BACKGROUND
memory is one of the earliest of the cognitive functions to show declines during the aging process  <cit> .
memory deficits associated with aging are seen in humans and non-human primates , dogs  <cit>  and rodents  <cit> .
one type of memory that is important for how individuals cope with their environment is spatial memory.
humans show 30% to 80% drops in performance in spatial memory tasks as they age  <cit> .
mice and rats also exhibit deficits in spatial memory performance during aging  <cit> .
aged c57bl/ <dig> mice show spatial reference memory problems when tested over  <dig> days in the morris water maze  <cit> .
we were interested in adopting a task that would show age-related deficits in fewer days in order to test drug interventions with the use of osmotic pumps .
the smallest pump available can deliver drug for 3– <dig> days, but, in addition to the time necessary for behavioral testing, time is also needed for recovery from surgery and pretraining.
berry and coworkers developed a one-day spatial memory task for rats, in which young rats show good improvement in performance  <dig> trials in one day  <cit> .
our initial attempts to use this one-day task with mice showed that young mice could not show a significant improvement within  <dig> trials in one day, but could with two days of testing .
the present study was designed to determine whether we could detect significant differences in performance between young and old mice in a spatial reference memory task with a two-day testing protocol .
the n-methyl-d-aspartate  receptor, a subtype of glutamate receptors, is particularly important in learning and memory functions  <cit> .
nmda antagonists inhibit memory performance  <cit>  and block the initiation of long-term potentiation in the hippocampus  <cit>  and neocortex  <cit> .
these studies suggest that detrimental changes to the nmda receptor during the aging process should impact negatively on memory functions.
aging animals do exhibit declines in nmda receptor binding densities and functions, including memory-related functions.
nmda-stimulated release of transmitters is decreased with increasing age  <cit> .
long-term potentiation is also altered in aged rodents  <cit> .
age-related declines in binding of glutamate and  propyl-1-phosphonic acid  to nmda binding sites have been reported in mice, rats, dogs, and monkeys  <cit> .
humans also exhibit declines with increased age in binding of mk <dig> to the nmda receptor complex in the frontal cortex  <cit> .
changes in nmda binding sites during aging have been correlated with poor performance in reference memory tasks, such as the morris water maze, in both prefrontal/frontal cortical regions  <cit>  and in the hippocampus  <cit> .
functional subunits of the nmda receptor complex have been cloned for rats  <cit>  and mice  <cit> .
the zeta <dig>  subunit has the same distribution as nmda-displaceable glutamate binding  <cit> .
the four members of the epsilon family of subunits, ε1– <dig> , in the mouse  <cit>  each provide different agonist/antagonist affinities to ζ1/ε heteromeric receptors  <cit> .
these ε subunits also produce different gating behaviors, responses to mg++, and i/v curves  <cit> .
there is a difference in the effects of aging on different subunits of the nmda receptor.
the density of mrna expression for the ε <dig> subunit declines with increasing age in the cerebral cortex and dentate gyrus of male c57bl/ <dig> mice  <cit> .
these changes in ε <dig> mrna expression correlate significantly with age-related changes in binding of agonist to nmda sites both across cortical and hippocampal regions  <cit>  and within prefrontal/frontal cortical regions  <cit> .
there is also an overall decrease in mrna expression of the ζ <dig> subunit across cortical and hippocampal regions, but to a lesser degree than seen with the ε <dig> subunit  <cit> .
although age-associated changes in ε <dig> mrna in the prefrontal/frontal cortex correlate with changes in nmda receptor binding and the binding correlates with spatial reference memory problems  <cit> , there has not been a direct relationship seen between the mrna expression of the nmda receptor and memory performance  <cit> .
c57bl/ <dig> mice show significant decreases with increasing age in the protein expression of ε <dig> and ζ <dig> subunits in homogenates from the whole cerebral cortex  <cit> .
significant declines during aging in protein expression in hippocampal homogenates are also seen in the ζ <dig> and ε <dig> subunits in both mice  <cit>  and fisher  <dig> rats  <cit> .
homogenates of only prefrontal/frontal cortex show a significant age-related decrease in the protein expression of the ε <dig> subunit alone  <cit> .
given the differences in pharmacological and electrophysiological properties that different combinations of subunits produce  <cit> , it is important to determine if any of these subunit changes relate to memory performance declines during aging.
it seems likely that the relationship between the receptor subunits and memory ability would be strongest with the protein expressed in the synaptic pool since this pool would be enriched in receptors that are functioning in neurotransmission.
we hypothesized that there would be a relationship between the ε <dig> and ζ <dig> subunit expression in the synaptic pool and reference memory performance.
the ε <dig> subunit does not show significant changes in mrna during aging and less change in protein expression than ε <dig> and ζ <dig>  <cit> , but ratios of high mrna expression of ε <dig> in the face of lower expression of ε <dig> or ζ <dig> do show a relationship to reference memory performance  <cit> .
it was expected that the protein expression of the ε <dig> subunit in the synaptic pool might not change significantly with age, but might show a relationship with reference memory performance.
the present study was designed to determine whether there was a relationship between nmda receptor subunit expression within crude synaptosomes and spatial reference memory ability in a two-day task.
the main study was done with the reference task performed prior to the cued task because this most closely resembled the longer reference memory tasks that have been used previously in this lab.
additional animals from the same shipment performed the cued task first in case some previous training in the mechanics of the water maze task  was necessary to see significant learning within  <dig> days in the reference memory task.
a crude synaptosome preparation was used because it should be enriched in nmda receptors that are in the synaptic pool  <cit> .
the analysis of the protein expression of syntaxin was intended to be used as a loading control for the western blot, but it showed significant changes during aging in the prefrontal/frontal cortex and could not be used for this purpose.
RESULTS
behavioral testing – place training trials followed by cued trials
place trials
there was a significant overall effect of age, f  =  <dig> , p = .
<dig>  and trial, f  =  <dig> , p = .
<dig>  on performance in place learning trials .
there was also a significant interaction between age and trial, f  =  <dig> , p = .
<dig>  on place learning performance.
the  <dig> months olds had significantly greater cumulative proximity measurements than 3-month-old mice when performance was averaged across all place trials .
the  <dig> month olds showed a significant reduction in cumulative proximity between the first place trial of day  <dig> and last trial of day  <dig> , but not between the first and eighth  place trials .
the 26-month-old mice showed only a near significant decrease  in cumulative proximity from the first to the last place trial and no significant change between place trials  <dig> and  <dig> .
there was no significant main effect of age, f  =  <dig> , p = .
<dig>  on cumulative proximity measurements across place learning trials  <dig> through  <dig> .
probe trials
probe trials  <dig> and  <dig> were performed on day  <dig> and probe trial  <dig> on day  <dig> .
there was a significant overall effect of age, f  =  <dig> , p = .
<dig>  and trial, f  =  <dig> , p = .
<dig>  on performance in probe trials .
the  <dig> months olds  tracker units) had significantly higher average proximity measurements than 3-month-old mice  when performance was averaged across all probe trials.
the old mice had significantly greater average proximity measurements than young on probe trials  <dig> and  <dig> , but there was no difference between the two age groups in performance on the initial probe trial .
the  <dig> month olds showed a significant reduction in average proximity between probe trials  <dig> and  <dig>  and probe trials  <dig> and  <dig> .
the 26-month-old mice showed no significant change in average proximity measurements between probe trials  <dig> and  <dig>  or probe trials  <dig> and  <dig> .
there was a significant main effect of age, f  =  <dig> , p = .
<dig>  on average proximity across probe trials  <dig> and  <dig> .
learning index scores were derived for each animal from the performance across the probe trials  <cit> .
there was a significant main effect of age on learning index scores f  =  <dig> , p = .
<dig> with old mice having higher scores than young .
cued trials
there was no significant overall effect of age, f  = .
<dig>  p = .
<dig>  but there was a significant overall effect of trial, f  =  <dig> , p < .
<dig>  on performance in cued trials when they occurred after the place training trials .
both the young and old mice exhibited a significant reduction  in cumulative proximity measurements between the first and last cued trials .
there were no significant differences between the age groups in any of the individual cued trials .
behavioral testing – cued trials prior to place training trials
cued trials
there was a significant overall effect of age, f  =  <dig> , p = .
<dig>  and there was a significant overall effect of trial, f  =  <dig> , p = .
<dig>  on performance in cued trials when they occurred prior to the place training trials .
the older mice had higher cumulative proximity measurements than the young overall in the cued trials .
the performance averaged across cued trials 2– <dig> for four of the old mice was greater than  <dig> standard deviations from the mean of the performance of young mouse group.
when these four mice were excluded from the analysis, there was no significant overall effect of age, f  = .
<dig>  p = .
<dig>  but there was still a significant overall effect of trial, f  =  <dig> , p = .
<dig>  on performance in cued trials that were performed prior to the place training trials .
place and probe trials following the cued trials
there was a significant main effect of aging on performance in place trials following the cued trials, both when the four old mice that did poorly in the cued trials were excluded, f  =  <dig> , p < .
<dig> , and when they were included in the analysis, f  =  <dig> , p < .
<dig>  .
the  <dig> month old mice had higher cumulative proximity measurements than the  <dig> month olds overall in the place trials, both when all mice were included  and when the four were excluded .
the  <dig> month old mice did not show a significant improvement in cumulative proximity between place trials  <dig> and  <dig> .
there was a significant main effect of aging on performance in probe trials when place training followed the cued trials, both when the four old mice that did poorly in the cued trials were excluded, f  =  <dig> , p = .
<dig>  and when they were included in the analysis, f  =  <dig> , p = .
<dig>  .
the old mice had higher average proximity measurements overall in the probe trials than the younger mice .
protein expression of nmda receptor subunits in crude synaptosomes
protein expression was assessed in the mice that underwent place training prior to cued training.
there was a significant decrease in caudal cortex equivalents per μg of protein loaded for the ζ <dig> and ε <dig> subunits of the nmda receptor  in crude synaptosomes from the prefrontal/frontal cortex of 26-month-old mice as compared to young .
there was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ε <dig>  subunit of the nmda receptor in the prefrontal/frontal cortex .
syntaxin also showed a significant decrease in expression between  <dig> and  <dig> months of age in the prefrontal/frontal cortex .
there was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ζ <dig> , ε <dig> , or ε <dig>  subunits of the nmda receptor in the hippocampus .
syntaxin showed a near-significant increase in expression between  <dig> and  <dig> months of age in the hippocampus .
correlations between learning performance and protein expression of nmda receptor subunits
there were significant negative correlations for both the overall cumulative proximity measurements in the place trials and the learning index scores derived from the probe trials and subunit equivalents/μg protein for the ζ <dig> and ε <dig> subunits in crude synaptosomes from the prefrontal/frontal cortex when all animals were considered .
the negative correlation coefficient indicated that high levels of protein expression were associated with low proximity measurements or learning index scores .
there were no significant correlations found for protein expression of the ε <dig> subunit or syntaxin in the prefrontal/frontal cortex and performance in either the place or probe trials across the ages .
none of the subunits nor syntaxin expression levels in the hippocampus showed a significant correlation with place or probe trial performance across the ages .
there was a near significant correlation between the protein expression of the ε <dig> subunit in the hippocampus of young alone  and old alone  and the learning index score.
there was a significant correlation between syntaxin expression in the hippocampus of the old group alone and the learning index score .
* p < .05; ** p < .01; correlations include individual mice from both age groups.
n =  <dig>  for prefrontal/frontal cortex; n =  <dig>  for hippocampus.
discussion
this study reports the detection of age-related changes in spatial reference memory performance in c57bl/ <dig> male mice with the use of a two-day reference memory task and changes in the protein expression of nmda receptors within crude synaptosomes during aging.
a significant decline in spatial reference learning ability in the old mice as compared to the young was detected with a two-day task, regardless of whether the cued trials were performed before or after the place training.
the young mice required two days in order to show significant improvement in the place-learning task.
there were significant decreases with increased age in the expression of the ζ <dig> and ε <dig> subunits of the nmda receptor in crude synaptosomes that were prepared from prefrontal/frontal cortex and these changes appeared to be related to declines in performance ability in the spatial reference memory task.
previous work in our laboratory utilized a reference memory task in the morris water maze that involved  <dig> days of testing in order to characterize learning abilities in different ages of c57bl/ <dig> mice  <cit> .
many other studies showing memory declines during aging in mice report the use of between  <dig> and  <dig> days to assess reference memory ability  <cit> .
frick and colleagues have used a one-day task with  <dig> place trials to show significant declines in memory performance between young  and middle-aged  male and female c57bl/ <dig> mice  <cit> .
it should be noted, however, that calhoun and co-workers found no effect of aging on reference memory performance in male c57bl/ <dig> mice performing a reference memory task in the water maze for  <dig> days  <cit> .
the present study demonstrated that declines in spatial memory ability in male c57bl/ <dig> mice between  <dig> and  <dig> months of age could be detected in both place learning trials and repeated probe trials with a 2-day protocol of place training.
these deficits in the reference memory task were seen in animals that showed no age-related differences in control cued testing, thus reducing the possibility that the performances in the reference memory task were due to problems with vision, motor ability or motivation.
the performance of the aged animals in the 2-day task that was performed before the cued task showed tendencies in the middle to late trials to plateau at a level higher than the young performance or to show a worsening of performance.
this pattern has been seen with c57bl/ <dig> mice in a 12-day reference memory task  <cit>  and in other studies using multiple training days for mice  <cit>  and age-impaired rats  <cit> .
thus, this two-day task appears to be as sensitive to reference memory declines as tasks involving more days of training.
the young mice in the present study that underwent place training first showed a significant improvement in performance in probe trial performance at the end of day one as compared to the naïve probe trials, but required two days to show significant improvement in the place learning trials.
the significant difference in performance between young and old mice was seen in the probe trials both during day  <dig> and across both days, but the age difference in place learning performance could only be detected across  <dig> days of training.
this suggests that two days is necessary to detect the greatest differences in spatial memory ability between young and old c57bl/ <dig> mice with a protocol of  <dig> place trials per day.
young female and male rats are able to show significant learning within eight place learning trials in a single day with the use of a similar protocol  <cit> .
our laboratory and others have also seen this necessity for using more trials for mice than rats in order to obtain significant improvement in performance in a working memory task  <cit> .
these studies suggest that mice need more rehearsal than rats do in order to perform both reference and working memory tasks.
mice have also been shown to use different strategies to perform water maze tasks  <cit> .
some aged mice showed deficits in performance of the cued trials as compared to the young when the cued trials were performed first, but these mice improved in performance by cued trial  <dig>  the majority of aged mice  that were tested in the cued trials first showed similar performances to the young mice throughout the cued trials.
in previous studies, both young and old mice have also been excluded from analysis after showing poor performance in cued trials that followed place training  <cit> .
this suggests that there are some mice that can't perform well in multiple tasks in the water maze.
regardless of whether the cued trials were conducted after or prior to the place training trials, those aged mice that showed no problems in the cued task exhibited significant deficits in the place and probe trials.
this suggests that the aged mice do have problems in performing the spatial reference memory task and it is not simply a problem with learning to acquire the platform, motivation, or sensory or motor abilities.
what cannot be ruled out is a problem with acquiring the specific rules for the hidden platform task.
there was a significant decline between young and old mice in the protein expression of the ζ <dig> and ε <dig> subunits in crude synaptosomes from the prefrontal/frontal cortex in the present study.
there was a 21% decrease in protein expression of the ζ <dig> subunit between  <dig> and  <dig> months of age in synaptosomes from prefrontal/frontal cortex in this study, but the protein expression of the ζ <dig> subunit showed a non-significant trend to decrease from  <dig>  months to  <dig> months of age in homogenates from the caudal portion of prefrontal/frontal cortex in a previous study  <cit> .
the protein expression of the ε <dig> subunit also showed a greater decrease with increased age in synaptosomes from whole prefrontal/frontal cortex  than previously reported in homogenates from the caudal portion of the prefrontal/frontal cortex   <cit> .
these results suggest that the synaptic pool of receptors is more susceptible to the effects of aging than the whole population of receptors within the neuron in the prefrontal/frontal cortex.
this may reflect an increased turnover rate in the subunits either before or after they are inserted into the synaptic pool.
these same subunits showed significant declines  between young and old mice in homogenates of the whole cerebral cortex from the same strain of mice  <cit> .
the ε <dig> subunit showed a significant decline between  <dig> and  <dig> months of age in homogenates from whole cerebral cortex  <cit> , but those ages and extra cortical regions were not included in the present study.
the crude synaptosomes prepared from the hippocampus showed a trend for an increase in protein expression of the nmda subunits examined.
a similar pattern is seen in wistar rat homogenates of hippocampus in which nr <dig> subunit proteins showed a significant increase and nr2a and nr2b subunit proteins showed a trend for an increase  <cit> .
this differs, however, from the results from homogenates prepared from the hippocampi of c57bl/ <dig> mice in which the ζ <dig> subunit showed a significant decrease in expression between  <dig> and  <dig> months of age  <cit> .
these differences in the effects of aging on nmda receptor expression in the synaptic pool versus the whole tissue are similar to those found in fisher  <dig> rats, in which the basal surface expression of nmda subunit proteins does not change between adult ages  <cit> , but expression of both nr <dig>  and nr2b  expression is decreased in homogenates of hippocampus from the same strain of rats  <cit> .
this may represent maintenance of the synaptic pool of nmda receptor subunits within the hippocampus during aging, but a reduction in the pool being produced.
this could happen if the turnover rate at the synapse was reduced.
there is also the possibility that the mice in the present study were influenced by the behavioral experience, although the rats in both of the studies mentioned above were naïve to behavioral training  <cit> .
regardless of the cause, there is a very different effect of aging on the nmda subunit expression patterns between the prefrontal/frontal cortex and the hippocampus in c57bl/ <dig> mice.
it should be noted that long-evans rats do not show significant effects of aging on nr <dig> protein expression either by western blotting of homogenates or immunofluorescence on tissue sections  <cit> , so there appear to be some strain and species differences in the effects of aging on nmda receptor expression in the hippocampus.
the protein expression of the ζ <dig> and ε <dig> subunits in the synaptosomal fraction of the prefrontal/frontal cortex correlated negatively with the proximity measurements in the place and the learning index score derived from the probe trials.
since high proximity measurements and scores indicate poor performance, the lower expression of ζ <dig> and ε <dig> in the old animals was associated with poorer performance, i.e., a positive correlation between protein and learning ability.
this suggests that the changes during aging in these subunits of the nmda receptor within the prefrontal/frontal cortex contribute to the memory problems seen in the older mice.
in support of this, the administration of the ε <dig> subunit antagonist ifenprodil into orbital regions of the prefrontal cortex of young mice inhibited learning in the same task, suggesting that ε <dig> subunits in that region are involved in performing this spatial reference memory task  <cit> .
the prefrontal cortex has been shown in both human and animal studies to be important in applying different strategies to the handling of memories that have spatial and temporal properties, in retrieval of the sources of information, and in adjusting to changing environmental conditions .
lesioning studies in rats show that orbital and medial prefrontal regions contribute to performance in spatial reference memory tasks in the morris water maze  <cit> .
the investigators provide arguments that this is due to a deficit in organization of the behaviours necessary to perform the task or problems with handling the spatial memory temporarily, as opposed to specific spatial guidance problems  <cit> .
insular cortex injections of tetrodotoxin also impair the retention of platform position in the water maze  <cit> .
more recent studies do not see an affect of lesions of the medial prefrontal cortex on performance of the spatial reference memory task in the water maze  <cit> , but the tissue dissections in this study were not limited to medial prefrontal regions.
they also included orbital and insular prefrontal regions, as well as some motor and somatosensory cortices.
the literature thus provides evidence that some prefrontal regions play a role in the performance of spatial reference memory tasks.
the current study suggests that declines in specific nmda receptor subunits in the prefrontal/frontal cortex contribute to declines in performing the task by aged mice.
the specific components of the performance that are affected will have to be determined.
the age-related declines seen in spatial reference memory performance of c57bl/ <dig> mice in a 12-day protocol show a relationship with decreases in binding of the transmitter glutamate to the nmda receptor in the prefrontal/frontal cortex and hippocampus  <cit>  and to the ratio of ε1/ζ <dig> and ε1/ε <dig> subunit mrnas within subregions of the hippocampus  <cit> .
changes in nr2b protein expression in homogenates from the hippocampus also show a correlation with spatial reference memory in aged fisher  <dig> rats  <cit> .
nr <dig> levels in the ca <dig> region correlate with water maze performance during aging in long-evans rats  <cit> .
there was the suggestion of a relationship between the ε <dig> subunit expression in the hippocampus within each age group and reference memory ability, but it did not reach significance.
there is a difference in how aging affects nmda receptor binding and subunit mrna expression between the dorsal and intermediate hippocampus in c57bl/ <dig> mice  <cit> , so using the whole hippocampus in this study may have diluted out some effects.
there was a significant decline in the protein expression of syntaxin, a presynaptic terminal membrane protein  <cit> , in the prefrontal/frontal cortex between  <dig> and  <dig> months of age in the c57bl/ <dig> mice in this study.
for this reason, syntaxin was not used to correct for loading differences in the present study.
the fact that there were differences in the effects of aging on the different nmda receptor subunit and syntaxin suggest that the effects were not due to different amounts of protein loaded.
although we were only able to control for loading differences in this study by loading known amounts of protein, the presence of the standards on every gel do provide control for variability between gels and transfers.
there was no significant effect of aging on syntaxin in the hippocampus, however, to be consistent in the reporting, the hippocampal subunits were also not normalized to syntaxin.
the present results are in agreement with studies on wistar rats that also show no declines in the protein expression of syntaxin in the hippocampus  <cit> , but differ from results of whole cerebral cortex from wistar rats in which syntaxin showed no aging change  <cit> .
there was evidence though in this study that syntaxin expression within the hippocampus of the old mice may contribute to the reference memory deficits seen in the aged animals.
CONCLUSIONS
age-associated deficits in spatial reference memory were detected in c57bl/ <dig> mice with the use of a two-day morris water maze task.
the most significant age-related declines in nmda receptor subunit expression in these mice involved the ε <dig> subunit within the synaptic pool of the prefrontal/frontal cortex.
the ζ <dig> subunit also showed a significant decrease during aging in this same subfraction of prefrontal/frontal cortical tissue.
lower expression of both the ζ <dig> and ε <dig> subunits across the ages was associated with poorer performance.
these results suggest that changes in the synaptic pool of nmda receptors containing ζ <dig> and ε <dig> subunits within the prefrontal/frontal cortex contributed to age-related declines in performance of a spatial reference memory task.

 memory is one of the earliest of the cognitive functions to show declines during the aging process. memory deficits associated with aging are seen in humans and non-human primates, dogs and rodents. one type of memory that is important for how individuals cope with their environment is spatial memory. humans show 30% to 80% drops in performance in spatial memory tasks as they age. mice and rats also exhibit deficits in spatial memory performance during aging. aged c57bl/ mice show spatial reference memory problems when tested over days in the morris water maze. we were interested in adopting a task that would show age-related deficits in fewer days in order to test drug interventions with the use of osmotic pumps. the smallest pump available can deliver drug for 3– days, but, in addition to the time necessary for behavioral testing, time is also needed for recovery from surgery and pretraining. berry and coworkers developed a one-day spatial memory task for rats, in which young rats show good improvement in performance trials in one day. our initial attempts to use this one-day task with mice showed that young mice could not show a significant improvement within trials in one day, but could with two days of testing. the present study was designed to determine whether we could detect significant differences in performance between young and old mice in a spatial reference memory task with a two-day testing protocol. the n-methyl-d-aspartate receptor, a subtype of glutamate receptors, is particularly important in learning and memory functions. nmda antagonists inhibit memory performance and block the initiation of long-term potentiation in the hippocampus and neocortex. these studies suggest that detrimental changes to the nmda receptor during the aging process should impact negatively on memory functions. aging animals do exhibit declines in nmda receptor binding densities and functions, including memory-related functions. nmda-stimulated release of transmitters is decreased with increasing age. long-term potentiation is also altered in aged rodents. age-related declines in binding of glutamate and propyl-1-phosphonic acid to nmda binding sites have been reported in mice, rats, dogs, and monkeys. humans also exhibit declines with increased age in binding of mk to the nmda receptor complex in the frontal cortex. changes in nmda binding sites during aging have been correlated with poor performance in reference memory tasks, such as the morris water maze, in both prefrontal/frontal cortical regions and in the hippocampus. functional subunits of the nmda receptor complex have been cloned for rats and mice. the zeta subunit has the same distribution as nmda-displaceable glutamate binding. the four members of the epsilon family of subunits, ε1–, in the mouse each provide different agonist/antagonist affinities to ζ1/ε heteromeric receptors. these ε subunits also produce different gating behaviors, responses to mg++, and i/v curves. there is a difference in the effects of aging on different subunits of the nmda receptor. the density of mrna expression for the ε subunit declines with increasing age in the cerebral cortex and dentate gyrus of male c57bl/ mice. these changes in ε mrna expression correlate significantly with age-related changes in binding of agonist to nmda sites both across cortical and hippocampal regions and within prefrontal/frontal cortical regions. there is also an overall decrease in mrna expression of the ζ subunit across cortical and hippocampal regions, but to a lesser degree than seen with the ε subunit. although age-associated changes in ε mrna in the prefrontal/frontal cortex correlate with changes in nmda receptor binding and the binding correlates with spatial reference memory problems, there has not been a direct relationship seen between the mrna expression of the nmda receptor and memory performance. c57bl/ mice show significant decreases with increasing age in the protein expression of ε and ζ subunits in homogenates from the whole cerebral cortex. significant declines during aging in protein expression in hippocampal homogenates are also seen in the ζ and ε subunits in both mice and fisher rats. homogenates of only prefrontal/frontal cortex show a significant age-related decrease in the protein expression of the ε subunit alone. given the differences in pharmacological and electrophysiological properties that different combinations of subunits produce, it is important to determine if any of these subunit changes relate to memory performance declines during aging. it seems likely that the relationship between the receptor subunits and memory ability would be strongest with the protein expressed in the synaptic pool since this pool would be enriched in receptors that are functioning in neurotransmission. we hypothesized that there would be a relationship between the ε and ζ subunit expression in the synaptic pool and reference memory performance. the ε subunit does not show significant changes in mrna during aging and less change in protein expression than ε and ζ, but ratios of high mrna expression of ε in the face of lower expression of ε or ζ do show a relationship to reference memory performance. it was expected that the protein expression of the ε subunit in the synaptic pool might not change significantly with age, but might show a relationship with reference memory performance. the present study was designed to determine whether there was a relationship between nmda receptor subunit expression within crude synaptosomes and spatial reference memory ability in a two-day task. the main study was done with the reference task performed prior to the cued task because this most closely resembled the longer reference memory tasks that have been used previously in this lab. additional animals from the same shipment performed the cued task first in case some previous training in the mechanics of the water maze task was necessary to see significant learning within days in the reference memory task. a crude synaptosome preparation was used because it should be enriched in nmda receptors that are in the synaptic pool. the analysis of the protein expression of syntaxin was intended to be used as a loading control for the western blot, but it showed significant changes during aging in the prefrontal/frontal cortex and could not be used for this purpose.  behavioral testing – place training trials followed by cued trials place trials there was a significant overall effect of age, f =, p =. and trial, f =, p =. on performance in place learning trials. there was also a significant interaction between age and trial, f =, p =. on place learning performance. the months olds had significantly greater cumulative proximity measurements than 3-month-old mice when performance was averaged across all place trials. the month olds showed a significant reduction in cumulative proximity between the first place trial of day and last trial of day, but not between the first and eighth place trials. the 26-month-old mice showed only a near significant decrease in cumulative proximity from the first to the last place trial and no significant change between place trials and. there was no significant main effect of age, f =, p =. on cumulative proximity measurements across place learning trials through. probe trials probe trials and were performed on day and probe trial on day. there was a significant overall effect of age, f =, p =. and trial, f =, p =. on performance in probe trials. the months olds tracker units) had significantly higher average proximity measurements than 3-month-old mice when performance was averaged across all probe trials. the old mice had significantly greater average proximity measurements than young on probe trials and, but there was no difference between the two age groups in performance on the initial probe trial. the month olds showed a significant reduction in average proximity between probe trials and and probe trials and. the 26-month-old mice showed no significant change in average proximity measurements between probe trials and or probe trials and. there was a significant main effect of age, f =, p =. on average proximity across probe trials and. learning index scores were derived for each animal from the performance across the probe trials. there was a significant main effect of age on learning index scores f =, p =. with old mice having higher scores than young. cued trials there was no significant overall effect of age, f =. p =. but there was a significant overall effect of trial, f =, p <. on performance in cued trials when they occurred after the place training trials. both the young and old mice exhibited a significant reduction in cumulative proximity measurements between the first and last cued trials. there were no significant differences between the age groups in any of the individual cued trials. behavioral testing – cued trials prior to place training trials cued trials there was a significant overall effect of age, f =, p =. and there was a significant overall effect of trial, f =, p =. on performance in cued trials when they occurred prior to the place training trials. the older mice had higher cumulative proximity measurements than the young overall in the cued trials. the performance averaged across cued trials 2– for four of the old mice was greater than standard deviations from the mean of the performance of young mouse group. when these four mice were excluded from the analysis, there was no significant overall effect of age, f =. p =. but there was still a significant overall effect of trial, f =, p =. on performance in cued trials that were performed prior to the place training trials. place and probe trials following the cued trials there was a significant main effect of aging on performance in place trials following the cued trials, both when the four old mice that did poorly in the cued trials were excluded, f =, p <., and when they were included in the analysis, f =, p <.. the month old mice had higher cumulative proximity measurements than the month olds overall in the place trials, both when all mice were included and when the four were excluded. the month old mice did not show a significant improvement in cumulative proximity between place trials and. there was a significant main effect of aging on performance in probe trials when place training followed the cued trials, both when the four old mice that did poorly in the cued trials were excluded, f =, p =. and when they were included in the analysis, f =, p =.. the old mice had higher average proximity measurements overall in the probe trials than the younger mice. protein expression of nmda receptor subunits in crude synaptosomes protein expression was assessed in the mice that underwent place training prior to cued training. there was a significant decrease in caudal cortex equivalents per μg of protein loaded for the ζ and ε subunits of the nmda receptor in crude synaptosomes from the prefrontal/frontal cortex of 26-month-old mice as compared to young. there was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ε subunit of the nmda receptor in the prefrontal/frontal cortex. syntaxin also showed a significant decrease in expression between and months of age in the prefrontal/frontal cortex. there was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ζ, ε, or ε subunits of the nmda receptor in the hippocampus. syntaxin showed a near-significant increase in expression between and months of age in the hippocampus. correlations between learning performance and protein expression of nmda receptor subunits there were significant negative correlations for both the overall cumulative proximity measurements in the place trials and the learning index scores derived from the probe trials and subunit equivalents/μg protein for the ζ and ε subunits in crude synaptosomes from the prefrontal/frontal cortex when all animals were considered. the negative correlation coefficient indicated that high levels of protein expression were associated with low proximity measurements or learning index scores. there were no significant correlations found for protein expression of the ε subunit or syntaxin in the prefrontal/frontal cortex and performance in either the place or probe trials across the ages. none of the subunits nor syntaxin expression levels in the hippocampus showed a significant correlation with place or probe trial performance across the ages. there was a near significant correlation between the protein expression of the ε subunit in the hippocampus of young alone and old alone and the learning index score. there was a significant correlation between syntaxin expression in the hippocampus of the old group alone and the learning index score. * p <.05; ** p <.01; correlations include individual mice from both age groups. n = for prefrontal/frontal cortex; n = for hippocampus. discussion this study reports the detection of age-related changes in spatial reference memory performance in c57bl/ male mice with the use of a two-day reference memory task and changes in the protein expression of nmda receptors within crude synaptosomes during aging. a significant decline in spatial reference learning ability in the old mice as compared to the young was detected with a two-day task, regardless of whether the cued trials were performed before or after the place training. the young mice required two days in order to show significant improvement in the place-learning task. there were significant decreases with increased age in the expression of the ζ and ε subunits of the nmda receptor in crude synaptosomes that were prepared from prefrontal/frontal cortex and these changes appeared to be related to declines in performance ability in the spatial reference memory task. previous work in our laboratory utilized a reference memory task in the morris water maze that involved days of testing in order to characterize learning abilities in different ages of c57bl/ mice. many other studies showing memory declines during aging in mice report the use of between and days to assess reference memory ability. frick and colleagues have used a one-day task with place trials to show significant declines in memory performance between young and middle-aged male and female c57bl/ mice. it should be noted, however, that calhoun and co-workers found no effect of aging on reference memory performance in male c57bl/ mice performing a reference memory task in the water maze for days. the present study demonstrated that declines in spatial memory ability in male c57bl/ mice between and months of age could be detected in both place learning trials and repeated probe trials with a 2-day protocol of place training. these deficits in the reference memory task were seen in animals that showed no age-related differences in control cued testing, thus reducing the possibility that the performances in the reference memory task were due to problems with vision, motor ability or motivation. the performance of the aged animals in the 2-day task that was performed before the cued task showed tendencies in the middle to late trials to plateau at a level higher than the young performance or to show a worsening of performance. this pattern has been seen with c57bl/ mice in a 12-day reference memory task and in other studies using multiple training days for mice and age-impaired rats. thus, this two-day task appears to be as sensitive to reference memory declines as tasks involving more days of training. the young mice in the present study that underwent place training first showed a significant improvement in performance in probe trial performance at the end of day one as compared to the naïve probe trials, but required two days to show significant improvement in the place learning trials. the significant difference in performance between young and old mice was seen in the probe trials both during day and across both days, but the age difference in place learning performance could only be detected across days of training. this suggests that two days is necessary to detect the greatest differences in spatial memory ability between young and old c57bl/ mice with a protocol of place trials per day. young female and male rats are able to show significant learning within eight place learning trials in a single day with the use of a similar protocol. our laboratory and others have also seen this necessity for using more trials for mice than rats in order to obtain significant improvement in performance in a working memory task. these studies suggest that mice need more rehearsal than rats do in order to perform both reference and working memory tasks. mice have also been shown to use different strategies to perform water maze tasks. some aged mice showed deficits in performance of the cued trials as compared to the young when the cued trials were performed first, but these mice improved in performance by cued trial the majority of aged mice that were tested in the cued trials first showed similar performances to the young mice throughout the cued trials. in previous studies, both young and old mice have also been excluded from analysis after showing poor performance in cued trials that followed place training. this suggests that there are some mice that can't perform well in multiple tasks in the water maze. regardless of whether the cued trials were conducted after or prior to the place training trials, those aged mice that showed no problems in the cued task exhibited significant deficits in the place and probe trials. this suggests that the aged mice do have problems in performing the spatial reference memory task and it is not simply a problem with learning to acquire the platform, motivation, or sensory or motor abilities. what cannot be ruled out is a problem with acquiring the specific rules for the hidden platform task. there was a significant decline between young and old mice in the protein expression of the ζ and ε subunits in crude synaptosomes from the prefrontal/frontal cortex in the present study. there was a 21% decrease in protein expression of the ζ subunit between and months of age in synaptosomes from prefrontal/frontal cortex in this study, but the protein expression of the ζ subunit showed a non-significant trend to decrease from months to months of age in homogenates from the caudal portion of prefrontal/frontal cortex in a previous study. the protein expression of the ε subunit also showed a greater decrease with increased age in synaptosomes from whole prefrontal/frontal cortex than previously reported in homogenates from the caudal portion of the prefrontal/frontal cortex. these results suggest that the synaptic pool of receptors is more susceptible to the effects of aging than the whole population of receptors within the neuron in the prefrontal/frontal cortex. this may reflect an increased turnover rate in the subunits either before or after they are inserted into the synaptic pool. these same subunits showed significant declines between young and old mice in homogenates of the whole cerebral cortex from the same strain of mice. the ε subunit showed a significant decline between and months of age in homogenates from whole cerebral cortex, but those ages and extra cortical regions were not included in the present study. the crude synaptosomes prepared from the hippocampus showed a trend for an increase in protein expression of the nmda subunits examined. a similar pattern is seen in wistar rat homogenates of hippocampus in which nr subunit proteins showed a significant increase and nr2a and nr2b subunit proteins showed a trend for an increase. this differs, however, from the results from homogenates prepared from the hippocampi of c57bl/ mice in which the ζ subunit showed a significant decrease in expression between and months of age. these differences in the effects of aging on nmda receptor expression in the synaptic pool versus the whole tissue are similar to those found in fisher rats, in which the basal surface expression of nmda subunit proteins does not change between adult ages, but expression of both nr and nr2b expression is decreased in homogenates of hippocampus from the same strain of rats. this may represent maintenance of the synaptic pool of nmda receptor subunits within the hippocampus during aging, but a reduction in the pool being produced. this could happen if the turnover rate at the synapse was reduced. there is also the possibility that the mice in the present study were influenced by the behavioral experience, although the rats in both of the studies mentioned above were naïve to behavioral training. regardless of the cause, there is a very different effect of aging on the nmda subunit expression patterns between the prefrontal/frontal cortex and the hippocampus in c57bl/ mice. it should be noted that long-evans rats do not show significant effects of aging on nr protein expression either by western blotting of homogenates or immunofluorescence on tissue sections, so there appear to be some strain and species differences in the effects of aging on nmda receptor expression in the hippocampus. the protein expression of the ζ and ε subunits in the synaptosomal fraction of the prefrontal/frontal cortex correlated negatively with the proximity measurements in the place and the learning index score derived from the probe trials. since high proximity measurements and scores indicate poor performance, the lower expression of ζ and ε in the old animals was associated with poorer performance, i.e., a positive correlation between protein and learning ability. this suggests that the changes during aging in these subunits of the nmda receptor within the prefrontal/frontal cortex contribute to the memory problems seen in the older mice. in support of this, the administration of the ε subunit antagonist ifenprodil into orbital regions of the prefrontal cortex of young mice inhibited learning in the same task, suggesting that ε subunits in that region are involved in performing this spatial reference memory task. the prefrontal cortex has been shown in both human and animal studies to be important in applying different strategies to the handling of memories that have spatial and temporal properties, in retrieval of the sources of information, and in adjusting to changing environmental conditions. lesioning studies in rats show that orbital and medial prefrontal regions contribute to performance in spatial reference memory tasks in the morris water maze. the investigators provide arguments that this is due to a deficit in organization of the behaviours necessary to perform the task or problems with handling the spatial memory temporarily, as opposed to specific spatial guidance problems. insular cortex injections of tetrodotoxin also impair the retention of platform position in the water maze. more recent studies do not see an affect of lesions of the medial prefrontal cortex on performance of the spatial reference memory task in the water maze, but the tissue dissections in this study were not limited to medial prefrontal regions. they also included orbital and insular prefrontal regions, as well as some motor and somatosensory cortices. the literature thus provides evidence that some prefrontal regions play a role in the performance of spatial reference memory tasks. the current study suggests that declines in specific nmda receptor subunits in the prefrontal/frontal cortex contribute to declines in performing the task by aged mice. the specific components of the performance that are affected will have to be determined. the age-related declines seen in spatial reference memory performance of c57bl/ mice in a 12-day protocol show a relationship with decreases in binding of the transmitter glutamate to the nmda receptor in the prefrontal/frontal cortex and hippocampus and to the ratio of ε1/ζ and ε1/ε subunit mrnas within subregions of the hippocampus. changes in nr2b protein expression in homogenates from the hippocampus also show a correlation with spatial reference memory in aged fisher rats. nr levels in the ca region correlate with water maze performance during aging in long-evans rats. there was the suggestion of a relationship between the ε subunit expression in the hippocampus within each age group and reference memory ability, but it did not reach significance. there is a difference in how aging affects nmda receptor binding and subunit mrna expression between the dorsal and intermediate hippocampus in c57bl/ mice, so using the whole hippocampus in this study may have diluted out some effects. there was a significant decline in the protein expression of syntaxin, a presynaptic terminal membrane protein, in the prefrontal/frontal cortex between and months of age in the c57bl/ mice in this study. for this reason, syntaxin was not used to correct for loading differences in the present study. the fact that there were differences in the effects of aging on the different nmda receptor subunit and syntaxin suggest that the effects were not due to different amounts of protein loaded. although we were only able to control for loading differences in this study by loading known amounts of protein, the presence of the standards on every gel do provide control for variability between gels and transfers. there was no significant effect of aging on syntaxin in the hippocampus, however, to be consistent in the reporting, the hippocampal subunits were also not normalized to syntaxin. the present results are in agreement with studies on wistar rats that also show no declines in the protein expression of syntaxin in the hippocampus, but differ from results of whole cerebral cortex from wistar rats in which syntaxin showed no aging change. there was evidence though in this study that syntaxin expression within the hippocampus of the old mice may contribute to the reference memory deficits seen in the aged animals.  age-associated deficits in spatial reference memory were detected in c57bl/ mice with the use of a two-day morris water maze task. the most significant age-related declines in nmda receptor subunit expression in these mice involved the ε subunit within the synaptic pool of the prefrontal/frontal cortex. the ζ subunit also showed a significant decrease during aging in this same subfraction of prefrontal/frontal cortical tissue. lower expression of both the ζ and ε subunits across the ages was associated with poorer performance. these results suggest that changes in the synaptic pool of nmda receptors containing ζ and ε subunits within the prefrontal/frontal cortex contributed to age-related declines in performance of a spatial reference memory task. 
mice were trained in a reference memory task for two days in a morris water maze. the presence of an age-related decline in performance of the reference memory task regardless of when the cued trials were performed suggests that the deficits were due to factors that were unique to the spatial reference memory task. these results also suggest that declines in specific nmda receptor subunits in the synaptic pool of prefrontal/frontal brain regions contributed to these age-related problems with performing a spatial reference memory task. the present study was designed to determine if age-related deficits could be detected with a shorter testing protocol and whether these deficits showed a relationship with nmda receptors. c57bl/ mice show a relationship during aging between nmda receptor expression and spatial reference memory performance in a 12-day task. the subunit changes showed a significant correlation with both place and probe trial performance. cued testing was performed either after or prior to reference memory testing. crude synaptosomes were prepared from prefrontal/frontal cortex and hippocampus of the mice that underwent reference memory testing first. young mice showed significant improvement in probe and place learning when reference memory testing was done prior to cued testing. a significant decrease in performance was seen between and months of age with the two-day reference task, regardless of whether cued testing was performed before or after reference memory testing. there was a significant decline in the protein expression of the ε and ζ subunits of the nmda receptor and syntaxin in prefrontal/frontal cortex. nmda receptor subunit and syntaxin proteins were analyzed with western blotting.
 c57bl/ mice show a relationship during aging between nmda receptor expression and spatial reference memory performance in a 12-day task. the present study was designed to determine if age-related deficits could be detected with a shorter testing protocol and whether these deficits showed a relationship with nmda receptors. mice were trained in a reference memory task for two days in a morris water maze. cued testing was performed either after or prior to reference memory testing. crude synaptosomes were prepared from prefrontal/frontal cortex and hippocampus of the mice that underwent reference memory testing first. nmda receptor subunit and syntaxin proteins were analyzed with western blotting.  young mice showed significant improvement in probe and place learning when reference memory testing was done prior to cued testing. a significant decrease in performance was seen between and months of age with the two-day reference task, regardless of whether cued testing was performed before or after reference memory testing. there was a significant decline in the protein expression of the ε and ζ subunits of the nmda receptor and syntaxin in prefrontal/frontal cortex. the subunit changes showed a significant correlation with both place and probe trial performance.  the presence of an age-related decline in performance of the reference memory task regardless of when the cued trials were performed suggests that the deficits were due to factors that were unique to the spatial reference memory task. these results also suggest that declines in specific nmda receptor subunits in the synaptic pool of prefrontal/frontal brain regions contributed to these age-related problems with performing a spatial reference memory task. 