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

脑源性神经营养因子（BDNF）对神经元的生长、分化和突触可塑性至关重要。BDNF从神经元的释放，以及BDNF对神经元和回路功能的影响，已经分别得到了很好的研究，其中BDNF从神经元转移到其他神经元已经被假设。但到目前为止，BDNF在细胞之间的转移还没有直接可视化和研究。使用四荧光团成像方法来识别细胞的起源和靶细胞，我们量化了BDNF在细胞之间的转移。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摄取由神经元释放的成熟（而不是原）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，表明TrkB水平决定神经元对星形胶质细胞BDNF的摄取。此外，增加神经元活性进一步增加星形胶质细胞（而不是神经元）的神经元表达的BDNF的摄取。最后，我们还发现，在初步实验中，星形胶质细胞并不简单地作为一个水槽或缓冲过量的BDNF，如前所述，而是BDNF采取了星形胶质细胞介导的生理效应，增加星形胶质细胞的领土。 该项目的目标是将这些初步观察扩展到，1）检查和量化内源性BDNF从神经元到星形胶质细胞的体内转移-使用CRISPR/Cas9标记脑中的内源性BDNF，2）确定星形胶质细胞对神经元BDNF的摄取以及随后的星形胶质细胞区域的增加，由TrkB.T1受体介导（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.T1敲除小鼠，以及3）确定BDNF从神经元到星形胶质细胞的转移对于突触和回路功能方面是否是必需的-使用突触强度的光学读数和电生理记录。共同提出的实验将确认和证实BDNF的一个意想不到的功能，从而它可以通过改变星形胶质细胞形态，通过星形胶质细胞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.