Significance: Excitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here, we report the development of an ultrafast genetically encoded glutamate sensor, iGlu _u , which allowed us to image glutamate clearance and synaptic depression during 100-Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f_u displaying fluorescence rise and decay times (t_1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6f_u revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t_1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6f_u is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.

Calcium signaling in neurons as in other cell types can lead to varied changes in cellular function. Neuronal Ca²⁺ signaling processes have also become adapted to modulate the function of specific pathways over a wide variety of time domains and these can have effects on, for example, axon outgrowth, neuronal survival, and changes in synaptic strength. Ca²⁺ also plays a key role in synapses as the trigger for fast neurotransmitter release. Given its physiological importance, abnormalities in neuronal Ca²⁺ signaling potentially underlie many different neurological and neurodegenerative diseases. The mechanisms by which changes in intracellular Ca²⁺ concentration in neurons can bring about diverse responses is underpinned by the roles of ubiquitous or specialized neuronal Ca²⁺ sensors. It has been established that synaptotagmins have key functions in neurotransmitter release, and, in addition to calmodulin, other families of EF-hand-containing neuronal Ca²⁺ sensors, including the neuronal calcium sensor (NCS) and the calcium-binding protein (CaBP) families, play important physiological roles in neuronal Ca²⁺ signaling. It has become increasingly apparent that these various Ca²⁺ sensors may also be crucial for aspects of neuronal dysfunction and disease either indirectly or directly as a direct consequence of genetic variation or mutations. An understanding of the molecular basis for the regulation of the targets of the Ca²⁺ sensors and the physiological roles of each protein in identified neurons may contribute to future approaches to the development of treatments for a variety of human neuronal disorders.

Faithful reporting of temporal patterns of intracellular Ca²⁺ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca²⁺ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca²⁺ affinities and accelerated kinetics by weakening the Ca²⁺-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3_fast is 40-fold faster than GCaMP3 with Ca²⁺ decay and rise times, t_1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3_fast revealed discreet transients with significantly faster Ca²⁺ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca²⁺ at 940 nm, GCaMP3_fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca²⁺ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.