Calcium dynamics associated with action potentials in single nerve terminals of pyramidal cells in layer 2/3 of the young rat neocortex

1. Calcium dynamics associated with a single action potential (AP) were stu died in single boutons of the axonal arbor of layer 2/3 pyramidal cells in the neocortex of young (P14-16) rats. We used fluorescence imaging with two -photon excitation and Ca2+-selective fluorescence indicators to measure vo lume-averaged Ca2+ signals. These rapidly reached a peak tin about 1 ms) an d then decayed more slowly (tens to hundreds of milliseconds). 2. Single APs and trains of APs reliably evoked Ca2+ transients in en passa nt boutons located on axon collaterals in cortical layers 2/3, 4 and 5, ind icating that APs propagate actively and reliably throughout the axonal arbo r. Branch point failures are unlikely to contribute to differences in synap tic efficacy and reliability in the connections made by layer 2/3 pyramidal cells. 3. AP-evoked Ca2+ transients in boutons were mediated by voltage-dependent Ca2+ channels (VDCCs), predominantly by the P/Q- and N-subtypes. 4. Ca2+ transients were, on average, of significantly larger amplitude in b outons than in the flanking segments of the axon collateral. Large amplitud e Ca2+ transients in boutons were spatially restricted Do within less than or equal to 3 mum of axonal length. 5. Single BP-evoked Ca2+ transients varied up to 10-fold across different b outons even if they were located on the same axon collateral. In contrast, variation of Ca2+ transients evoked by successive APs in a given single bou ton was small (coefficient of variation, c.v less than or equal to 0.21). 6. Amplitudes of AP-evoked Ca2+ signals did not correlate with the distance of boutons from the soma. In contrast, AP-evoked Ca2+ signals in spines of basal dendrites decreased slightly (correlation coefficient, r(2) = -0.27) with distance from the soma. 7. Measurements with the low-affinity Ca2+ indicator Magnesium Green sugges t that the volume-averaged residual free [Ca2+], in a bouton increases on a verage by 500 nM following a single AP. Higher concentrations of indicator caused, on average, a decrease in the amplitude and an increase in the deca y time constant of Ca2+ transients. Assuming a single-compartment model the concentration dependence of decay time constants suggests a low endogenous Ca2+ binding ratio close to 140, indicating that of the total Ca2+ influx (similar to2 fC) less than 1 % remained free. 8. The indicator concentration dependence of decay time constants further s uggests that the residual free Delta [Ca2+](1) associated with an AP decays with a time constant of about 60 ms (35 degreesC) reflecting a high Ca2+ e xtrusion rate of about 2600 s(-1) 9. The results show that BP-evoked volume-averaged Ca2+ transients in singl e boutons are evoked reliably and, on average, have larger amplitudes than Ca2+ transients in other subcellular compartments of layer 2/3 pyramidal ce lls. A major functional signature is the large variation in the amplitude o f Ca2+ transients between different boutons. This could indicate that local interactions between boutons and different target cells modify the spatiot emporal Ca2+ dynamics in boutons and cause target cell-specific differences in their transmitter release properties.