Visualizing BDNF cell-to-cell transfer and its effects on synapse and circuit function

脑源性神经营养因子（BDNF）对神经元生长、分化和突触可塑性至关重要。从神经元中释放BDNF，以及BDNF对神经元和神经回路功能的影响，都已经分别得到了很好的研究，其中假设BDNF从神经元转移到其他神经元。但到目前为止，BDNF在细胞间的转移还没有被直接观察和研究。使用四荧光成像方法来识别源细胞和靶细胞，我们量化了细胞间BDNF的转移。令人惊讶的是，在初步数据中，我们发现星形胶质细胞似乎是神经元表达的BDNF的主要受体。我们进一步发现星形胶质细胞特异性地吸收由神经元释放的成熟（非亲）BDNF。神经元中TrkB受体的过度表达将释放的BDNF从星形胶质细胞重定向到神经元，这表明TrkB水平决定了神经元和星形胶质细胞对BDNF的摄取。此外，神经元活动的增加进一步增加了星形细胞（而不是神经元）对神经元表达的BDNF的摄取。最后，我们还在初步实验中发现，星形胶质细胞并不像之前提出的那样，仅仅作为过量BDNF的吸收或缓冲，而是星形胶质细胞吸收BDNF通过增加星形胶质细胞的范围来调节生理效应。该项目的目标是将这些初步观察扩展到：1)检查和量化体内内源性BDNF从神经元到星形胶质细胞的转移——使用CRISPR/Cas9标记大脑中的内源性BDNF； 2)确定星形胶质细胞对神经元BDNF的摄取以及随后在星形胶质细胞领域的增加是否由TrkB介导。T1受体（在星形胶质细胞中表达的主要BDNF受体）-使用星形胶质细胞特异性TrkB。3)确定BDNF从神经元到星形胶质细胞的转移对于突触和电路功能方面是否必要-使用突触强度的光学读数和电生理记录。总之，提出的实验将证实并证实BDNF的一种意想不到的功能，即它可以通过改变星形胶质细胞的形态，影响大脑中的突触和电路功能。

Brain-derived neurotrophic factor (BDNF) is essential for neuronal growth, differentiation, and synaptic plasticity. The release of BDNF from neurons, and the effects of BDNF on neuron and circuit function, have both been well-studied separately, where transfer of BDNF from neurons to other neurons has been assumed. But to date, the transfer of BDNF between cells has not been directly visualized and investigated. Using a four-fluorophore imaging approach to identify both the cell of origin and target cells, we quantified the transfer of BDNF between cells. Surprisingly, in preliminary data, we found that astrocytes appear to be the main recipient of neuronally expressed BDNF. We further found that astrocytes specifically take up mature (not pro) BDNF that is released by neurons. Over-expression of the TrkB receptor in neurons redirected released BDNF from astrocytes to neurons, suggesting that TrkB levels determine neuronal versus astrocytic BDNF uptake. In addition, increased neuronal activity further increased astrocytic (but not neuronal) uptake of neuronally expressed BDNF. Finally, we also found in preliminary experiments, that astrocytes do not simply act as a sink or buffer for excess BDNF, as previously proposed, but rather that BDNF taken up by astrocytes mediates physiological effects by increasing astrocytic territory. The goal of this project is to extend these initial observations to, 1) examine and quantify the transfer of endogenous BDNF from neurons to astrocytes in vivo - using CRISPR/Cas9 to label endogenous BDNF in the brain, 2) determine if astrocytic uptake of neuronal BDNF, and subsequent increases in astrocytic territory, are mediated by the TrkB.T1 receptor (the main BDNF receptor expressed in astrocytes) - using astrocyte-specific TrkB.T1 knockout mice, and 3) determine if the transfer of BDNF from neurons to astrocytes is necessary for aspects of synapse and circuit function - using optical readouts of synaptic strength and electrophysiological recordings. Together the proposed experiments will confirm and substantiate an unexpected function of BDNF, whereby it can affect synapse and circuit function in the brain via astrocytes, by changing astrocyte morphology.