TED, a new method to analyze dynamics of intracellular Ca2+ stores in hippocampal networks and to analyze the role of Ca2+ stores in neurotrophin signalling cascades

• Applicant: Professor Dr. Robert Blum
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Molecular and Cellular Neurology and Neuropathology
• Term: from 2011 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 194101929

Calcium signals induced by the neurotrophin brain-derived neurotrophic factor (BDNF) are critically involved in synaptic plasticity, learning and memory. However, the interplay of BDNF-induced calcium signaling cascades is not accurately defined. These signaling cascades include ER calcium store release events, fast calcium influx events, and the modulation of neuronal excitability. In the preceding funding period, we improved the technique targeted-esterase induced dye loading (TED). This method was established by our group to enable the analysis of fast ER calcium dynamics in neurons. TED is based on over-expression of a carboxylesterase (CES2) targeted to the ER lumen. The esterase activity in the ER cleaves off hydrophobic side chains from the acetoxymethylester form of low-affinity synthetic calcium indicators. Thereupon a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. The method enables the direct imaging of ER calcium transients with a high temporal and spatial resolution. We developed a first transgenic mouse model, tg(Thy1-RedCES2), to enable TED-based ER calcium imaging in the hippocampal circuit. The objective of the proposed project is to optimize TED-based ER calcium imaging with help of the mouse model tg(Thy1-RedCES) in order to define the temporal and spatial interplay of BDNF-dependent calcium signaling in the hippocampus and to characterize the underlying homeostatic calcium fluxes. We put the focus on CA3 pyramidal neurons and the stratum lucidum, a model system to investigate pre- and postsynaptic components of fast and slow BDNF calcium signaling during synaptic transmission. Electrical stimulation and acute agonist application experiments will clarify the temporal and spatial properties of the neuronal ER calcium store. Signaling experiments in hippocampal neurons will provide basic information about the properties of the fast versus slow BDNF signaling pathway. The working hypothesis is that BDNF induces somato-dendritic calcium release events via the TrkB-PLCgamma pathway (seconds), but induces fast (milliseconds) BDNF/TrkB/NaV1.9-dependent calcium influx at postsynapses. TrkB, the BDNF receptor, is highly abundant in presynaptic mossy fibre terminals. Therefore, we ask whether presynaptic ER calcium release in mossy fibre terminals is induced by BDNF/TrkB. Finally, we follow our recent finding that TrkB/Nav1.9 are not only involved in fast BDNF-mediated responses at postsynapses, but are mediators of axonal or presynaptic excitability. The project shall offer fundamental information about the properties of the ER calcium store of neurons. Furthermore, we expect a better understanding of the BDNF-dependent calcium signaling in neurons and at synapses. This is important because BDNF signaling cascades are a major target in research for a better understanding and treatment of both neurological and psychiatric disorders.

• DFG Programme: Research Grants