The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves Ca 2+ and NMDA receptor-dependent mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitudinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal plasticity.

Synaptic plasticity correlates with the local dendritic translocation of CaMKII in a Ca2+- and microtubule-dependent manner.

Understanding how brief synaptic events can lead to sustained changes in synaptic structure and strength is a necessary step in solving the rules governing learning and memory. Activation of ERK1/2 (extracellular signal regulated protein kinase 1/2) plays a key role in the control of functional and structural synaptic plasticity. One of the triggering events that activates ERK1/2 cascade is an NMDA receptor (NMDAR)-dependent rise in free intracellular Ca 2ϩ concentration. However the mechanism by which a short-lasting rise in Ca 2ϩ concentration is transduced into long-lasting ERK1/2-dependent plasticity remains unknown. Here we demonstrate that although synaptic activation in mouse cultured cortical neurons induces intracellular Ca 2ϩ elevation via both GluN2A and GluN2B-containing NMDARs, only GluN2B-containing NMDAR activation leads to a long-lasting ERK1/2 phosphorylation. We show that ␣CaMKII, but not ␤CaMKII, is critically involved in this GluN2B-dependent activation of ERK1/2 signaling, through a direct interaction between GluN2B and ␣CaMKII. We then show that interfering with GluN2B/␣CaMKII interaction prevents synaptic activity from inducing ERKdependent increases in synaptic AMPA receptors and spine volume. Thus, in a developing circuit model, the brief activity of synaptic GluN2B-containing receptors and the interaction between GluN2B and ␣CaMKII have a role in long-term plasticity via the control of ERK1/2 signaling. Our findings suggest that the roles that these major molecular elements have in learning and memory may operate through a common pathway.

Abbreviations used in this paper: AMPAR, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CaM, calmodulin; CaMKII, Ca 2+ /calmodulindependent protein kinase II; cLTP, long-term potentiation in culture; Glu, glutamate; Gly, glycine; NMDAR, N-methyl-d-aspartate receptor; WT, wild type.

1The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical 2 rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but 3 only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in 4 mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively 5 their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach 6 of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the 7 quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal ac-8 tivity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into 9 longitudinal fibers. We show that this activity-dependent remodeling involves Ca 2+ and NMDA-dependent 10 mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitu-11 dinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal 12 plasticity. 13 Introduction 14One of the hallmark discoveries made possible by fluorescence nanoscopy methods is the existence of a 15 periodical lattice of F-actin, spectrin, and associated proteins under the surface membrane of neuronal 16 processes. This lattice, containing F-actin rings periodically spaced 180-190nm apart, was initially discovered 17 in axons [1]. The lattice was later observed in dendrites of multiple types of neurons, albeit to a lesser extent 18 compared to axons [2][3][4]. Several isoforms of spectrin have been observed in the lattice, with variable 19 prevalence in axons compared to dendrites and during development [3, 4]. In addition to forming periodical 20 rings, other F-actin structures have been described at the nanoscale in axons and dendritic shaft, including 21 longitudinal fibers [2,[5][6][7]. 22 The role and regulatory mechanisms of the periodical submembrane skeletal structure remain unclear. It 23 has been shown to be regulated during development in axons, dendrites, and dendritic spines [2][3][4] 8]. It was 24 1 demonstrated that the submembrane lattice destabilization triggers axonal degeneration [9, 10]. A recent 25 study provided evidence that it serves as a signaling platform for receptor tyrosine kinase transactivation in 26 neurons [11]. 27 The more variable appearance and sporadic presence of the F-actin/spectrin lattice in dendrites compared 28 to axons suggests that the structure is differently regulated in these distinct processes [2][3][4] 12]. A clear 29 discrimination between spatially overlapping axons and dendrites, using a combination of specific markers, 30 has been lacking in studies comparing the properties of the lattice, which may have impacted the analyses 31 of its prevalence in dendrites. Furthermore, the greater diversity of F-actin nanostructures in dendrites...