Alomone Labs Ltd polyclonal anti-L-type CaV1.3 (ACC-0050) antibodies labeled a high molecular weight band of ~230?kDa and a low Rabbit Polyclonal to MEN1 molecular weight band of ~200?kDa (top panel). dendrites at all ages studied (P1C72). Immunohistochemical studies on one-week-old hippocampi demonstrated significantly more colocalization of GABAB receptors with CaV1.2 than with CaV1.3, suggesting that modulation of L-type calcium current in early development is mediated through CaV1.2 channels. 1. Introduction Calcium is an ideal signaling molecule within neurons because the intracellular concentration is kept very low by calcium binding proteins as well as transporters that sequester calcium in intracellular organelles. Therefore, very small changes in the intracellular calcium concentration can act as a molecular switch, controlling a variety of cellular processes such as regulation of gene expression, neurotransmitter release, propagation of action potentials, synaptic plasticity, neurite outgrowth, cell death, and muscle contraction. Increases in free intracellular calcium can be mediated through release from intracellular stores or by influx through ligand gated or voltage gated channels within the cell membrane. There are 5 broad classes of voltage dependent calcium channels (L, N, P/Q, R, T) characterized by their respective kinetics, voltage dependence, and pharmacological sensitivity (for review, see [1, 2]). The different physiological characteristics of these channels allow for diverse function. In addition to the biophysical properties of the channels, individual channels are located in different regions of neurons reflecting their role in cellular function. For example, the N- and P/Q-type channels are found mainly on the presynaptic terminals of neurons where they control neurotransmitter release [3, 4]. In contrast, L-type calcium channels are typically found on the soma or dendrites of neurons where they regulate enzymatic activity, excitability, and gene expression [5, 6]. Voltage dependent channels are heteromeric structures composed of 4 to 5 different subunits. The = 3 or 4 4). Antibodies against CaV1.3 labeled two bands of ~200?kDa and ~230?kDa on the Western blots (Figure 2(a)). The specificity of the antibodies used against CaV1.3 was previously determined by Fossat et al.  by probing the spinal cord for CaV1.3 expression following channel knockdown using a peptide nucleic acid- (transportan 10-PNA conjugates) based antisense strategy. In contrast to the CaV1.2 bands, these two bands were relatively even in their expression over time with neither dominating significantly over the other. Thus, the integrated optical density of both bands were added together for the final analysis of steady-state expression (Figure 2(b)). The steady-state expression level of CaV1.3 was very low on P1 and rose steadily to reach approximate adult levels of expression by P15. Open in a separate window Figure 2 Steady-state expression of L-type CaV1.3 channels in the superior region of rat hippocampus. (a) Representative European blot analysis of proteins extracted from your superior region of hippocampi from postnatal day time 1 through 72 rats. Alomone Labs Ltd polyclonal anti-L-type CaV1.3 (ACC-0050) antibodies labeled a high molecular weight band of ~230?kDa and a low molecular weight band of ~200?kDa (top panel). Antibodies against = 3 Quinidine or 4 4). 3.2. Localization of CaV1.2 and 1.3 Utilizing Confocal Microscopy In order to distinguish between the different hippocampal regions stained with antibodies against CaV1.2 and CaV1.3, all the sections were stained with the nuclear stain DAPI (4,6-diamidino-2-phenylindole) to allow visualization of the cell layers within the hippocampus. Number 3 demonstrates the various regions of the hippocampus (CA1, dentate gyrus, etc.) using a low-magnification image of a hippocampal section taken from a P8 rat. High-magnification images of the CA1 Quinidine region including the pyramidal cell coating with either the stratum oriens or stratum radiatum were utilized to analyze the distribution of the channels across the layers of the superior hippocampus. Since sections processed on different days may differ in intensity due to bleaching of fluorophores or minor variations in protocol, Quinidine we chose not to determine changes.