Free calcium concentration within the ER lumen ([Ca²⁺]_L) was monitored in cultured neurones isolated from dorsal root ganglia obtained from newborn (1-3 days old) Sprague-Dawley rats, killed humanely according to Schedule 1. For [Ca²⁺]_L recordings we have used mag-fura-2 (K_D ~ 25-50 µM) suitable for detecting high intraluminal [Ca²⁺] levels. The cells were loaded with Ca²⁺ probe by incubation with the membrane-permeable form of mag-fura-2 (5 µM for 20 min at 37 °C), so that the probe was trapped within both intracellular organelles and the cytoplasm. To remove the cytoplasmic portion of the dye we perfused the cells with normal, dye-free, intrapipette solution under whole-cell voltage-clamp conditions. To measure simultaneously [Ca²⁺]_L and cytoplasmic calcium concentration ([Ca²⁺]_i) the intrapipette solution was supplemented with 0.05 mM high-affinity Ca²⁺ indicator fluo-3K₅. Cell perfusion with fluo-3K₅ did not significantly affect mag-fura-2 fluorescence. Mag-fura-2 and fluo-3 signals were separated using their distinct excitation properties (ex. 340/380 nm for mag-fura-2 and 488 nm for fluo-3). All data are presented as means ± S.D.

The resting Ca²⁺ concentration within the ER varied between 60 and 270 µM (average level 177 ± 58 µM, n = 63). Brief (5 s) application of 20 mM caffeine triggered a rapid fall in [Ca²⁺]_L. The amplitude of caffeine-induced [Ca²⁺]_L decrease varied between 20 and 120 µM; however, we never observed a complete depletion of the store in response to short caffeine application. On average caffeine diminished the resting [Ca²⁺]_L value by 39 ± 11 % (n = 41).

When the voltage-clamped neurone was depolarised from -70 mV to 0 mV for 3 s we observed a significant [Ca²⁺]_i elevation that developed in parallel with transient decrease in [Ca²⁺]_L. The amplitude of [Ca²⁺]_L decrease was smaller than that evoked by caffeine varying between 5 and 30 µM; and it correlated linearly with the resting [Ca²⁺]_L: i.e. the larger the resting [Ca²⁺]_L, the higher the amplitude of [Ca²⁺]_L drop in response to depolarisation. This transient depolarisation-induced response of [Ca²⁺]_L was potentiated by 1 mM caffeine and completely blocked by 50 µM of ryanodine. Therefore these observations demonstrate directly that plasmalemmal Ca²⁺ entry triggers Ca²⁺ release from the ER lumen. To characterise in more detail the relations between Ca²⁺ entry and Ca²⁺ release from the ER we measured [Ca²⁺]_i and [Ca²⁺]_L while grading Ca²⁺ entry. First, we stimulated the DRG neurones by a standard I-V protocol, when the cell was depolarised from -70 mV by series of 400 ms steps with an increasing amplitude. This stimulation resulted in characteristic bell-shaped patterns of [Ca²⁺]_i transients, which were precisely mirrored by transient [Ca²⁺]_L decreases.

Similar direct relations between Ca²⁺ entry and Ca²⁺ release were also observed when we graded Ca²⁺ entry by varying the duration of Ca²⁺ current (I_Ca) between 50 and 3000 ms. When stimulating the neurones with such a protocol we found that the shortest I_Ca (50 ms) already induced Ca²⁺ release as indicated by transient [Ca²⁺]_L decrease. The increase in the duration of the I_Ca resulted in an almost linear increase in the amplitude of Ca²⁺ release, which saturated when Ca²⁺ current length exceeded 1 s.

Therefore, for the first time we were able to directly visualise physiological Ca²⁺-induced Ca²⁺ release (CICR) in neuronal preparations and demonstrate the linear relations between Ca²⁺ entry and the degree of CICR activation.

This research was supported by a BBSRC grant.