord. BB–DD: Cav2.3 coexpression in the (BB) midbrain, (CC) hindbrain, and (DD) spinal cord. For the assistance of color-blind readers, a magenta–green copy of this figure is provided as Supplementary Figure 4. Scale bar = 100 μm in A (applies to A–DD).
To determine whether VGCC α1 subunits are coexpressed with inhibitory neurotransmitter markers, multiplex FISH analysis was performed, probing for VGCCs and xVIAAT. To a lesser extent, VGCC α1 subunits are coexpressed with the inhibitory neural marker (Table3). Forebrain and midbrain coexpression is restricted to ventral regions and occurs most prominently with Cav1.2, Cav2.1, and Cav2.2 (Fig. 3A,B,K,L,P,Q). Hindbrain coexpression is located dorsally and occurs with Cav1.2, Cav1.3, and Cav2.2 (Fig. 3C,H,R). In the spinal cord, coexpression is found with Cav1.2, Cav1.3, Cav2.1, and Cav2.2 in the inhibitory commissural reciprocal interneurons (cINs) and ascending recurrent interneurons (aINs) (Fig. 3D,I,N,S). Retina coexpression occurs in the inner nuclear layer (INL) with Cav2.1 and Cav2.2 (Fig. 4D,E).