Changes in Synaptic Morphology Accompany Actin Signaling during LTP

Abstract

Stabilization of long-term potentiation (LTP) is commonly proposed to involve changes in synaptic morphology and reorganization of the spine cytoskeleton. Here we tested whether, as predicted from this hypothesis, induction of LTP by theta-burst stimulation activates an actin regulatory pathway and alters synapse morphology within the same dendritic spines. TBS increased severalfold the numbers of spines containing phosphorylated (p) p21-activated kinase (PAK) or its downstream target cofilin; the latter regulates actin filament assembly. The PAK/cofilin phosphoproteins were increased at 2 min but not 30 s post-TBS, peaked at 7 min, and then declined. Double immunostaining for the postsynaptic density protein PSD95 revealed that spines with high pPAK or pCofilin levels had larger synapses (ϩ60 -70%) with a more normal size frequency distribution than did neighboring spines. Based on these results and simulations of shape changes to synapse-like objects, we propose that theta stimulation markedly increases the probability that a spine will enter a state characterized by a large, ovoid synapse and that this morphology is important for expression and later stabilization of LTP.

Preferential loss of dorsal-hippocampus synapses underlies memory impairments provoked by short, multi-modal stress

“…[28][29][30] We used wide-field imaging followed by 3-dimensional deconvolution tomography, a method enabling analysis of hundreds of thousands of synapses. 43,44 We sampled systematically dorsal (septal) and ventral (temporal) hippocampus, and compared the effects of restraint and multimodal stresses (Figures 2a and d). PSD-95 localized to the heads of GFP-expressing dendritic spines of hippocampal pyramidal neurons, (Figure 2c), so that counts of PSD-95 puncta provided a measure of dendritic spines and synapses.…”

Experiential learning in rodents: past experience enables rapid learning and localized encoding in hippocampus

“…We tested this by counting synapses associated with a high concentration of pCofilin. This constitutively active protein severs developing actin filaments unless inactivated by phosphorylation, a step that is critical to the production of the cytoskeletal changes required for LTP consolidation (Chen et al 2007;Rex et al 2009). Previous studies showed that unsupervised learning in a simple open field by rats with no prior experience increases the percentage of hippocampal synapses containing relatively high levels of pCofilin within hippocampal field CA1; an NMDA receptor antagonist blocked both the synaptic changes and memory encoding (Fedulov et al 2007).…”

“…For both analyses, rats were then immediately euthanized by decapitation under isoflurane inhalation anesthesia. Brains were fast frozen and cryostat sectioned; slide mounted sections were fixed in methanol and processed for dual immunofluorescence as described (Chen et al 2007) using mouse anti-PSD-95 (1:1000; ThermoFisher Scientific #MA1-045) and rabbit pCofilin Ser3 (1:1000 Abcam #12866) and secondary antisera including AlexaFluor488 anti-mouse IgG and AlexaFluor594 anti-rabbit IgG (both 1:1000, Invitrogen). Some sections were processed without one or the other primary antisera to verify an absence of secondary antisera cross-reactivity or channel bleed-through.…”

“…(Rex et al 2009;Lynch et al 2013). Phosphorylation (inactivation) of the actin severing protein cofilin is a critical step in the actin polymerization phase of this process (Chen et al 2007); therefore we used phosphorylated (p) Cofilin as a marker for synapses having recently undergone potentiation.…”

“…Four to five spaced tissue sections through rostral hippocampus were processed for dual immunolabeling for PSD-95 and pCofilin Ser3 as in previous work ( Fig. 5A; Chen et al 2007). For this analysis, entire hippocampal cross sections were captured in several hundred digital images each, and the numbers and pCofilin immunolabeling densities of double-labeled synapses were determined for 66 contiguous sampling zones, composing 11 primary anatomical divisions covering apical and basal dendritic fields (Fig.…”