Despite the many studies regarding the development of the signalling components present in the plasma membrane of neurons, few have addressed the development of intracellular calcium signalling machinery. In this study, we investigated the effects of spontaneous calcium signals, resulting from synaptogenesis, on the maturation of metabotropic responses.
Post-natal (1-2 days old) Porton-Wistar rat pups were killed according to Home Office guidelines. Cultures of rat hippocampal neurons were prepared as described previously (Koizumi et al. 1999) and cells were studied between 4 and 21 days in culture. Functional synaptic networks were obtained by culturing neurons at 35 000 cm-2 (‘high-density’ neurons) and were compared against synaptically inactive neurons cultured at 5000 cm-2 (‘low-density’ neurons). Changes in intracellular calcium concentration were measured using video imaging of fura-2 (acetoxymethyl ester loading). Caffeine (40 mM) and carbachol (10 µM) were employed as agonists of ryanodine receptors and muscarinic acetylcholine receptors, respectively. All data are given as means ± S.E.M.
Responses to acute caffeine stimulation were absent in low-density neurons at any stage of the cultures. In contrast, high-density neurons began to show calcium release in response to caffeine following 9 days in culture. These caffeine-evoked calcium signals progressively increased in amplitude from day 9 to 21, reaching a peak amplitude of 286 ± 34 nM (n = 33 cells). Similar results were obtained using carbachol: low-density neurons did not respond to carbachol at any stage, whereas high-density neurons responded to carbachol from day 9. The amplitude of the carbachol responses peaked at 264 ± 60 nM (n = 26 cells) on day 15.
Superfusion with 25 mM KCl depolarised the neurons, giving calcium signals that served to increase the loading of the intracellular calcium stores. Such store loading allowed similar amplitude caffeine-evoked calcium signals in low- and high-density neuronal cultures. KCl application also promoted the response of low-density neurons to carbachol. However, there was a significant difference in the amplitude of the carbachol-evoked calcium signals between low- and high-density neurons. This indicates that the development of metabotropic signalling, but not ryanodine receptor expression, was dependent on neuronal density.
The development of caffeine- and carbachol-evoked responses from day 9 onwards in the high-density neurons correlated with the onset of spontaneous calcium oscillations, most probably arising from the formation of synaptic contacts between cells. Such oscillations were rare in the low-density neuronal cultures. The spontaneous activity serves a dual purpose of increasing the loading of intracellular calcium stores and also promoting the expression of proteins involved in metabotropic responses.