The role of neurotrophins in the maintenance and function of MF-CA3 synapses - PROJECT SUMMARY/ABSRACT The hippocampus has a well-established role in the encoding and recall of spatial memory. At the center of the hippocampal circuit are dentate gyrus (DG) granule cell mossy fiber (MF) to CA3 pyramidal neuron synapses. MF-CA3 synapses have been found to play a key role in learning and memory. However, little is known about the regulation of development and function of these highly plastic synapses. Neurotrophins, such as BDNF and NT3, play a critical role in neuronal development and function. BDNF is well characterized in its role in the CNS however, NT3 has long been forgotten. Both BDNF and NT3 and their receptors, TrkB and TrkC, respectively are located at MF-CA3 synapses. Results from our lab suggest the ablation of NT3, encoded by Ntf3, in the DG alone is sufficient to reduce synaptic transmission and induce contextual fear memory deficits. Despite their colocalization in the DG, initial results suggest independent or synergistic roles of BDNF and NT3 and MF-CA3 synapses. It is left to be determined what are the independent or coordinated roles of BDNF and NT3 in development and function at MF-CA3 synapses. My data indicates the ablation of Bdnf in the DG results in basal synaptic transmission deficits. Currently, knowledge about neurotrophins at MF-CA3 synapses is limited to BDNF's role in long-term potentiation, nearly nothing is known about how NT3 acts on MF-CA3 synapses. Thus, my preliminary observation offers insight into the possible mechanism underlying synaptic plasticity induced learning and memory. The overarching hypothesis is that BDNF and NT3 work independently or synergistically at MF CA3 synapses to promote development and function. To investigate this hypothesis, I will employ three integrated aims to dissect in vivo alterations at MF-CA3 synapses caused by the ablation of Bdnf or Bdnf and Ntf3 from DG granule cells. From Aim 1, I will determine how BDNF and NT3 regulate structure at MF-CA3 synapses. I have generated DG specific knock out of Bdnf (Bdnf-cKO) and Bdnf and Ntf3 (Bdnf/Ntf3-dcKO) using the Pomc-Cre mouse line. To analyze MF axon terminals and CA3 dendric spines called thorny excrescence (TE), I utilize AAV stereotaxic injection to label neuronal structures. Additionally, I utilize expansion sample preparation to image synapse immunostaining at super resolution and perform 3D reconstruction to analyze individual synapses. Finally, I will perform electron microscopy to reveal detailed structure at MF-CA3 synapses. From Aim 2, I will characterize how the ablation of Bdnf affects the function of MF-CA3 synapses by employing behavioral assay to understand to what extent of spatial and contextual memory deficits in Bdnf-cKO mice. From Aim 3, I will perform single nuclei RNA sequencing (snRNA-seq) on DGGCs to elucidate the mechanism by which BDNF regulates the active zone of MF axon terminals. The outcomes of the proposed research will elucidate the roles of two neurotrophins in the function of synaptic transmission and memory.
