Spaced training rescues memory and ERK1/2 signaling in fragile X syndrome model mice

Abstract

Recent studies have shown that short, spaced trains of afferent stimulation produce much greater long-term potentiation (LTP) than that obtained with a single, prolonged stimulation episode. The present studies demonstrate that spaced training regimens, based on these LTP timing rules, facilitate learning in wild-type (WT) mice and can offset learning and synaptic signaling impairments in the fragile X mental retardation 1 (Fmr1) knockout (KO) model of fragile X syndrome. We determined that 5 min of continuous training supports object location memory (OLM) in WT but not Fmr1 KO mice. However, the same amount of training distributed across three short trials, spaced by one hour, produced robust long-term memory in the KOs. At least three training trials were needed to realize the benefit of spacing, and intertrial intervals shorter or longer than 60 min were ineffective. Multiple short training trials also rescued novel object recognition in Fmr1 KOs. The spacing effect was surprisingly potent: just 1 min of OLM training, distributed across three trials, supported robust memory in both genotypes. Spacing also rescued training-induced activation of synaptic ERK1/2 in dorsal hippocampus of Fmr1 KO mice. These results show that a spaced training regimen designed to maximize synaptic potentiation facilitates recognition memory in WT mice and can offset synaptic signaling and memory impairments in a model of congenital intellectual disability.

Keywords: Fmr1 KO; hippocampus; massed training; novel object recognition; object location memory.

Fig. 1.

Long-term object location memory (OLM) and novel object recognition (NOR) are impaired in Fmr1 KOs. (A) For OLM, mice were given 5 min of continuous (massed) training with two identical objects (A1 and A2). For retention testing 24 h later, one object was in the familiar location (A3) and one was placed in a novel location (A4); for control (“con”) mice, objects were in the familiar location. (B) With 5-min training, OLM was robust in WT mice but absent in KOs (***P < 0.001; n ≥ 8 per group). (C) Short-term memory (STM) was comparable between genotypes (P = 0.57; n ≥ 10 per group). (D) KOs and WTs expressed long-term OLM when trained for 10 min (P = 0.88; n ≥ 8 per group). (E) Fmr1 KOs on the C57BL/6 background had deficient long-term OLM (*P < 0.05; n ≥ 7 per group) but control level short-term OLM (P = 0.68; n ≥ 7 per group) with 5-min massed training. (F) KOs traveled greater distance (meters) than WTs on habituation days 2–5 (P < 0.01 for each day), but not during training or retention trials (P > 0.50 both; n ≥ 8 per group). (G) KOs trained and tested in their dark cycle did not express OLM (***P < 0.001; n ≥ 10 per group). (H) KO mice given 5-min massed training on two or six successive days failed to express long-term OLM although memory was robust in WTs (***P < 0.001 vs. WTs; n ≥ 8 per group). (I) With 5-min training, WTs showed robust long-term NOR whereas KOs did not (***P < 0.001; n ≥ 12 per group).

Fig. 2.

Spaced training facilitates long-term memory in WTs and normalizes learning in Fmr1 KOs. (A) Training was distributed across 3 trials so that total training time was equivalent to massed trial durations that did not elicit long-term memory in WTs (3 min) or Fmr1 KOs (5 min). Training trials were spaced by 60 min; mice were tested 24 h after training. (B) WTs did not learn object location given one, 3 min long (“massed”) training trial, but did exhibit robust OLM after three 60-s trials “spaced” by 60 min (***P < 0.001; n ≥ 7 per group). (C) WT and KO mice trained in three 100-s trials, spaced by 60 min, exhibited long-term OLM (n ≥ 8 per group p); KOs given the same total training en masse did not (Fig. 1B). (D) Spaced training rescued long-term novel object recognition (NOR) in KOs (P = 0.33; n ≥ 12 per group). (E) With spaced training, KOs spent less time (seconds) exploring objects and traveled less distance (meters) than in a 5 min massed trial (**P < 0.01, ***P < 0.001; n ≥ 9 per group). (F) In both Fmr1 KO and WT mice, OLM was not supported by three 100-s trials spaced by 20 or 120 min, or by two 100-s– or 150-s–long trials spaced by 60 min. Five 60-s trials separated by 60 min supported long-term OLM in both genotypes. (G) WT and KO mice trained in three 20-s trials, spaced by 60 min, for 1 min total training, exhibited robust and comparable long-term OLM (P = 0.83 WT vs. KO; n ≥ 10 per group).

Fig. 3.

Increases in synaptic ERK1/2 activation are associated with object location memory (OLM) in WT mice and absent in KOs given massed training. (A–C) Deconvolved images of immunolabeling for PSD95 (green) and p-ERK1/2 (red) in CA1; overlap appears yellow (arrow). (Scale bars: 5 µm, 1.25 µm, and 0.5 µm in A, B, and C, respectively.) (D) In CA1 stratum radiatum (SR), “Total” numbers of densely p-ERK1/2+ elements did not differ between genotypes (P = 0.73; normalized to WT means) but those colocalized with PSD95 (PSD95+) were more numerous in KOs than WTs (*P < 0.05; N ≥ 10 per group). (E) Mice were handled and habituated and then given 5-min massed training with (“train”) and without (“con”) objects present; p-ERK1/2+ PSDs was quantified for CA1 SR sample fields (Fig. S6). (F) Numbers of densely p-ERK1/2+ PSDs were greater in section 7, ∼2.16 mm posterior to Bregma (2 way ANOVA P = 0.002, interaction between section and group; P < 0.001, section 7 vs. others) of WT mice given spaced training. The dashed box around images shows the approximate region of further analysis (images from Allen Institute for Brain Science). (G) After 5-min massed training, numbers of densely p-ERK1/2+ PSDs were increased in WTs (**P < 0.01) but decreased in Fmr1 KOs (*P < 0.05, **P < 0.01; n ≥ 10 per group; normalized to WT control mean). Note: KOs had constitutively greater numbers of p-ERK1/2 enriched contacts. (H) Ten minutes of massed training increased numbers of densely p-ERK1/2+ PSDs in KOs (*P < 0.05; n ≥ 10 per group: normalized to KO controls).

Fig. 4.

Synaptic ERK1/2 is activated with spaced training, and required for OLM in Fmr1 KOs. (A) Experimental Fmr1 KOs were given three 100 s OLM training trials separated by 1 h; control (“con”) mice were placed in the apparatus without objects present. Brains were harvested after the third arena session. (B) When quantified for the three planes (“massed region”) evaluated in WTs, numbers of p-ERK1/2+ PSDs in KO spaced training mice were not different from those in KO control mice (P = 0.22). However, numbers of p-ERK1/2+ PSDs were elevated in the caudal two-thirds of this span (*P < 0.05; n ≥ 9 per group; normalized to KO control mean). (C) Intensity frequency distribution for p-ERK1/2+ PSDs in CA1 planes activated by spaced training shows a rightward-shift in immunolabeling intensities of p-ERK1/2+ contacts in trained KOs. (D) Paradigm for testing effects of ERK1/2 activation inhibitor SL327 (50 mg/kg) on OLM with spaced training in KOs: injections were given 30 min before arena sessions on days 5–10 and 30 min before the last trial on the training day. (E) In KOs, OLM was blocked by SL327 but was robust with vehicle-treatment (*P < 0.05; n = 10 per group). (F) Exploration times (seconds) were comparable between vehicle- and SL327-treated KOs during spaced training and retention testing (P > 0.05 all vehicle vs. SL327 comparisons; n = 10 per group).