Abstracts

Rationale: Multiple lines of evidence implicate activation of the receptor tyrosine kinase TrkB as a critical mediator of epileptogenesis following status epilepticus. Because of the seminal role of TrkB in this disorder, understanding the mechanisms underlying its activation and the resulting functional consequences should facilitate development of targeted therapies. Unfortunately though, our abilities to fully probe TrkB's function have been hindered by current technology. Specifically, unlike ionotropic receptors whose activity can be dynamically monitored in a defined neuron with a patch pipette, measuring TrkB activity requires a surrogate measure, namely phospho-specific antibodies used in brain extracts analyzed by Western Blot or in fixed tissue using immunohistochemistry. The non-dynamic nature of these readouts limits insights into the spatiotemporal patterns of TrkB activation. To address these limitations, we set out to develop a biosensor that enables imaging of TrkB activation in living cells and in real time. Methods: We combined recent advances in fluoresence resonance energy transfer (FRET) technology with two-photon fluorescence lifetime microscopy (2pFLIM) to successfully desgin and implement a dynamic sensor for TrkB activation. We evaluated the sensitivity and specificity of the sensor by expressing it in HeLa cells and cultured rat embyronic cortical neurons and then measuring its response to various TrkB-activating (BDNF, Zinc, and H₂O₂) and incativating (K252a) stimuli. Additionally, we combined biolistic transfection of the sensor in cultured hippocampal slices with 2pFLIM and focal two-photon glutamate uncaging to TrkB activation in single dendritic spines of CA1 pyramidal neurons during glutamate uncaging evoked structural plasticity. Results: In HeLa cells and cultured cortical neurons, the sensor reported rapid and specific TrkB activation in response to BDNF, zinc, and hydrogen peroxide that could be reversed with K252a - results consistent with conventional methods of examining TrkB activation, namely Western Blotting. Further, by expressing the sensor in cultured hippocampal slices, we found that TrkB rapidly and persistently activates in a single spine following glutamate uncaging in an NMDA receptor and CamKII dependent manner. Conclusions: The development and utilization of this sensor in a variety of preparations - HeLa cells, cultured cortical neurons, and cultured hippocampal slices - makes possible the dynamic imaging of the spatiotemporal pattern of receptor tyrosine kinase activation within living cells in real time. Continued use and development of this tool promises to further inform our understanding of TrkB's functional role in various aspects of neuronal physiology and pathology and ultimately translate into the development of novel therapies.