Developmental Changes In The α 1 Subunit Composition Of L-type Voltage-gated Calcium Channel In Rat Hippocampal Subregions

Voltage-dependent calcium channels (VDCCs) play an important role in neuronal development and are essential for converting electrical activity into biochemical events. Both voltage-dependent calcium channels and calcium entry have been implicated in many processes of immature neurons including neurite outgrowth, neuronal migration, axon and dendrite extension and synaptic plasiticy. Calcium channels have also been implicated in the initiation of developmental gene expression.Multiple VDCCs (N-, P/Q-, L-, and R-type) are expressed in neuronal tissues .The α1 subunit, of which there are at least six genes, resides in the membrane and forms the pore of the channel. L-type voltage-sensitive Ca2+ channels (L-VSCCs) are composed of up to four distinct subunits (α1,β,α2-δ and γ), and two al subunits(CaV1.2α1c and CaV1.3α1D.) have been identified in the brain. By the use of gene-targeted null mutant mice and heterologous expression systems, researchers have previously demonatrated that CaV1.3α1D subunit was activated at more negative potential level and less sensitive to dihydropyridine (DHP) than CaV1.2α1c subunit, which hints that they might possess different biological functions.Several families of ligand-gated ion channels evidence developmental changes in their subunit composition. However, few reports have investigated the developmental expression of calcium channel subunits. By the use of immunohistochemical studies at the light microscopy level, we found that α1-subunits exhibit differential subcellular distribution and relocalization within hippocampus after postnatal development in rat.We examined DAB-developed hippocampal sections from P1 to adult rats. At P1, both CaV1.2α1C and CaV1.3α1D-antibody positively stained neurons were equallydiscernible in any hippocampal subfield and the dentate gyrus (DG). However, from P7 to adult, Within the hippocampal formation, we observed a wide distibution of neurons which are immunoreactive for L-type calcium channels recognized by the CaV1.3aio antibody. Cell bodies of pyramidal neurons in the CA1-CA3 areas and granule cells in DG are smoothly stained with the CaV1.3aiD antibody. Intemeurons in the stratum oriens, stratum radiatum, and lacunosum moleculare of the hippocampus and intemeurons in the pexiform layer of DG are also immunoreactive for the CaV1.3aiD subunit of L-type calcium channels. In each case, there is dense immunoreactivity in neuronal cell bodies and often an accumulation of immunoreactivity in the very region where the basal and apical dendrites emerge from the cell body. In contrast, there is relatively weak immunoreactivity for the CaV1.3aio antibody in the dendrites of nurons which diminishes progressively in more distal portions. Yet, at P7, five of the seven rats showed that CaV1.2aic was mainly distributed in area CA3 of hippocampus with very slight labeling within the CA1 region and DG, only the other two showed similar immunoreactive staining to that of the CaV1.3aio antibody. From P14 to adult, The CaV1.2aic antibody shows immunoreactivity which is quite different in its general distribution within the CA1 region of hippocampus and DG to that of the CaV1.3aio antibody: slightly distributed in neuronal cell bodies while strongly labeled in the dendritic fields. In area CA3 of hippocampus, strong staining of both the cell bodies and the proximal dendritic fields of pyramidal cells is found. However, the class C L-type calcium channels are strikingly clustered rather than smoothly distributed along the cell surface, and staining is clearly detectable within stratum oriens, stratum radistum of hippocampus and molecular layer and pexiform layer of DG The immunoreactivity is abolished when the CaVI.2aicantibody is blocked by the CaV1.2aic peptide, but is unaltered when the CaV1.2alC antibody is preincubated with the CaV1.3aio peptide, indicating that the CaV1.2alC antibody is specific for the CaV1.2aic subunit and does not cross react with the corresponding CaV1.3aio sequence, and vice versa. For quantitative analysis positive expression, relative protein abundance gray density of coarse granular immunoproducts of hippocampus and DG from each animal were selected and quantitated.By quantitative comparision on relative protein abundance-grey density of coarse granular immunoproducts of CaV1.2aic and CaV1.3aiD subunits, we found that temporal distributon of CaV1.2aic subunits in neuronal cell bodies within CA1 area of hippocampus increased at P7(P<0.05,n=7), and remained stable afterwards. In areaCA3 of hippocampus, however; the CaV1.2aiC antibody shows immunoreactivity which increased at both P7and AD group (P<0.05,n=7) . In area DG of hippocampus, the expression of CaV1.2aic remained at a relatively low level, with a slightly increasing at AD group (P<0.05,n=8) . Immunoreactivity of CaV1.3aio in cell bodies of pyramidal neurons in the CA1 area and granule cells in DG area increased significantly at P7 and P14 (P<0.05,n=7), while in cell bodies of pyramidal neurons in the CA3 area, immunoreactive staining for CaV1.3aID gradually increased from P7 to AD (P<0.05) . As far as the dendritic staining for CaV1.2aic and CaV1.3aio subunits is concerned, we checked those from CA1 and CA3 area of hippocampus. In CA1 area, CaV1.2aic mainly located in the dendritic area and CaVOaiD showed little dendritic staining. Since two different types of staining for CaV1.2aic from the apical dendrite were observed, we named apical dendrite within 50 um from the soma as ADI, and apical dendrite 50um away from the soma as AD2. Immunoreactivity of CaV1.2aic increased significantly at P14 in both AD2 and basal dendrite (P<0.05,n=7) , however; the expression of CaV1.2aic in ADI area remained a relatively low level, and increased only at AD group (P<0.05,n=7 ) . The immunoreactive staining for CaVOctio showed no significant change from P7 to AD in ADI > AD2andBD.By using patch clamp techniques, we recorded the whole-cell L-type calcium currents of actually dissociated hippocampal CA1 and CA3 neurons. We identified L-type voltage-gated calcium channel by it's voltage-dependence > high selectivity for calcium ions > fast-activating , long-lasting and highly sensitive to nifedipine and Bay K 8644. We used 5mM Ba as charge carrier and whole-cell Ca2+ current recordings elicited by a series of depolarizing voltage-clamp steps from a holding potential of-50mV were performed in hippocampal CA1 and CA3 neurons at PI and AD. The activation thresholds peak potential, maximum average current density and reverse potential were observed for cells at each group. The activation threshold, peak potential and reverse potential in hippocampal CA1 (and CA3) neurons at PI were -30mV> + 10mV, and +50mV, respectively. The maximum average current density in hippocampal CA1 and CA3 neurons at PI were 9.93 + 0.63 pA/pF(n=ll) and 8.94 + 0.58 pA/pF(n=8) respectively. The activation threshold, peak potential and reverse potential in hippocampal CA1 (and CA3) neurons at AD were -30mV, + 10mV, and + 50mV, respectively. The maximum average current density in hippocampal CA1 and CA3 neurons at AD were 28.35 + 2.56 pA/pF(n=8) and 27.32 +2.54 pA/pF(n=8), respectively.Then, we recorded whole-cell Ca2+ current in hippocampal slices by the use of lmM Ca as charge carrier, O.OOlmM TTX and lOmM TEA-Cl as blockers of sodium and potassium channels, respectively. Again, the activation threshold > peak potential maximum average current density and reverse potential were observed for cells at each group. The activation thresholds peak potential and reverse potential in hippocampal CA1 (and CA3) neurons at PI were -30mV> +10m"V\ and +50mV, respectively. The maximum average current density in hippocampal CA1 and CA3 neurons at PI were 4.02 + 0.32 pA/pF(n=12) and 3.94 ± 0.45 pA/pF(n=10), respectively. The activation threshold n peak potential and reverse potential in hippocampal CA1 (and CA3) neurons at P14 (and AD as well) were -30mV> + 10mV\ and + 50mV, respectively. The maximum average current density in hippocampal CA1 and CA3 neurons at P14 were 8.33 + 0.79 pA/pF(n=5) and 8.25 + 0.91 pA/pF(n=4), respectively. At AD group, the maximum average current density in hippocampal CA1 and CA3 neurons were 10.09 + 0.87 pA/pF(n=4) and 11.35 + 1.10 pA/pF(n=4), respectively.These results demonstrate that al-subunits of L-type calcium channels exhibit differential subcellular distribution and relocalization within hippocampus after postnatal development in rat. The maximum of the current-voltage relationship as well as activation threshold and the maximum average current density was shifted significantly to hyperpolarized potentials in both CA1 and CA3 neurons acutely dissociated from hippocampus or from hippocampal slices during postnatal development. Thus, our results may help researchers better understand the functions of CaV1.2aic and CaV1.3aio subunits of L-type calcium channels.