Synaptic evidence for the efficacy of spaced learning

Abstract

The superiority of spaced vs. massed training is a fundamental feature of learning. Here, we describe unanticipated timing rules for the production of long-term potentiation (LTP) in adult rat hippocampal slices that can account for one temporal segment of the spaced trials phenomenon. Successive bouts of naturalistic theta burst stimulation of field CA1 afferents markedly enhanced previously saturated LTP if spaced apart by 1 h or longer, but were without effect when shorter intervals were used. Analyses of F-actin-enriched spines to identify potentiated synapses indicated that the added LTP obtained with delayed theta trains involved recruitment of synapses that were "missed" by the first stimulation bout. Single spine glutamate-uncaging experiments confirmed that less than half of the spines in adult hippocampus are primed to undergo plasticity under baseline conditions, suggesting that intrinsic variability among individual synapses imposes a repetitive presentation requirement for maximizing the percentage of potentiated connections. We propose that a combination of local diffusion from initially modified spines coupled with much later membrane insertion events dictate that the repetitions be widely spaced. Thus, the synaptic mechanisms described here provide a neurobiological explanation for one component of a poorly understood, ubiquitous aspect of learning.

Fig. 1.

Timing determines the efficacy of a second theta train in eliciting additional potentiation. (A) A second theta burst train (TBS2) does not produce additional potentiation when applied 10 min (Left) or 30 min (Center) post-TBS1 but is effective when applied after a 60 min delay (Right) [y axis: fold change in the slope of field (f)EPSP relative to the pre-TBS1 baseline: means ± SEMs]. (B) Traces for LTP1 and LTP2. (C) Percentage facilitation of the composite (four sequential fEPSPs) responses triggered by theta bursts 2–5 delivered at the indicated times after TBS1. The y axis summarizes the areas of the responses expressed as a percent increase above the area of the first burst response in the train (P = 0.92). (D) Using 60-min intertrain intervals, TBS3 elicits further potentiation, whereas TBS4 does not. (E) Mean potentiation at 60 min after TBS2 applied at indicated post-TBS1 intervals (**P < 0.01). (F) LTP2 is reversed by low-frequency (5-Hz) stimulation applied 60 s (Left), but not 60 min (Right), after TBS2.

Fig. 2.

A second theta burst train expands the pool of F-actin-enriched spines. (A) Fluorescent phalloidin labeling in CA1 stratum radiatum following control stimulation (CON) (three test pulses per min) or TBS2 (reverse contrast). (Scale bar = 10 μm). (B) Counts of densely phalloidin-positive spines in slices collected 15 or 75 min after TBS1 (gray bars) or 15 min after TBS2 delayed by 60 min (black bar). (C) Traces show responses to two successive bursts separated by 200 ms (red for second response) delivered in the presence or absence of AMPA receptor modulator CX614. (D) Counts of TBS1-induced phalloidin labeling for vehicle (gray) and CX614-treated (blue) slices. (E) Pretreatment with CX614 (blue line) caused an ∼70% increase in the magnitude of LTP induced by TBS1; this was accompanied by a loss of TBS2–induced potentiation. (***P < 0.001; **P < 0.01.)

Fig. 3.

Induction of single spine enlargement (SSE) in acute hippocampal slices. (A) All targeted spines from young and adult eGFP-expressing (47) mice showed significant enlargement following SSGU (arrow). Neighboring (nontargeted) spine volumes were stable for the entire experiment [young: F(_1,27) = 12.6; P = 0.001 (n = 23 targeted, n = 5 neighboring); adult: F(_1,32) = 6.8; P = 0.014 (n = 28 targeted, n = 5 neighboring)]. (Scale bar, 1 μm.) (B) Comparison of enlarged spine volumes at 2 and 45 min after uncaging. Spines were persistently enlarged relative to their preuncaging baseline in young (n = 19) and adult (n = 12) spines (P < 0.01; one-sample t test). The magnitude of enlargement between groups was not different at either time point. (C) Single spine enlargement occurs at a higher frequency in slices from younger animals (young: 19 spines enlarged out of 23 targeted spines; adult: 12 spines enlarged out of 28 targeted spines; P < 0.005; Fisher exact test). (D) Relative volumes were compared for young (n = 23) and adult (n = 28) spine populations by taking the spine head-to-shaft intensity for each targeted spine at the indicated time points and expressing this value relative to that obtained immediately preceding SSGU (P > 0.05). (E) Left, After initial induction, a second SSGU applied 60 min later does not produce additional enlargement (P > 0.05, paired t test; n = 12). Right, As a control, SSGU was performed following a 60-min delay. Error bars are SEMs.

Fig. 4.

Spine actin polymerization after TBS2 occurs on the same dendritic segments as after TBS1. (A) 3D reconstructions of GFP-labeled dendrites (47) (green) show the localization of phalloidin-labeled F-actin aggregates (red) after TBS1. (B) Left, Percentage of all spines that were phalloidin-positive after TBS1 alone or after TBS2 applied 60 min after TBS1 (*P < 0.05). Right, Percentage of 5-μm dendritic segments that contained two or more phalloidin-positive spines was greater after TBS2 than after TBS1 (**P < 0.005). (C) Frequency distribution for dendritic segments containing zero to six labeled spines after TBS1 or TBS2; after TBS2, more of the labeling occurred in clusters. (D) Left, Brefeldin A had no effect on LTP induced by TBS1. Right, Brefeldin applied after TBS1 caused LTP1 to decay to baseline and completely blocked induction of LTP2.