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

摘要

class="enumerated" style="list-style-type:decimal">Calcium dynamics associated with a single action potential (AP) were studied 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 volume-averaged Ca²⁺ signals. These rapidly reached a peak (in about 1 ms) and then decayed more slowly (tens to hundreds of milliseconds).Single APs and trains of APs reliably evoked Ca²⁺ transients in en passant boutons located on axon collaterals in cortical layers 2/3, 4 and 5, indicating that APs propagate actively and reliably throughout the axonal arbor. Branch point failures are unlikely to contribute to differences in synaptic efficacy and reliability in the connections made by layer 2/3 pyramidal cells.AP-evoked Ca²⁺ transients in boutons were mediated by voltage-dependent Ca²⁺ channels (VDCCs), predominantly by the P/Q- and N-subtypes.Ca²⁺ transients were, on average, of significantly larger amplitude in boutons than in the flanking segments of the axon collateral. Large amplitude Ca²⁺ transients in boutons were spatially restricted to within ≤ 3 m of axonal length.Single AP-evoked Ca²⁺ transients varied up to 10-fold across different boutons even if they were located on the same axon collateral. In contrast, variation of Ca²⁺ transients evoked by successive APs in a given single bouton was small (coefficient of variation, c.v. ≤ 0.21).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, r² = -0.27) with distance from the soma.Measurements with the low-affinity Ca²⁺ indicator Magnesium Green suggest that the volume-averaged residual free [Ca²⁺]i in a bouton increases on average by 500 nM following a single AP. Higher concentrations of indicator caused, on average, a decrease in the amplitude and an increase in the decay 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 (∼2 fC) less than 1 % remained free.The indicator concentration dependence of decay time constants further suggests that the residual free Δ[Ca²⁺]i associated with an AP decays with a time constant of about 60 ms (35°C) reflecting a high Ca²⁺ extrusion rate of about 2600 s⁻¹.The results show that AP-evoked volume-averaged Ca²⁺ transients in single boutons are evoked reliably and, on average, have larger amplitudes than Ca²⁺ transients in other subcellular compartments of layer 2/3 pyramidal cells. A major functional signature is the large variation in the amplitude of Ca²⁺ transients between different boutons. This could indicate that local interactions between boutons and different target cells modify the spatiotemporal Ca²⁺ dynamics in boutons and cause target cell-specific differences in their transmitter release properties.