L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of l-lactate are currently hampered by the limited selection and performance of l-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular l-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular l-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular l-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of l-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular l-lactate dynamics in mice.

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues.

Brain glycogen metabolism is known to be involved in the learning and memory processes. Protein targeting to glycogen (PTG) is a crucial molecule for glycogenesis, and its expression level is shown to be increased in the dorsal hippocampus during fear memory acquisition and recall, suggesting that PTG may contribute to the memory process. However, its detailed role in the dorsal hippocampus remains unclear. Therefore, we knocked down the expression of PTG in the dorsal hippocampus and attempted to analyze its function behaviorally. PTG expression was found to be enriched in astrocytes. Furthermore, short hairpin RNA against PTG suppressed the expression of PTG in astrocytes. Mice with knockdown of PTG in the dorsal hippocampus showed suppressed alternation behavior in the Y-maze test and reduced memory recall at the first hour after acquisition in the passive avoidance test. Knockdown of mouse dorsal hippocampal astrocyte-specific PTG also impaired working memory in the Y-maze test. GluR1, GluR2, and NR2a subunits expressions were significantly down-regulated in the dorsal hippocampus of mice in which PTG was knocked down. These results indicate that PTG in the dorsal hippocampal astrocytes may contribute to working and short-term memories by maintaining the expression of glutamate receptor subunits.