Structural plasticity of dendritic secretory compartments during 1 LTP-induced synaptogenesis 2 3

37 38 Long-term potentiation (LTP), an increase in synaptic efficacy following high-frequency 39 stimulation, is widely considered a mechanism of learning. LTP involves local 40 remodeling of dendritic spines and synapses. Smooth endoplasmic reticulum (SER) and 41 endosomal compartments could provide local stores of membrane and proteins, 42 bypassing the distant Golgi apparatus. To test this hypothesis, effects of LTP were 43 compared to control stimulation in rat hippocampal area CA1 at postnatal day 15 (P15). 44 By two hours, small spines lacking SER increased after LTP, whereas large spines did 45 not change in frequency, size, or SER content. Total SER volume decreased after LTP 46 consistent with transfer of membrane to the added spines. Shaft SER remained more 47 abundant in spiny than aspiny dendritic regions, apparently supporting the added 48 spines. Recycling endosomes were elevated specifically in small spines after LTP. 49 These findings suggest local secretory trafficking contributes to LTP-induced 50 synaptogenesis and primes the new spines for future plasticity. 51 52 Impact Statement 53 The secretory and recycling components of neuronal dendrites, smooth endoplasmic 54 reticulum and endosomes, were discovered to support synaptogenesis underlying a 55 cellular mechanism of learning and memory in the developing brain. 56 Introduction 57 As the longest and most architecturally complex cells in the body, neurons face 58 the unique challenge of regulating membrane and protein levels in distal compartments. 59 Neurons have highly elaborate dendritic arbors. These dendrites possess synapses, 60 points of contact where electrochemical transmission of information occurs. Most of the 61 excitatory synapses are situated on dendritic spines, tiny protrusions with a head and 62 neck comprising a geometry that is essential for shaping electrical signals (Yuste and 63 Denk 1995, Hering and Sheng 2001, Yuste 2011, Harnett, Makara et al. 2012, Harris 64 and Weinberg 2012, Yuste 2013) and providing biochemical compartmentalization 65 (Harris and Stevens 1989, Bourne and Harris 2008, Colgan and Yasuda 2014). For 66 synapses to function appropriately, the levels of receptor proteins at the postsynaptic 67 density must also be finely tuned. Synapses are often located hundreds of micrometers 68 away from the neuronal cell body. Adding to this spatial problem is the challenge of 69 regulating protein abundance on the membrane in a temporally precise manner, as 70 demanded by fast-acting processes such as synaptic potentiation. 71 Integral membrane proteins destined for the cell surface are canonically thought 72 to be synthesized in the somatic rough endoplasmic reticulum, transported to the Golgi 73 apparatus, and then secreted into the plasma membrane via exocytosis. It is now 74 known that many proteins are translated locally in dendrites, a highly regulated process 75 essential for normal development and plasticity (Sutton and Schuman 2006, Bramham 76 and Wells 2007, Hanus and Schuman 2013). Endoplasmic reticulum (ER) extends into 77 dendrites, forming a continuous tubular network with regions of varying structural 78 complexity and occasional entry into spines (Spacek and Harris 1997, Cooney, Hurlburt 79 et al. 2002, Cui-Wang, Hanus et al. 2012). Together with endosomes, the ER is 80 perfectly positioned to provide a local source of membrane and integral membrane 81 proteins, such as glutamate receptors. However, the Golgi apparatus is absent in most 82 distal dendrites. This puzzling observation has been resolved by recent work 83 demonstrating that dendritic and somatic protein trafficking are highly segregated, and 84 that glutamate receptors are trafficked through a specialized Golgi apparatus85 independent pathway from the dendritic ER to the plasma membrane via recycling 86 endosomes (Bowen, Bourke et al. 2017). Structural changes in ER contribute to normal 87 synaptogenesis during development and maturation (Cui-Wang, Hanus et al. 2012). 88 The involvement of this system in activity-induced synaptogenesis is unknown. 89 Long-term potentiation (LTP), the long-lasting enhancement of synaptic strength 90 due to repetitive activity, is thought to underlie learning and memory. This process has 91 been studied extensively in the hippocampus, a key brain region responsible for new 92 memory formation. Insertion of glutamate receptors from an extrasynaptic reserve pool 93 into the postsynaptic compartment is required for LTP in hippocampal area CA1 94 (Granger, Shi et al. 2013). LTP is also accompanied by structural changes in dendritic 95 spines (Bourne and Harris 2012, Bailey, Kandel et al. 2015). In the young rat 96 hippocampus, LTP produces new dendritic spines (Watson, Ostroff et al. 2016), 97 contrasting with adult rat hippocampus where new spine outgrowth is stalled in favor of 98 synapse enlargement (Bourne and Harris 2011, Bell, Bourne et al. 2014). While Golgi 99 apparatus-independent trafficking has not been studied directly in the context of lasting 100 LTP, recycling endosomes (RE) are known to supply AMPA receptors (Park, Penick et 101 al. 2004), and recycling endosome exocytosis is required for spine formation and growth 102 shortly after the induction of LTP (Park, Salgado et al. 2006). Expanded knowledge 103 about the involvement of Golgi apparatus-independent pathways in developmental 104 synaptic plasticity could provide new targets for rescuing dysregulated synaptogenesis 105 in cases of profound developmental disorders (Fiala, Spacek et al. 2002). 106 Here, three-dimensional reconstruction from serial section electron microscopy 107 (3DEM) revealed morphological changes in SER and endosomal compartments 2 hours 108 following the induction of LTP. The findings are consistent with the involvement of the 109 Golgi-bypass secretory system in supporting synaptic plasticity in the developing 110 hippocampus. 111 112 Results 113 An acute within-slice experimental protocol (Watson, Ostroff et al. 2016) was used to 114 compare the effects of TBS and control stimulation on subcellular membranous 115 compartments in dendrites. In brief, two stimulating electrodes were positioned ~800 μm 116 apart with a recording electrode halfway in between them in CA1 stratum radiatum of 117 P15 rat hippocampus in one slice from each of two animals (Figure 1A). Baseline 118 responses were collected from both electrodes. TBS was delivered at one stimulating 119 electrode and control stimulation was delivered at the other stimulating electrode, 120 counterbalanced in position relative to CA3 for each experiment. There was a significant 121 increase in the field excitatory postsynaptic potential (fEPSP) slope immediately after 122 TBS (Figure 1B,C). Slices were fixed 120 minutes later. EM image volumes were 123 collected from tissue on a diagonal ~120 μm below and to the side of each stimulating 124 electrode. Segments of spiny dendrites, synapses, and all subcellular membrane 125 compartments were reconstructed in three dimensions (see Methods for details). 126