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 memory training. This reduction in GluN10XX splice variant mRNA did not significantly affect cognitive flexibility in the reversal trials or associative memory in cued trials.
Young mice treated with siRNA specific for GluN10XX splice variants of the NMDA receptors showed delays in learning early on in training, as compared to the mice treated with control siRNA and vehicle treated animals. There was a 10–19% decline in mRNA expression for the GluN10XX splice variants in superficial and deep layers of ventro-lateral orbital region of the brain after treatment with the specific siRNA. Specificity of knockdown of GluN10XX splice variants was verified by the absence of any alterations in hybridization density of the splice variants containing the N1 cassette, GluN11XX or a control mRNA, GAPDH, following GluN10XX specific siRNA treatment in the same brain regions. These results suggest that the delay in learning seen in the reference memory task was likely due to the reduction in expression of the GluN10XX splice variants in the ventro-lateral orbital regions of the brain.
Mice treated with GluN10XX
specific siRNA exhibited a worse performance in the very beginning of training in the reference memory task, as compared to control siRNA injected animals. However, the lack of difference from the vehicle-injected animals in the same block of trials made this difficult to interpret. At the beginning of the second day (Block 3), the group treated with siRNA specific for the GluN10XX
splice variants performed worse than each of the control groups in one or more measures of place trial performance. These GluN10XX
siRNA injected mice then showed improved performance, similar to all other groups, by block 4, suggesting that GluN10XX
splice variants may play a role primarily in early learning. The results were similar between the more traditional measures of pathlength and latency and the proximity score, although not always reaching significance, with p values ranging from .009–.07. The proximity measure has been shown to be the most sensitive at measuring spatial bias for mice in the water maze [32
]. It is less affected by swim speed or floating behavior observed in mice and better reflects spatial bias than the traditional measures [29
]. The near-significant differences at p = .07 between the control siRNA and GluN10XX
siRNA-treated mice in pathlength and latency measures may suggest that this is a modest effect for those measures of performance, but it does appear that the GluN10XX
siRNA-treated mice were impaired in their search for the platform as compared to those receiving control siRNA.
Reference memory tasks involving several trials per day for several days in the Morris water maze involve both acquisition and consolidation of memory and it was not possible to determine which was affected in this study. Memory consolidation in mice has been shown to occur within several hours to days after training for a task [33
]. Leon and coworkers have shown, with the use of protein kinase inhibitors, that memory consolidation can occur 2 hours after acquisition of single day learning in the Morris water maze [34
]. With the use of inhibitors of protein synthesis, Artinian and co-workers [35
] observed initiation of memory consolidation as early as 4 hours after training in the Morris water maze. The major differences in performance following treatment with the GluN10XX
siRNA in this study were seen during the first half of training, particularly in the beginning of day 2, and further improvements in performance occurred later. Based on the above findings, it is possible that the deficit in memory seen in animals treated with GluN10XX
specific siRNA during the first half of training may be due to problems with early acquisition and/or early consolidation.
In the present study, we observed 10–19% declines in mRNA expression for the GluN10XX
subunit splice variants across the ventro-lateral orbital region of the prefrontal cortex following treatment with GluN10XX
specific siRNA. The GluN1 subunit of the NMDA receptor has been identified as a necessary subunit for proper functioning of the NMDA receptor [36
]. The GluN10XX
subunit splice variants which lack the N1 cassette, have reduced affinity for agonist by almost five fold as compared to the ones with the N1 cassette [39
]. These GluN10XX
subunit splice variants show increased mRNA expression in orbital, insular and medial prefrontal cortices of the brain in old mice after they have been subjected to behavioral testing experience [21
]. This increase in GluN10XX
subunit splice variant mRNA expression in the orbital region also had a near-significant (corrected p = .08) association with performance in reference memory in old mice, with higher expressers showing better memory [21
]. Inducible and region specific knockout of all GluN1 subunits has revealed involvement of the subunit in consolidation of hippocampal-independent nondeclarative taste memory [40
]. The prefrontal cortex has been shown to be important for reference memory function including formation of recent memory [41
] and recall of stored information [43
]. Tests of spatial memory using Morris water maze with multiple distant cues and varied number of trials have been shown to involve prefrontal cortex for acquisition of memory [45
]. The prefrontal cortex has also been shown to be involved in consolidation and recall of recent spatial memory after training in the Morris water maze [34
]. Therefore, a reduction in GluN10XX
subunit splice variants by GluN10XX
siRNA treatment in the ventro-lateral orbital cortex might be responsible for the problem in early acquisition and/or consolidation of long term memory after training with Morris water maze.
We only observed differences in reference memory between the animals left untreated and the animals receiving GluN10XX
siRNA in the pathlength measure for the present study. The mice that were left untreated did not receive any acetaminophen, codeine or isoflurane. Prolonged exposure to isoflurane during development can cause neurodegeneration in rodents [46
] but does not have this same effect on aged rodents [47
]. A study by Ishida and coworkers [48
] on the effects of acetaminophen on memory performance in Morris water maze shows that a high dose (302.3 mg/kg) causes memory impairment, but a low dose (15.1 mg/kg) facilitates memory performance. The dose of acetaminophen in the present study was close to the low dose of acetaminophen in the Ishida et al. [48
] study and so may have facilitated memory performance in mice that underwent surgery and received acetaminophen.
Reduction of the mRNA for GluN10XX
subunit splice variants in the ventro-lateral orbital regions did not seem to inhibit performance in the probe trials of the reference memory task, in which the escape platform was missing. Instead there appeared to be a trend for improved performance in the mice injected with GluN10XX
specific siRNA, as compared to the mice injected with control siRNA at the end of the second day of training. The probe trials were used to measure the bias towards a previously learned location of the escape platform. If there was no difference among the different treatment groups, it would indicate a similar bias among the treatment groups for the previously learned platform position. The greatest difference between injected animals in the place trials occurred at the beginning of the second training day. The measurement of bias was at the end of that day, after the mice receiving GluN10XX
siRNA showed equal or better performance to the other treatment groups in place trials. Thus, the bias may have been developed by the time the probe trial was performed. Young animals possess redundant systems for different functions of the body, which deteriorates over time [3
]. Better performance in place and probe trials towards the end of the second training day observed in the mice injected with GluN10XX
specific siRNA could be due to use of other redundant systems that are available to these young animals.
The lack of a larger deficit throughout water maze training may suggest that the GluN10XX
subunit splice variants within the prefrontal cortex do not play a major role in spatial reference memory. However, a reduction in mRNA expression of only 10–19% within young animals did produce a significant decrease in performance within the early part of training. Overexpression of the GluN2B subunit within the cortex and hippocampus of transgenic mice [49
] and induced within the prefrontal cortex of aged mice (unpublished observation) both showed significantly improved performance in the water maze, but only primarily in the middle of water maze training. This suggests that NMDA receptors are more essential within specific phases of learning in this task, as opposed to throughout training.
Lesions in the prefrontal cortex of rodents in some studies have been shown to cause impairment in memory performance during water maze training [45
]. Interestingly Hoh and coworkers have shown that the lesions are not effective if the rodents are pretrained prior to the lesion, suggesting no role of the prefrontal cortex in retrieval and storage of memory information [50
]. Lesions in the prefrontal cortex in other studies failed to impair performance in water maze training [51
]. This might partly be due to non-uniformity of the lesions among the various studies and use of various water maze training protocols. However, this suggests that the role of prefrontal cortex in spatial reference memory is complex, which also may account for the modest changes seen in this study. Studies involving lesions to the hippocampus have been more consistent with respect to impairing performance in water maze training [45
]. The hippocampus expresses a high density of NMDA receptors and GluN1 subunits [54
] and these receptors are important for spatial memory in the water maze [56
]. Thus, it is possible that the application of the GluN10XX
siRNA to the hippocampus may produce a more robust disruption of water maze performance and provide more information about the role of these splice variants in spatial reference memory. In the current study, the focus was on the prefrontal cortex because of previous results showing that the upregulation of GluN10XX
splice variant within this region in aged mice is associated with better performance in reference memory tasks of Morris water maze training [21
There was no overall significant difference in performance of mice between different treatment groups when the escape platform was moved to the opposite quadrant. However, a trend of mice injected with GluN10XX
specific siRNA to perform poorly early in the task was observed. This might suggest a role for GluN10XX
splice variants within the ventro-lateral orbital region in flexibility in animals. Prefrontal cortex has been shown to be involved in reversal training in both non-human primates [57
] and rodents [10
]. The role of the orbital prefrontal cortex in reversal learning has shown inconsistent results across different tasks. Object reversal learning was impaired in monkeys with orbital prefrontal lesions [57
] and reversal learning in attentional set shifting was impaired by lesion in orbital prefrontal cortex in rats [59
], but tasks involving serial reversal learning and response extinction were not affected by lesion in the same area in rats [60
]. Damage to medial prefrontal cortex, however, shows compelling evidence for problems in reversal learning during spatial learning tasks in Morris water maze [10
]. From the above findings it may be inferred that the GluN10XX
subunit splice variants in the ventro-lateral orbital region may not be important for cognitive flexibility or that a 10–19% reduction of mRNA for these splice variants was not sufficient to cause significant impairment in a young animal.
In conclusion, reducing the expression of the GluN10XX subunit splice variants of the NMDA receptor in orbital cortex appeared to interfere in the early phase of spatial reference learning. In contrast cognitive flexibility and associative memory did not appear to be altered by the reduction of the GluN10XX subunit splice variants in the ventro-lateral orbital region. Overall, this study suggested that there may be a role for GluN10XX subunit splice variants within orbital cortex in early acquisition and/or consolidation of spatial long-term memory.