The existence of functional InsP₃-induced Ca²⁺ release in neurones is firmly established. Several studies have demonstrated that metabotropic agonists trigger Ca²⁺ release in various types of nerve cells (see Verkhratsky & Petersen, 1998, for review). The InsP₃-driven Ca²⁺ release was also observed in Purkinje neurones in brain slices in response to synaptic stimulation (Rose & Konnerth, 2001) suggesting its functional importance. Yet beside its proven existence, the dynamics of intra-ER Ca²⁺ concentration changes in response to InsP₃ remain unknown.

Free calcium concentration within the ER lumen ([Ca²⁺]_L) was monitored in cultured neurones isolated from dorsal root ganglia obtained from neonatal (1Ð3 days old) Sprague-Dawley rats, killed according to UK legislation. The cells were loaded with Ca²⁺ probe by incubation with the membrane-permeable form of mag-fura-2 (5 mM 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 the cellular membrane was permeabilised by brief (7Ð10 s) application of saponin (0.001 %) in ‘intracellular’ solution (mM: KCl 140, ATP 3, MgCl₂ 2, CaCl₂ 0.4, EGTA 1, Hepes/KOH 20; pH 7.4, free Ca²⁺ concentration 100 nM). Fluorescence images were captured using an Olympus IX70 inverted microscope ( X 40 UV objective) equipped with a charge-coupled device cooled intensified camera (Pentamax Gene IV, Roper Scientific, UK). The specimen was alternately illuminated at 340 and 380 by a monochromator (Polychrom IV, TILL Photonics, Germany) at a cycle frequency of 1-5 Hz. Control over the experiment, image storage and off-line analysis was performed by use of MetaFluor/MetaMorph software (Universal Imaging Corporation, USA) running on a Windows 98 workstation.

Treatment with saponin triggered a rapid decrease in fluorescence signal at both excitation wavelengths, which was associated with a progressive increase in 340/380 nm ratio. The resting [Ca²⁺]_L varied between 200 and 500 µM. Brief (15Ð30 s) application of InsP₃ in concentrations between 1 and 10 mM triggered decrease in the [Ca²⁺]_L, which recovered after the removal of the drug. Stimulation of ryanodine receptors by short (5 s) administration of 20 mM caffeine triggered a much faster transient fall in [Ca²⁺]_L to 70Ð90 mM. Prolonged incubation with 5-10 mM InsP₃ led to a progressive slow depletion of [Ca²⁺]_L which stabilised at the same level. Application of caffeine immediately after prolonged incubation with InsP₃ did not produce an additional decrease in [Ca²⁺]_L. Similarly if the stores were depleted by prolonged application of caffeine, InsP₃ was not able to trigger further Ca²⁺ release. We conclude that Ca²⁺-induced Ca²⁺ release (Solovyova et al. 2002) and InsP₃-induced Ca²⁺ release mechanisms share a common ER Ca²⁺ store in DRG neurones.

This research was supported by a BBSRC research grant (ref. 34/C12751).

All procedures accord with current UK legislation.