Neurons in the nervous system can change their functional properties over time. At present, there are no techniques that allow reliable monitoring of changes within identified neurons over repeated experimental sessions. We increased the signal strength of troponin C-based calcium biosensors in the low-calcium regime by mutagenesis and domain rearrangement within the troponin C calcium binding moiety to generate the indicator TN-XXL. Using in vivo two-photon ratiometric imaging, we show that TN-XXL exhibits enhanced fluorescence changes in neurons of flies and mice. TN-XXL could be used to obtain tuning curves of orientation-selective neurons in mouse visual cortex measured repeatedly over days and weeks. Thus, the genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease.

Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.

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Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.

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Tools from molecular biology, in combination with in vivo optical imaging techniques, provide new mechanisms to noninvasively observe brain processing. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. Here we introduce CNiFERs, cell-based neurotransmitter fluorescent engineered reporters, to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, make use of the Gq protein-coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca2+], and report [Ca2+] with a genetically encoded fluorescent Ca2+ sensor. The initial realization of CNiFERs detects acetylcholine release via activation of M1 muscarinic receptors. Chronic implantation of M1-CNiFERs in frontal cortex of the adult rat is used to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We show that these drugs potently inhibit in situ muscarinic receptor activity.

In the visual system of Drosophila, photoreceptors R1-6 relay achromatic brightness information to five parallel pathways. Two of them, the lamina monopolar cells L1 and L2, represent the major input lines to the motion detection circuitry. Here we devised a new method for the optical recording of visually evoked changes in intracellular Ca 2+ in neurons, using targeted expression of a genetically encoded Ca 2+ indicator. Ca 2+ in single terminals of L2 neurons in the medulla carries no information about the direction of motion. However, the observed strong increase in intracellular Ca 2+ induced by light-OFF and only small changes induced by light-ON suggest halfwave rectification of the input signal. Thus, L2 predominantly transmits brightness decrements to downstream circuits that then compute the direction of image motion.2

Coupling of excitation to secretion, contraction, and transcription often relies upon Ca2+ computations within the nanodomain—a conceptual region extending tens of nanometers from the cytoplasmic mouth of Ca2+ channels. Theory predicts that nanodomain Ca2+ signals differ vastly from the slow submicromolar signals routinely observed in bulk cytoplasm. However, direct visualization of nanodomain Ca2+ far exceeds optical resolution of spatially distributed Ca2+ indicators. Here we couple an optical genetically encoded Ca2+ indicator (TN-XL) to the carboxyl tail of CaV2.2 Ca2+ channels, enabling nearfield imaging of the nanodomain. Under TIRF microscopy, we detect Ca2+ responses indicative of large-amplitude pulses. Single-channel electrophysiology reveals a corresponding Ca2+ influx of only 0.085 pA, and FRET measurements estimate TN-XL distance to the cytoplasmic mouth at ~55 Å. Altogether, these findings raise the possibility that Ca2+ exits the channel through the analog of molecular portals, mirroring the crystallographic images of side windows in voltage-gated K channels.