SummaryThe structural and functional plasticity of synapses is critical for learning and memory. Long-term potentiation (LTP) induction promotes spine growth and AMPAR accumulation at excitatory synapses, leading to increased synaptic strength. Glutamate initiates these processes, but the contribution from extracellular modulators is not fully established. Wnts are required for spine formation; however, their impact on activity-mediated spine plasticity and AMPAR localization is unknown. We found that LTP induction rapidly increased synaptic Wnt7a/b protein levels. Acute blockade of endogenous Wnts or loss of postsynaptic Frizzled-7 (Fz7) receptors impaired LTP-mediated synaptic strength, spine growth, and AMPAR localization at synapses. Live imaging of SEP-GluA1 and single-particle tracking revealed that Wnt7a rapidly promoted synaptic AMPAR recruitment and trapping. Wnt7a, through Fz7, induced CaMKII-dependent loss of SynGAP from spines and increased extrasynaptic AMPARs by PKA phosphorylation. We identify a critical role for Wnt-Fz7 signaling in LTP-mediated synaptic accumulation of AMPARs and spine plasticity.

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These authors contributed equally to this manuscript 2 The specialised blood barriers of the nervous system are important for protecting the neural environment but can hinder therapeutic accessibility 1,2 . Studies in the central nervous system (CNS) have shown the importance of the cellular components of the neuro-vascular unit for blood-brain barrier (BBB) function. Whilst the endothelial cells (ECs) confer barrier function with specialised tight junctions (TJs) and low levels of transcytosis, pericytes and astrocytes provide complete coverage of the ECs and both deliver essential signals for the development and maintenance of the BBB 3-9 . In contrast, the blood-nerve barrier (BNB) of the peripheral nervous system (PNS) remains poorly defined 10 . Here, we show that the vascular unit in the PNS has a distinct cellular composition with only partial coverage of the BNB-forming ECs. Using a mouse model, in which barrier function can be controlled 11 , we show the BNB, while less tight than the BBB, is maintained by low levels of transcytosis and the TJs of the ECs, with opening of the barrier associated with increased transcytosis. Importantly, we find that while ECs of the PNS have higher transcytosis rates than those of the CNS, the barrier is reinforced by resident macrophages that specifically engulf leaked material. This identifies a distinct role for macrophages as an important component of the BNB acting to protect the PNS environment with implications for improving therapeutic delivery to this tissue.To investigate the cellular structures comprising the BNB in the PNS, we took complementary approaches. To determine the total cellular coverage of the ECs in peripheral nerve in a nonbiased fashion, we performed 3D EM of endoneurial blood vessels that can detect all cellular contacts (Fig. 1a, Extended Data Fig. 1a, Movie1). In contrast to the CNS 12 , we found cellular coverage of PNS endoneurial blood vessels was incomplete, a finding confirmed by higherresolution 2D EM images (Fig1b and Extended Data Fig. 1b). Pericytes could be identified by their tight association with the ECs within the basal lamina, however, other cell types were observed, which made looser contacts with the ECs. In a separate study, we have used immunostaining to identify all cell-types within the endoneurium of peripheral nerve 13 and we used these markers and others, to determine the cell-types that interact with ECs in the PNS and verified these findings using CLEM. We found 3 different populations of cells that directly

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