Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity1, 2, 3, 4, 5, 6, including structural long-term potentiation (sLTP)7, 8, which is a correlate of an animal’s learning7, 9, 10, 11, 12. However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy13, 14, 15, 16, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction9, 14, 15, 16, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl-d-aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin17, 18, 19. We demonstrate that this postsynaptic BDNF–TrkB signalling pathway is necessary for both structural and functional LTP20. Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR–CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

SUMMARY CaMKII plays a critical role in decoding calcium (Ca2+) signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ signals in spines remain elusive. Here, we measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each pulse during repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. Thr286 phosphorylation slows the decay of CaMKII and thus, lowers the frequency required to induce spine plasticity by several fold. In the absence of Thr286 phosphorylation, increasing the stimulation frequency results in high peak mutant CaMKIIT286A activity that is sufficient for inducing plasticity. Our findings demonstrate that Thr286 phosphorylation plays an important role in induction of LTP by integrating Ca2+ signals, and it greatly promotes, but is dispensable for the activation of CaMKII and LTP.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed fret donor, monomeric cyan-excitable red fluorescent protein (mCyRFP), which has a large stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity.

Presynaptic GABA(B) receptor (GABA(B)R) heterodimers are composed of GB(1a)/GB(2) subunits and critically influence synaptic and cognitive functions. Here, we explored local GABA(B)R activation by integrating optical tools for monitoring receptor conformation and synaptic vesicle release at individual presynaptic boutons of hippocampal neurons. Utilizing fluorescence resonance energy transfer (FRET) spectroscopy, we detected a wide range of FRET values for CFP/YFP-tagged GB(1a)/GB(2) receptors that negatively correlated with release probabilities at single synapses. High FRET of GABA(B)Rs associated with low release probability. Notably, pharmacological manipulations that either reduced or increased basal receptor activation decreased intersynapse variability of GB(1a)/GB(2) receptor conformation. Despite variability along axons, presynaptic GABA(B)R tone was dendrite specific, having a greater impact on synapses at highly innervated proximal branches. Prolonged neuronal inactivity reduced basal receptor activation, leading to homeostatic augmentation of release probability. Our findings suggest that local variations in basal GABA concentration are a major determinant of GB(1a)/GB(2) conformational variability, which contributes to heterogeneity of neurotransmitter release at hippocampal synapses.