Research report
Reducing expression of GluN10XX subunit splice variants of the NMDA receptor interferes with spatial reference memory

https://doi.org/10.1016/j.bbr.2012.02.014Get rights and content

Abstract

The GluN1 subunit of the N-methyl-d-aspartate (NMDA) receptor shows age-related changes in its expression pattern, some of which correlate with spatial memory performance in mice. Aged C57BL/6 mice show an age-related increase in mRNA expression of GluN1 subunit splice variants that lack the N terminal splice cassette, GluN10XX (GluN1-a). This increase in expression is associated with good performance in reference and working memory tasks. The present study was undertaken to determine if GluN10XX splice variants are required for good performance in reference memory tasks in young mice. Mice were bilaterally injected with either siRNA specific for GluN10XX splice variants, control siRNA or vehicle alone into ventro-lateral orbital cortices. A fourth group of mice did not receive any injections. Starting five days post-injection, mice were tested for their performance in spatial reference memory, associative memory and cognitive flexibility tasks over four days in the Morris water maze. There was a 10–19% reduction in mRNA expression for GluN10XX splice variants within the ventro-lateral orbital cortices in mice following GluN10XX siRNA treatment. Declines in performance within the first half of reference memory testing were seen in the mice receiving siRNA against the GluN10XX splice variants, as compared to the mice injected with control siRNA, vehicle and/or no treatment. These results suggest a role for the GluN10XX splice variants in orbital regions for early acquisition and/or consolidation of spatial reference memory.

Highlights

► Injection of GluN10XX specific siRNA into mice brain showed localized reduction of its mRNA expression. ► Mice with reduced GluN10XX expression showed impairment in reference memory testing. ► GluN10XX splice variants might be involved with memory acquisition and/or consolidation.

Introduction

The aging process has been shown to cause functional declines in many different processes [1]. Memory is one function that is affected early in the aging process. One type of memory, spatial memory, which is important for navigation of organisms within their environment, is particularly affected by the aging process [2], [3]. Brain regions that are important for acquisition, consolidation and retrieval of spatial memory include the prefrontal cortex and hippocampus [2], [4], [5]. Animals, such as rodents and primates, have been used to model different aspects of spatial memory involving both the hippocampus and prefrontal cortex [6], [7], [8]. The prefrontal cortex has also been shown to be important for flexibility of learning within different memory tasks [9], [10]. The aging process is known to hamper performance in reversal tasks, which are used to assess flexibility [11], [12].

Prefrontal cortex and hippocampus have a high concentration of N-methyl-d-aspartate (NMDA) receptors, a type of glutamate receptor involved in learning and memory [13], [14]. NMDA receptors are particularly important in spatial memory [15], [16], [17]. NMDA receptors are heteromeric tetramers composed of subunits from one or more of three different families of subunits, identified as GluN1, GluN2 and GluN3 [18]. There exist eight splice variants in the GluN1 subunit family due to the presence of one N-terminal and two C-terminal splice cassettes [18]. These eight splice variants are heterogeneous in their expression patterns, both during development [19] and aging [20], [21]. The presence or absence of splice cassettes N1, C1 and C2 in individual splice variants will be indicated throughout this article by a series of three subscripts following GluN1, with 0 indicating absence; 1 indicating presence and X indicating either presence or absence of the cassettes [18]. For example, GluN1X10 indicates presence or absence of N1 cassette, presence of C1 cassette and absence of C2 cassette. The absence of the C2 cassette also implies the presence of a new terminal sequence, C2′ [18].

Evidence shows that NMDA receptor binding density and the expression of some of the subunits decline with increasing age in both the hippocampus and prefrontal cortex [22], [23], [24]. The GluN1 subunit of the NMDA receptor has been shown to decline in expression of both protein and mRNA during aging in the prefrontal cortex of C57BL/6 mice [25], [26]. The individual splice forms of this subunit, representing only N or C terminal sequences, are heterogenous with respect to changes in their expression pattern during aging. Our earlier studies have shown that the mRNA expression of sequences found in GluN1X11 (GluN1–1) and GluN1X10 (GluN1–3) splice variants of the GluN1 subunit in the prefrontal/frontal cortex and hippocampus decline during aging [20], but there is an increase in the mRNA of the GluN10XX (GluN1-a) splice variants in response to behavioral testing experience in the prefrontal cortex of old mice [21]. Significant and near significant associations have been seen between higher expression of GluN1X10 and GluN10XX mRNAs within the orbital cortex and better performance in reference and working memory tasks in aged mice [21]. It is however, not known how important these splice forms are to memory. It is also not known how much influence the GluN1 subunits of the NMDA receptor have on observed cognitive flexibility in animals. The present paper utilized in vivo siRNA administration to determine whether decreased expression of GluN10XX splice variants in orbital cortices play a significant role in spatial memory and/or flexibility in young C57Bl/6 mice. The use of young animals has the advantage of not having all of the confounds of other changes that can occur during aging.

Section snippets

Animals

A total of 48 male three-month-old C57BL/6 mice (The Jackson Laboratory, Maine) were used for the study. They were fed ad libitum and housed in cages under 12 h light and 12 h dark cycle. The animals were randomly divided into four treatment groups of twelve animals each; siRNA specific for GluN10XX splice variants (GluN10XX siRNA), control siRNA, vehicle and no treatment. After behavioral testing was performed, all animals were euthanized with exposure to CO2, followed by decapitation. The

Spatial reference memory

There was no overall effect of treatment on cumulative proximity in the blocks of four place trials of the reference memory task (F(3,44) = .66, p = .58), but there was a significant treatment by block interaction (F(15,220) = 1.79, p = .038). When individual blocks were analyzed separately for effects of treatment, there was a significantly higher cumulative proximity in the reference memory task in mice injected with GluN10XX siRNA as compared to mice injected with control siRNA in block 1 (p = .02,

Discussion

The present study provided evidence for a role for the GluN10XX subunit splice variants of the NMDA receptor within the prefrontal cortex of the brain in spatial reference memory in young mice. Reduction of the GluN10XX splice variant mRNA expression in the ventral and lateral orbital regions of the brain in young mice lead to poor performance in the first half of training for spatial reference memory. There was no difference in behavior between any treatments in the later phases of reference

Acknowledgment

Funding sources: This research was supported in part by NIH grants AG016322 (KRM).

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