LTP Induction Translocates Cortactin at Distant Synapses in Wild-Type But NotFmr1Knock-Out Mice

Abstract

Summary Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present studies tested if this involves activity-driven effects on the actin-regulatory protein cortactin, and if such effects are disturbed in the Fmr1-knockout (KO) model of Fragile X Syndrome in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin while basal levels were reduced by nocodazole, indicating that cortactin’s movements into and away from synapses are respectively regulated by microtubule and actomyosin motors. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1-KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin’s interactions with actin and co-precipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein’s use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

Long-Term Memory Deficits are Associated with Elevated Synaptic ERK1/2 Activation and Reversed by mGluR5 Antagonism in an Animal Model of Autism

“…Brains were fast frozen in À 50 1C 2-methylbutane and cryostat sectioned (20 mm, coronal) to generate multiple series of evenly spaced sections through hippocampus. The slide-mounted tissue was processed for dual immunofluorescence (Seese et al, 2012) including overnight incubation in primary antisera cocktail containing mouse anti-PSD95 (1:1000, Pierce) and rabbit antisera to p-ERK1/2 Thr202/Tyr204 (1:500, Cell Signaling), ERK1/2 (1:500, Cell Signaling), or p-CREB Ser133 (1:250, Millipore). Secondary antisera included donkey anti-mouse AlexaFluor488 and donkey anti-rabbit AlexaFluor594.…”

“…Z-stacks were processed for restorative deconvolution and construction of 3D montages of each 42 840 mm 3 sample field (Volocity 5.0, Perkin Elmer). Automated systems then counted and measured the fluorescence intensities of single-and double-labeled elements that met size and eccentricity constraints of synapses (Seese et al, 2012;Seese et al, 2013). Contacts were counted as double labeled if there was any overlap in their immunolabeled boundaries as evaluated in 3D.…”

“…Synaptoneurosomes were isolated from male and female C57BL/6 WT and BTBR mice at 14-16 days of age (Bernard-Trifilo et al, 2005;Seese et al, 2012). Both sexes from these pre-pubertal animals were used to increase yield.…”

“…There is now a large literature demonstrating disturbances in spine morphology and signaling cascades at excitatory synapses in animal models of cognitive disability (Belichenko et al, 2007;Kramar et al, 2012), including disorders on the autism spectrum (Chen et al, 2010;Hung et al, 2008;Seese et al, 2012;Tropea et al, 2009). Learning and executive functions are impaired in BTBR mice (Amodeo et al, 2012;Lipina and Roder, 2013;MacPherson et al, 2008;Ribeiro et al, 2013;Rutz and Rothblat, 2012;Silverman et al, 2013;Yang et al, 2012), but thorough analyses of associated synaptic abnormalities and evidence for pharmacological normalization of these measures are lacking.…”

“…We used the hippocampus-dependent object location memory (OLM) task to compare memory in BTBRs and C57BL/6 mice, and fluorescence deconvolution tomography to determine whether strain effects on cognitive performance are associated with abnormalities in synaptic signaling of extracellular signal-regulated kinase (ERK) 1/2, a protein critical for long-term memory (Thomas and Huganir, 2004) and disturbed in forms of autism and its models (Hou et al, 2006;Michalon et al, 2012;Samuels et al, 2009;Seese et al, 2012;Zou et al, 2011). The results show that poor learning performance is associated with disturbances in ERK1/2 signaling in BTBRs and that both measures are amenable to normalization with pharmacological treatments.…”