Activity-evoked capacitative Ca2+ entry: implications in synaptic plasticity

Abstract

The Ca2+ influx controlled by intracellular Ca2+ stores, called store-operated Ca2+ entry (SOC), occurs in various eukaryotic cells, but whether CNS neurons are endowed with SOC capability and how they may operate have been contentious issues. Using Ca2+ imaging, we present evidence for the presence of SOC in cultured hippocampal pyramidal neurons. Depletion of internal Ca2+ stores by thapsigargin caused intracellular Ca2+ elevation, which was prevented by SOC channel inhibitors 2-aminoethoxydiphenyl borate (2-APB), SKF96365, and La3+. Interestingly, these inhibitors also accelerated the decay of NMDA-induced Ca2+ transients without affecting their peak amplitude. In addition, SOC channel inhibitors attenuated tetanus-induced dendritic Ca2+ accumulation and long-term potentiation at Schaffer collateral-CA1 synapses in hippocampal slice preparations. These data suggest a novel link between ionotropic receptor-activated SOC and neuroplasticity.

Figure 1.

Both hippocampal and dentate neurons display SOC. A, Representative traces of somatic [Ca²⁺]_i dynamics obtained from a pyramidal neuron. A [Ca²⁺]_i rise was evoked by pretreatment with 1 μm thapsigargin (Tg (+)) for 5 min in Ca²⁺-free conditions (open bars) and subsequent bath addition of 1.8 mm Ca²⁺ (closed bar). B, The [Ca²⁺]_i rise was prevented by 30 μm 2-APB, 100 μm La³⁺, but not by 5 μm nicardipine. These agents were continuously applied from 5 min before thapsigargin. C, Summary of the effects of 2-APB, La³⁺, 5 μm nicardipine, and 10 μm verapamil on SOC in cultured pyramidal and granule neurons. The ordinate indicates the average amplitude of capacitative [Ca²⁺]_i plateaus as an increase in F_340/360 ratios (%). ^*p < 0.05, ^**p < 0.01 versus corresponding control: Fisher's protected least significant difference after one-way ANOVA (n = 11-34 neurons from 3-8 independent experiments).

Figure 2.

NMDA receptor-activated SOC in hippocampal pyramidal neurons. A, Representative traces of[Ca²⁺]_itransients evoked by local application of NMDA (10μm for 10 sec) in the absence (top) or presence (bottom) of 30μm 2-APB, which were obtained from each one pyramidal (left) or granule (right) cell. After the initial increase, the somatic[Ca²⁺]_i decayed with a monoexponential time course with the mean time constantτ_f (dotted lines) in granule cells, whereas pyramidal cells exhibited double-exponential decay kinetics with the time constants τ_f (dotted lines) and τ_s (broken lines). B, Summary of the effects of 30 μm 2-APB, 3 μmSKF96365 (SKF), 100 μmLa³⁺, and 1 μm thapsigargin (Tg) on NMDA responses in cultured Ammon's horn and dentate gyrus neurons. The ordinate indicates the average coefficients of the fast and slow components in double-exponential[Ca^{2 +}]_i decay kinetics of NMDA responses(A_f and A_s, respectively). All the drugs were continuously perfused from 5 min before NMDA exposure. ^**p < 0.01 versus control, ^#p < 0.05 versus Tg: Fisher's protected least significant difference following one-way ANOVA (n = 7-64 neurons from 3-10 independent experiments). C, Effect of extracellular Ca²⁺ removal and 30 μm 2-APB on the A_s component. D, No [Ca²⁺]_i rise occurred when NMDA (10 μm for 10 sec) was applied in the absence of external Ca²⁺ (n = 7).

Figure 3.

Synaptic activation induces SOC in hippocampal pyramidal cells. A, Example of the NMDA channel activity in an outside-out patch isolated from a cultured pyramidal neuron before or after application of 30μm 2-APB. B, Image of a fura-2-loaded neuron. C, Representative optical recordings of tetanus-elicited [Ca²⁺]_i changes in a region of the neuron delimited by the box shown inB. Electrical field stimulation(100 Hz for 1 sec, 60 V, 200 μsecduration) was applied in the absence or presence of 30μm 2-APB or 50μm AP-5. D, Summary of the effects of AP-5 and 2-APB on tetanus-elicited [Ca²⁺]_i elevation. The ordinate shows the average area under the curve (AUC) of ΔF/F_340/360 during and after the tetanus (n = 7 neurons). ^**p < 0.01 versus control: Fisher's protected least significant difference after one-way ANOVA.

Figure 4.

SOC channel inhibitors attenuate hippocampal CA1 LTP. A, Representative time course of changes in fEPSPs evoked at Schaffer collateral-CA1 synapses after tetanic stimulation (100 Hz for 1 sec) applied in the absence (open circles) or presence (closed circles) of 2-APB. 2-APB was applied during time -15 to 5. The insets indicate field potentials recorded at times 0 (pre) and 60(post). The fEPSP slopes are expressed as a percentage of changes from baseline. B, Summary of the effects of 30μm 2-APB and 3μmSKF96365 (SKF) on the LTP magnitude. The ordinate shows the average changes in fEPSP slopes at time 55-60 (n = 6-16 recordings). ^*p < 0.05 versus control: Student's t test.