Presynaptic GCaMP expression decreases vesicle release probability at the calyx of Held

Abstract

Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs’ ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.

Imaging and Analysis of Presynaptic Calcium Influx in Cultured Neurons Using synGCaMP6f

“…Different mouse lines with stable transfection of GCaMP-expression, in which the entire development was affected by the presence of the indicator, show modified neuronal activity with interictal spikes up to epileptiform events (Steinmetz et al, 2017). In AAV-infected mice moderately reduced vesicle release was described in the calyx of Held synapse more than 18 days after transfection (Singh et al, 2018) and accumulation of GCaMP6 in the nucleus occurred in the third week of transfection (Yang et al, 2018). GCaMP6m modifies the gating of Cav1.3 channels, that show enhanced voltage gated activation and calcium-dependent inactivation, which in part resembles the effect of additional apo-calmodulin, but Cav2.2 (N-type) calcium channels were not significantly affected (Yang et al, 2018).…”

“…With GCaMP6f, the fast version of the GCaMP6 family, the dynamic range, i.e., the fluorescence increase factor from calcium-free to calcium-saturated, is now above 50 (Chen et al, 2013), and thus rivals the range of the small-molecule synthetic indicators. Originally intended to observe spontaneous activity in living animals, the GCaMP6 indicators may cause some problems when permanently expressed in transgenic models in vivo , inducing increased buffering in the targeted compartments (Singh et al, 2018) and aberrant electrical activity similar to interictal spikes (Steinmetz et al, 2017). Very recently, modifications of Cav1.3 calcium currents by GCaMP6f in transient transfection of HEK cells are described next to the in vivo problems and a GCaMP-X that claims to overcome these problems was introduced (Yang et al, 2018), but this is not tested here.…”

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

“…Evaluated in cultured neurons, GCaMP 6s and 6m variants exhibited even larger improvements in response amplitudes, but this was at the cost of kinetics (see Table 3) [98]. Variants of GCaMP based on rise and delay kinetics-GCaMP 6s (slow), 6m (medium), and 6f (fast)-typically have a high affinity, with K d values between 100 and 300 nM [108,109], which results in slow Ca 2+ dissociation rates and signal decay occurring at 1-5 s −1 at 20 • C [110]. This has acted as a constraint against rapid tracking processes in excitable cells, posing a difficulty for monitoring rapid Ca 2+ transients that require millisecond resolution and fluorescence decay and rise rates up to 1000 s −1 [103].…”

Methods of measuring presynaptic function with fluorescence probes

“…GCaMP was developed to more accurately measure presynaptic calcium changes in living neurons (Brockhaus et al 2019). However, animals expressing GCaMP displayed aberrant current activity and suppression of SV probability (Singh et al 2018;Steinmetz et al 2017), suggesting that artifacts derived from GCaMP overexpression in vivo should be assessed.…”