Two-Photon FRET/FLIM Imaging of Cerebral Neurons.

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.

Optogenetic Manipulation of Postsynaptic cAMP Using a Novel Transgenic Mouse Line Enables Synaptic Plasticity and Enhances Depolarization Following Tetanic Stimulation in the Hippocampal Dentate Gyrus.

cAMP is a positive regulator tightly involved in certain types of synaptic plasticity and related memory functions. However, its spatiotemporal roles at the synaptic and neural circuit levels remain elusive. Using a combination of a cAMP optogenetics approach and voltage-sensitive dye (VSD) imaging with electrophysiological recording, we define a novel capacity of postsynaptic cAMP in enabling dentate gyrus long-term potentiation (LTP) and depolarization in acutely prepared murine hippocampal slices.

Crucial Role of Postsynaptic Syntaxin 4 in Mediating Basal Neurotransmission and Synaptic Plasticity in Hippocampal CA1 Neurons.

Trafficking of neurotransmitter receptors on postsynaptic membranes is critical for basal neurotransmission and synaptic plasticity, yet the underlying mechanisms remain elusive. Here, we investigated the role of syntaxin 4 in postsynaptic hippocampal CA1 neurons by analyzing conditional knockout (syntaxin 4 cKO) mice. We show that syntaxin 4 cKO resulted in reduction of basal neurotransmission without changes in paired-pulse ratios.

A Temporary Gating of Actin Remodeling during Synaptic Plasticity Consists of the Interplay between the Kinase and Structural Functions of CaMKII.

The structural modification of dendritic spines plays a critical role in synaptic plasticity. CaMKII is a pivotal molecule involved in this process through both kinase-dependent and independent structural functions, but the respective contributions of these two functions to the synaptic plasticity remain unclear. We demonstrate that the transient interplay between the kinase and structural functions of CaMKII during the induction of synaptic plasticity temporally gates the activity-dependent modification of the actin cytoskeleton.

A Far-Red Emitting Probe for Unambiguous Detection of Mobile Zinc in Acidic Vesicles and Deep Tissue.

Imaging mobile zinc in acidic environments remains challenging because most small-molecule optical probes display pH-dependent fluorescence. Here we report a reaction-based sensor that detects mobile zinc unambiguously at low pH. The sensor responds reversibly and with a large dynamic range to exogenously applied Zn in lysosomes of HeLa cells, endogenous Zn in insulin granules of MIN6 cells, and zinc-rich mossy fiber boutons in hippocampal tissue from mice.

Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices.

Mossy fiber termini in the hippocampus accumulate Zn(2+), which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn(2+) at the synaptic level is challenging owing to the difficulty of visualizing Zn(2+) at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn(2+) dynamics mediated by NMDA receptor-dependent long-term potentiation induction at single mossy fiber termini of dentate gyrus neurons in adult mouse hippocampal slices.