Abstract
Neuronal activity results in the release of [Formula: see text] into the extracellular space (ECS). Classically, measurements of extracellular [Formula: see text] ([Formula: see text]) are carried out using [Formula: see text]-sensitive microelectrodes, which provide a single point measurement with undefined spatial resolution. An imaging approach would enable the spatiotemporal mapping of [Formula: see text]. Here, we report on the design and characterization of a fluorescence imaging-based [Formula: see text]-sensitive nanosensor for the ECS based on dendrimer nanotechnology. Spectral characterization, sensitivity, and selectivity of the nanosensor were assessed by spectrofluorimetry, as well as in both wide-field and two-photon microscopy settings, demonstrating the nanosensor efficacy over the physiologically relevant ion concentration range. Spatial and temporal kinetics of the nanosensor responses were assessed using a localized iontophoretic [Formula: see text] application on a two-photon imaging setup. Using acute mouse brain slices, we demonstrate that the nanosensor is retained in the ECS for extended periods of time. In addition, we present a ratiometric version of the nanosensor, validate its sensitivity in brain tissue in response to elicited neuronal activity and correlate the responses to the extracellular field potential. Together, this study demonstrates the efficacy of the [Formula: see text]-sensitive nanosensor approach and validates the possibility of creating multimodal nanosensors.
Highlights
During the repolarization phase of action potentials, neurons release potassium (Kþ), causing an increase of the extracellular Kþ concentration (1⁄2Kþo)
We demonstrate that this Asante Potassium Green 4 (APG4)-based Kþ sensitive nanosensor is suitable for imaging 1⁄2Kþo in acute brain slices and can be used to monitor the spatiotemporal dynamics of 1⁄2Kþo in the brain
A second dye, AlexaFluor 568 (AF568) was incorporated, with a final 1.2∶1.2∶1 APG4 to AF568 to dendrimer ratio
Summary
During the repolarization phase of action potentials, neurons release potassium (Kþ), causing an increase of the extracellular Kþ concentration (1⁄2Kþo). Since Kþ is one of the key membrane potential determinants and, fundamentally affects electrical signaling, the central nervous system has several homeostatic mechanisms to control 1⁄2Kþo. In addition to passive diffusion through the extracellular space (ECS), Kþ is taken up and released by both glia and neurons, which is accomplished by various Kþ selective channels and transporters.[1] In addition to its fundamental effects on neuronal excitability, changes in 1⁄2Kþo have been shown to influence long-term potentiation,[2] vascular tone,[3,4] and glial morphology.[5] Impaired 1⁄2Kþo homeostasis is associated with a wide range of pathological states, such as epilepsy,[6,7] cortical spreading depression,[8] and ischemic stroke/ anoxia.[9] It is yet unclear whether pathological changes in 1⁄2Kþo are a result or a cause of some of these diseases.
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