Quantitative ultrastructural localization of voltage-gated calcium channel subunits in the mouse brain

My thesis deals with the localization of T-type calcium channel are R-type calcium channel in the brain. T-type calcium channels play a pivotal role in regulating neural membrane excitability. However, the precise subcellular distributions of T-type channel subunits are not well understood. Here, I investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited gradient such that the labeling density was the highest in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about three-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells.R-type calcium channels (RTCC) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, I characterized the regional and subcellular localization of Cav2.3 at both light and electron microscopic levels. Cav2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained < 20 immunogold particles, with an average density of 260 immunoparticles per µm3 spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Cav2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites