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. 1999 Oct 1;19(19):8674-84.
doi: 10.1523/JNEUROSCI.19-19-08674.1999.

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

E M Blalock  1 , N M Porter, P W Landfield
Affiliations

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

E M Blalock et al. J Neurosci. .

Abstract

The membrane density of L-type voltage-sensitive Ca(2+) 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 Ca(2+) channel current density (picoamperes per picofarad), mediated by each Ca(2+) 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 Ca(2+) 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 Ca(2+) influx, which could importantly influence function and vulnerability during development and/or aging.

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Figures

Fig. 1.
Fig. 1.
Ca2+ channel current increase over age in culture. A, Whole-cell current averaged for each age group (n = 7, 24, 23, and 21 for 3, 7, 14, and 21 DIV, respectively) revealed a dramatic increase in peak current amplitude with age in culture. B,I–V experiments showed that maximum inward current was generated by similar voltage steps in all ages tested.C, Capacitance-normalized current revealed a large increase in density between 3 and 7 DIV with a trend toward increasing current density at later time points.
Fig. 2.
Fig. 2.
ω-CTX and nimodipine inhibited nonoverlapping components of the current record. The fraction of mean Ca2+ channel current inhibited by nimodipine (Nim; 10 μm) and ω-CTX (CTX; 1 μm) was unchanged by switching the order of drug application from nimodipine first to ω-CTX first. Two-Way ANOVA on repeated measures (RM), p< 0.01 for drug type; p > 0.5 for both presentation order and interaction.
Fig. 3.
Fig. 3.
Pharmacological dissection of whole-cell Ca2+ channel current: a different profile of functional Ca2+ channel current types with age in culture. A, Averaged current records of cells aged 3, 7, 14, and 21 DIV recorded in vehicle (0.1% EtOH and 0.1 mg/ml cytochrome c), after the addition of 10 μmnimodipine, and after the further addition of 1 μmω-conotoxin GVIA. B, Capacitance-normalized nimodipine-sensitive current (L), ω-conotoxin GVIA-sensitive current (N), and current unaffected by these blockers (P/Q + R) revealed different age-dependent profiles at peak (left panel) and late (right panel) portions of the current record.
Fig. 4.
Fig. 4.
Age in culture did not alter concentration dependence of nimodipine inhibition of Ca2+ channel current. Semilog concentration–response graph is shown for six or seven cells per age group exposed to control and five concentrations of nimodipine. Nimodipine-sensitive late current density is plotted as a function of the log of nimodipine concentration. Nonlinear fits (see Materials and Methods) and means ± SEM for each age group are plotted. Resulting fit parameters are shown in Table 1.
Fig. 5.
Fig. 5.
Baclofen-mediated inhibition of current through G-protein-dependent mechanisms. A, Cells treated 24 hr with 0.1 mg/ml BSA (n = 7) showed normal response to baclofen. Sister cultures treated 24 hr with 0.1 mg/ml PTX (n = 6) failed to respond to baclofen. Two-way ANOVA RM: baclofen, p < 0.05; PTX,p > 0.1; interaction, p < 0.05) B, Time course of percentage-normalized responses. In control cells (▪) baclofen inhibition was reversible by wash; Ca2+ channel current in PTX-treated cells (□) was not significantly altered by baclofen, and Ca2+channel current in cells pretreated with nimodipine (▴) (n = 6) was significantly inhibited (p < 0.001, paired t test) to a relatively greater degree than control cells.
Fig. 6.
Fig. 6.
Decline in baclofen-sensitive current with age in culture. A, Averaged waveforms from 7 (n = 12), 14 (n = 11), and 21 (n = 10) DIV neurons before and after 100 μm baclofen application. Baclofen significantly inhibited current in all groups (two-way ANOVA RM; *post hocTukey’s comparison, p < 0.05). B, Baclofen-sensitive current density was significantly reduced with age in culture (one-way ANOVA, p < 0.001). To facilitate comparison with previous data, N-type peak current density (gray bars) from Figure 3B is replotted.
Fig. 7.
Fig. 7.
GTPγS-sensitive current decreases with age in culture. Top, Averaged whole-cell current from control and GTPγS-treated cells at 7 and 21 DIV (n = 8–9 per group). Bottom, Peak current density (left) and percent of control peak current (right) inhibited by intracellular GTPγS both demonstrate a significant inhibition of current by GTPγS in 7 but not 21 DIV cultured cells (p < 0.05; two-way ANOVA). To facilitate comparisons with Figure 6, the amount and percent of current inhibited was calculated relative to the mean of the untreated control group.
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