Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non‐fast‐spiking GABAergic hippocampal interneurons using two‐photon laser microscopy and high‐ and low‐affinity dyes
Authors: Kisfali Máté , Lőrincz Tibor , Vizi E. Sylvester 
Published: August 2013

Key points
Previously, the use of two‐photon scanning microscopy to characterize Ca2+ transients at the individual bouton level has been primarily limited to glutamatergic terminals.
No previous study has attempted to record Ca2+ dynamics in individual boutons in response to somatic stimulation or to compare them with the Ca2+ dynamics recorded in the dendrites of the same GABAergic interneurons located in the CA1 area of the rat hippocampus. In addition, the endogenous buffer capacity that affects the decay of transients was also estimated.
This study was limited to interneurons that responded to somatic current stimulation at frequencies <60 Hz and that had cell bodies located in the stratum radiatum.
The amplitudes of the Ca2+ transients and changes in [Ca2+]i that occurred in response to somatic single or burst stimulation were much higher in boutons (428 nm/AP) than in dendrites (49 nm/AP). These data were calculated as unperturbed values, which excluded the modulatory effect of the dye's buffer capacity and the possible failure to reach equilibrium in dye concentrations in dendrites and boutons. Therefore, the higher density of Ca2+ channels expressed in boutons might account for the nine‐fold difference in Δ[Ca2+]i observed in boutons and dendrites.
Our results indicate that care should be taken when using high‐ and low‐affinity dyes to measure [Ca2+]i in boutons to avoid saturation and the erroneous calculation of [Ca2+]i.
Abstract  Using two‐photon laser microscopy, high‐ and low‐affinity dyes and patch clamp electrophysiology, we successfully measured somatic stimulation‐evoked Ca2+ transients simultaneously in the dendrites and axonal boutons of the same non‐fast‐spiking GABAergic interneurons in acute slice preparations obtained from hippocampal area CA1. The advantage of the acute preparation is that both neuronal connections and anatomy are maintained. Calculated as unperturbed values, the amplitudes of Ca2+ transients and changes in [Ca2+]i in response to somatic single or burst stimulation were much higher in boutons (428 nm/AP) than in dendrites (49 nm/AP), leading to the conclusion that the much greater influx of Ca2+ observed in terminals might be due to a higher density of N‐type voltage‐sensitive Ca2+ channels compared to the L‐type channels present in dendrites. Whereas the decay of Ca2+ transients recorded in dendrites was primarily mono‐exponential, the decay in boutons was bi‐exponential, as indicated by an initial fast phase, followed by a much slower reduction in fluorescence intensity. The extrusion of Ca2+ was much faster in boutons than in dendrites. To avoid saturation effects and the flawed conversion of fluorescence measures of [Ca2+]i, we assessed the limits of [Ca2+] measurements (which ranged between 6 and 82% of the applied dye saturation) when high‐ and low‐affinity dyes were applied at different concentrations. When two APs were delivered at a high frequency (>3 Hz) of stimulation, the low‐affinity indicators OGB‐6F (KD= 3.0 μm) and OGB‐5N (KD= 20 μm) were able to accurately reflect the changes in ΔF/F produced by the consecutive APs. There was no difference in the endogenous buffer capacity (κE), which can shape Ca2+ signals, calculated in dendrites (κE= 354) or boutons (κE= 458).