Calcium channel density and hippocampal cell death with age in long-term culture - PubMed (original) (raw)

Calcium channel density and hippocampal cell death with age in long-term culture

N M Porter et al. J Neurosci. 1997.

Abstract

The expression of voltage-gated calcium (Ca2+) channel activity in brain cells is known to be important for several aspects of neuronal development. In addition, excessive Ca2+ influx has been linked clearly to neurotoxicity both in vivo and in vitro; however, the temporal relationship between the development of Ca2+ channel activity and neuronal survival is not understood. Over a period spanning 28 d in vitro, progressive increases in high voltage-activated whole-cell Ca2+ current and L-type Ca2+ channel activity were observed in cultured hippocampal neurons. On the basis of single-channel analyses, these increases seem to arise in part from a greater density of functionally available L-type Ca2+ channels. An increase in mRNA for the alpha1 subunit of L-type Ca2+ channels occurred over a similar time course, which suggests that a change in gene expression may underlie the increased channel density. Parallel studies showed that hippocampal neuronal survival over 28 d was inversely related to increasing Ca2+ current density. Chronic treatment of hippocampal neurons with the L-type Ca2+ channel antagonist nimodipine significantly enhanced survival. Together, these results suggest that age-dependent increases in the density of Ca2+ channels might contribute significantly to declining viability of hippocampal neurons. The results also are analogous to patterns seen in neurons of aged animals and therefore raise the possibility that long-term primary neuronal culture could serve as a model for some aspects of aging changes in hippocampal Ca2+ channel function.

PubMed Disclaimer

Figures

Fig. 1.

Photomicrographs of rat primary hippocampal neurons in culture. A, B, and_C_ show neurons of ages 1, 3, and 6 DIV, respectively. An increase in the size of the soma and the length of neurites during the first week in culture is accompanied by a rapid decline in cell number. Examples of hippocampal neurons 10, 15, and 28 DIV in culture are represented in D, E, and_F_, respectively. Scale bar (shown in A): 100 μm.

Fig. 2.

HVA whole-cell Ca2+ currents increase with age in culture. Representative whole-cell Ca2+ current traces from hippocampal neurons are shown at different ages in culture. Currents were evoked during a 150 msec depolarization step from _V_h = −70 mV to V_c = +10 mV. RCs, recorded at −70 mV after the depolarization step, typically do not appear until cells are 3 DIV. The voltage step and calibration bars are shown at the_bottom.

Fig. 3.

Rate of increase of HVA whole-cell Ca2+ current exceeds that of the capacitance of the cell with age in culture. A, Peak whole-cell Ca2+ current (•) and corresponding cell capacitance (♦) measured over time in culture. Currents were evoked during a 150 msec depolarization from _V_h = −70 mV to _V_c = +10 mV. Cell capacitance was estimated from a capacitive transient evoked by a 5 mV hyperpolarizing step from V_h = −70 mV. B, Ca2+ current density for cells shown in_A was obtained by dividing the peak whole-cell current by the cell capacitance. Current density increased rapidly during the first 1–3 DIV, remained stable until 10 DIV, and then continued to rise until 28 DIV. Values are mean ± SEM.

Fig. 4.

I–V curves for hippocampal neurons of different ages in culture. Average I–V curves from cells recorded at different times in culture are superimposed to show the relative change in HVA whole-cell Ca2+ current amplitude with age. Voltage dependence is shifted to slightly more negative potentials as cells age in culture.

Fig. 5.

Single L-type Ca2+ channel activity in hippocampal neurons increases with age in culture. Representative current traces of L-type Ca2+channels in cell-attached, multichannel patches recorded at different ages in culture. Currents were evoked during a 150 msec depolarization from _V_h = −70 mV to_V_c = +10 mV. ROs, recorded at −70 mV after the depolarization step, rarely appear before 3 DIV.

Fig. 6.

Maximal patch current (_I_max) and Ca2+channel density (N/μm2) increase with age in culture. A, _I_max(•) evoked in cell-attached patches from hippocampal cells of different age in culture. Recordings were evoked during a 150 msec depolarization from _V_h = −70 mV to_V_c = +10 mV. Patch-pipette resistance (♦) did not vary over the course of the study. B, Slope conductance of L-type Ca2+ channels was not altered as a function of age in culture. Amplitudes (i) of clearly resolvable single L-type Ca2+ channels were measured during depolarization to multiple test voltages [depolarization openings (DOs)] or on repolarization (ROs) to −70 mV in cell-attached patches from hippocampal cells 2 (•), 10 (▪), and 28 (▴) DIV. The slope conductance was determined from the regression line obtained by dividing the average amplitude by the indicated voltages for four to six patches per time point. C, L-type Ca2+ channel density increases as hippocampal neurons age in culture. As described in Materials and Methods,N (the number of channels per patch) was calculated by dividing _I_max by i; the area of the patch membrane, _μm_2, was calculated from the pipette resistance.

Fig. 7.

Phases of neuronal death correlate closely with increases in Ca2+ current density. A, Two phases of neuronal death in culture are observed: an initial rapid phase that occurs by 6 DIV and then a slower more gradual phase that continues until 28 DIV. In the cell survival studies, the same groups of cells were followed in long-term culture (see Materials and Methods). Whole-cell current density (B) is plotted again to show the relationship of current density to cell survival (A) of sister cultures. Ca2+ current density rises rapidly between 1 and 3 DIV, stabilizes, and then rises again from 10 to 28 DIV.

References

1. Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron. 1995;15:961–973. - PubMed
1. Armstrong CM, Gilly WF. Access resistance and space clamp problems associated with whole-cell patch clamping. In: Rudy B, Iverson LE, editors. Methods in enzymology Vol 207. Academic; San Diego, CA: 1992. pp. 100–122. - PubMed
1. Banker GA, Cowan WM. Rat hippocampal neurons in dispersed cell culture. Brain Res. 1977;126:397–425. - PubMed
1. Banker GA, Cowan WM. Further observations on hippocampal neurons in dispersed cell culture. J Comp Neurol. 1979;187:469–494. - PubMed
1. Barnes CA. Memory changes with age: neurobiological correlates. In: Martinez JR Jr, Kesner RP, editors. Learning and memory: a biological view. Academic; New York: 1991. pp. 259–296.

Calcium channel density and hippocampal cell death with age in long-term culture - PubMed (original) (raw)