Decreased G-protein-mediated regulation and shift in calcium channel types with age in hippocampal cultures

The membrane density of L-type voltage-sensitive Ca²⁺ channels (L- VSCCs) of rat hippocampal neurons increases over age [days in vitro (DIV)] in long-term primary cultures, apparently contributing both to spontaneous cell death and to enhanced excitotoxic vulnerability. Similar increases in L-VSCCs occur during brain aging in vivo in rat and rabbit hippocampal neurons. However, unraveling both the molecular basis and the functional implications of these age changes in VSCC density will require determining whether the other types of high-threshold VSCCs (e.g., N, P/Q, and R) also exhibit altered density and/or changes in regulation, for example, by the important G-protein-coupled, membrane-delimited inhibitory pathway. These possibilities were tested here in long-term hippocampal cultures. Pharmacologically defined whole-cell currents were corrected for cell size differences over age by normalization with whole-cell capacitance. The Ca²⁺ channel current density (picoamperes per picofarad), mediated by each Ca²⁺ channel type studied here (L, N, and a combined P/Q + R component), increased through 7 DIV. Thereafter, however, only L-type current density continued to increase, at least through 21 DIV. Concurrently, pertussis toxin-sensitive G-protein- coupled inhibition of non-L-type Ca²⁺ channel current induced by the GABA(B) receptor agonist baclofen or by guanosine 5'-3-O-(thio)triphosphate declined dramatically with age in culture. Thus, the present studies identify selective and novel parallel mechanisms for the time-dependent alteration of Ca²⁺ influx, which could importantly influence function and vulnerability during development and/or aging.