Research reportTemporal dynamics of immediate early gene expression during cellular consolidation of spatial memory
Introduction
Cellular consolidation refers to the initial stabilisation of a memory trace in the hours following learning [1]. Protein synthesis inhibitors administered during this time dirsupt memory formation, implicating de novo protein synthesis in this process [2]. These proteins are involved in the remodelling of synapses, resulting in a more enduring and stable memory [3]. The expression of Immediate Early Genes (IEGs) consitutes the cell's earliest genomic response to stimulation, and these proteins can either directly modify the structure and function of a cell to stabilise a memory, or activate late-response genes to facilitate cellular consolidation [4]. Synaptic plasticity in the hippocampus is disrupted in mice lacking these genes, with Zif268 Knockout (KO) mice showing impaired late-long term potentiation (LTP) in the dentate gyrus, while late-LTP and long term depression (LTD) is impaired in CA1 of Arc KO mice. A corresponding deficit in learning is also observed in these animals; Zif268 KO mice fail to reactivate the same place cell ensemble in CA1 24 h after exploration of a novel environment [5]. The use of antisense oligodeoxynucleotides (AS-ODNs) which block the translation of mRNA into protein, has also revealed the necessity of IEG expression for memory formation. c-Fos antisense-ODNs infused into CA3 before training in the radial arm maze has been shown to increase both working and reference memory errors [6]. Arc AS-ODNs administered into the hippocampus either before or immediately after training in the water maze task disrupts retention 48 h later without affecting acquisition, whereas administration 8 h after training had no effect [7], highlighting the time-sensitive role of these proteins in the early stages of consolidation.
However, there is some evidence that the role of IEG expression in consolidation is not just limited to the first few hours. During resting periods following spatial exploration, IEG expression is observed in the majority of CA1 neurons initially active during learning, suggesting a re-activation of the neural ensemble [8]. Upregulation of Arc and Zif268 is also observed in the cortex during REM sleep 4 h after spatial exploration [9]. Ramirez-Amaya, Vazdarjanova [10] provided further evidence of multiple “waves” of consolidation, where at 8 h, but not 4 h, following spatial exploration, CA1, CA3 and the parietal cortex showed elevated Arc expression, which was again observed in CA3 and the parietal cortex at 24 h. Double labelling of Arc mRNA and protein confirmed that 81% of neurons activated at 8 h, and 82% of neurons activated at 24 h belonged to the original ensemble active during exploration. However, while this multiple wave of IEG expression has been induced through electroconvulsive shock in mice, where a second wave of Arc expression has been observed in CA1 at 8 h, but not 4 or 6 h following treatment [11], this phenomenon was not replicated under naturalistic conditions of spatial exploration. Furthermore, Arc, Zif268 and c-Fos mRNA expression in the hippocampus following place learning in the water maze drops to caged control levels or below at 6 h [12], corresponding approximately to an 8 h time-point for IEG protein. Therefore the evidence for multiple waves of IEG expression over the course of consolidation of spatial memory remains inconclusive, and may differ according to the IEG used and region under investigation.
In order to resolve this ambiguity in the literature and conduct a more comprehensive investigation into temporal dynamics of IEGs in cellular consolidation, we examined the expression of Zif268, c-Fos and Arc protein in the rat hippocampus, medial prefrontal, entorhinal, perirhinal, retrosplenial, and parietal cortices, at 90 min, 4 h and 8 h after three days of training in the reference memory version of the water maze.
Section snippets
Subjects
Twenty-six male Wistar rats, obtained from Charles River Laboratories, UK, were used as subjects in this experiment. Subjects were approximately three months old and weighed 200–300 g at the beginning of experimentation. All animals were housed three per cage in plastic-bottomed cages (56 cm × 38 cm wide and 22 cm high; NKP Cagers, UK) with a 3 cm layer of woodchip bedding, paper strip nesting material and cardboard tubes for environmental enrichment. Animals were housed in a conventional facility
Morris water maze acquisition
An overall significant effect was found in time taken to escape the maze across the three days of training by all animals (one-way repeated measures ANOVA, F(2,40) = 3.84, p = 0.030), with escape latencies lower on day three compared to day 1 (p = 0.041, see Fig. 1). No difference in water maze escape latencies was observed across the 90 min, 4 h or 8 h groups, (mixed ANOVA, F(2,18) = 0.29, p > 0.05) indicating performance across the three groups was comparable.
Zif268
A significant effect was found between groups
Discussion
This experiment examined the changes in IEG expression in 11 brain regions at three separate time-points in the hours following learning in the Morris water maze in order to resolve the inconsistency in the literature regarding multiple waves of IEG-facilitated consolidation in the hours following spatial learning. The most notable finding was the apparent absence of a second wave of IEG expression at 8 h in any of the brain regions studied. In some regions the complete opposite was observed,
Conclusions
This study did not find evidence for multiple waves of IEG expression following spatial learning, and reaffirm the initial couple of hours following learning as the critical time window for IEG-facilitated consolidation. However, the muted expression of IEG protein in the anterior cingulate, prelimbic and infralimbic cortices at 8 h following learning may be indicative of important neural changes in the late stages of cellular consolidation which pave the way for subsequent systems-level
Conflict of interests
The authors declare no competing financial interests
Acknowledgements
This work was funded in part by the Irish Research Council, and an NUIM John and Pat Hume scholarship held by D.N.B.
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