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

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 ( approximately 2 fC) less than 1% remained free. The indicator concentration dependence of decay time constants further suggests that the residual free Delta[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.

NMDA spikes in basal dendrites of cortical pyramidal neurons.

Basal dendrites are a major target for synaptic inputs innervating cortical pyramidal neurons. At present little is known about signal processing in these fine dendrites. Here we show that coactivation of clustered neighbouring basal inputs initiated local dendritic spikes, which resulted in a 5.9 +/- 1.5 mV (peak) and 64.4 +/- 19.8 ms (half-width) cable-filtered voltage change at the soma that amplified the somatic voltage response by 226 +/- 46%. These spikes were accompanied by large calcium transients restricted to the activated dendritic segment. In contrast to conventional sodium or calcium spikes, these spikes were mediated mostly by NMDA (N-methyl-D-aspartate) receptor channels, which contributed at least 80% of the total charge. The ionic mechanism of these NMDA spikes may allow 'dynamic spike-initiation zones', set by the spatial distribution of glutamate pre-bound to NMDA receptors, which in turn would depend on recent and ongoing activity in the cortical network. In addition, NMDA spikes may serve as a powerful mechanism for modification of the cortical network by inducing long-term strengthening of co-activated neighbouring inputs.

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.

Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials.

We compared the transient increase of Ca2+ in single spines on basal dendrites of rat neocortical layer 5 pyramidal neurons evoked by subthreshold excitatory postsynaptic potentials (EPSPs) and back-propagating action potentials (APs) by using calcium fluorescence imaging. AP-evoked Ca2+ transients were detected in both the spines and in the adjacent dendritic shaft, whereas Ca2+ transients evoked by single EPSPs were largely restricted to a single active spine head. Calcium transients elicited in the active spines by a single AP or EPSP, in spines up to 80 micro(m) for the soma, were of comparable amplitude. The Ca2+ transient in an active spine evoked by pairing an EPSP and a back-propagating AP separated by a time interval of 50 ms was larger if the AP followed the EPSP than if it preceded it. This difference reflected supra- and sublinear summation of Ca2+ transients, respectively. A comparable dependence of spinous Ca2+ transients on relative timing was observed also when short bursts of APs and EPSPs were paired. These results indicate that the amplitude of the spinous Ca2+ transients during coincident pre- and postsynaptic activity depended critically on the relative order of subthreshold EPSPs and back-propagating APs. Thus, in neocortical neurons the amplitude of spinous Ca2+ transients could encode small time differences between pre- and postsynaptic activity.