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Age-related declines in a two-day reference memory task are associated with changes in NMDA receptor subunits in mice.

Magnusson KR, Scruggs B, Zhao X, Hammersmark R - BMC Neurosci (2007)

Bottom Line: NMDA receptor subunit and syntaxin proteins were analyzed with Western blotting.A significant decrease in performance was seen between 3 and 26 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 epsilon2 and zeta1 subunits of the NMDA receptor and syntaxin in prefrontal/frontal cortex.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA. Kathy.Magnusson@oregonstate.edu

ABSTRACT

Background: C57BL/6 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.

Results: 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 3 and 26 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 epsilon2 and zeta1 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.

Conclusion: 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.

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Effects of age on protein expression of NMDA receptor subunits in crude synaptosomes. A: Representative bands from crude synaptosomes prepared from cortex and labelled with an antibody specific for the ε2 subunit. To the right are bands from different μg loads of caudal cortex used to obtain a standard curve. Five different μg amounts of caudal cortex were loaded as standards for each blot. To the left are representative bands from the prefrontal/frontal (P/F) cortex of a 3 (3 mo) and 26 (26 mo) month old mouse. B: Representative bands for all other proteins analyzed in crude synaptosomes from prefrontal/frontal cortex and hippocampus. Columns show labelling of different proteins from the same animal and well. Each well was loaded with 3 μg of crude synaptosomes. C, D: Graphs of protein expression of the ζ1, ε1, and ε2 subunits of the NMDA receptor and syntaxin in 3 and 26 month old mice, expressed as μg caudal cortex equivalents/μg protein loaded, in prefrontal/frontal cortex (C) and hippocampus (D). * p < .05 for difference from 3 month old mice (analysis of variance and Fisher's protected least significant difference post-hoc analysis). n = 8 for 3 month olds and n = 6 for 26 month old mice for prefrontal/frontal cortex. n = 6 for both ages for hippocampus. Error bars represent SEM.
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Figure 4: Effects of age on protein expression of NMDA receptor subunits in crude synaptosomes. A: Representative bands from crude synaptosomes prepared from cortex and labelled with an antibody specific for the ε2 subunit. To the right are bands from different μg loads of caudal cortex used to obtain a standard curve. Five different μg amounts of caudal cortex were loaded as standards for each blot. To the left are representative bands from the prefrontal/frontal (P/F) cortex of a 3 (3 mo) and 26 (26 mo) month old mouse. B: Representative bands for all other proteins analyzed in crude synaptosomes from prefrontal/frontal cortex and hippocampus. Columns show labelling of different proteins from the same animal and well. Each well was loaded with 3 μg of crude synaptosomes. C, D: Graphs of protein expression of the ζ1, ε1, and ε2 subunits of the NMDA receptor and syntaxin in 3 and 26 month old mice, expressed as μg caudal cortex equivalents/μg protein loaded, in prefrontal/frontal cortex (C) and hippocampus (D). * p < .05 for difference from 3 month old mice (analysis of variance and Fisher's protected least significant difference post-hoc analysis). n = 8 for 3 month olds and n = 6 for 26 month old mice for prefrontal/frontal cortex. n = 6 for both ages for hippocampus. Error bars represent SEM.

Mentions: 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 ζ1 and ε2 subunits of the NMDA receptor (p = .0014 and .004, respectively) in crude synaptosomes from the prefrontal/frontal cortex of 26-month-old mice as compared to young (Figure 4A–C). There was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ε1 (p = .10) subunit of the NMDA receptor in the prefrontal/frontal cortex (Figure 4B, C). Syntaxin also showed a significant decrease in expression between 3 and 26 months of age in the prefrontal/frontal cortex (p = .0221; Figure 4B, C). There was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ζ1 (p = .5441), ε1 (p = .0715), or ε2 (p = .2568) subunits of the NMDA receptor in the hippocampus (Figure 4B, D). Syntaxin showed a near-significant increase in expression between 3 and 26 months of age in the hippocampus (p = .0603; Figure 4D).


Age-related declines in a two-day reference memory task are associated with changes in NMDA receptor subunits in mice.

Magnusson KR, Scruggs B, Zhao X, Hammersmark R - BMC Neurosci (2007)

Effects of age on protein expression of NMDA receptor subunits in crude synaptosomes. A: Representative bands from crude synaptosomes prepared from cortex and labelled with an antibody specific for the ε2 subunit. To the right are bands from different μg loads of caudal cortex used to obtain a standard curve. Five different μg amounts of caudal cortex were loaded as standards for each blot. To the left are representative bands from the prefrontal/frontal (P/F) cortex of a 3 (3 mo) and 26 (26 mo) month old mouse. B: Representative bands for all other proteins analyzed in crude synaptosomes from prefrontal/frontal cortex and hippocampus. Columns show labelling of different proteins from the same animal and well. Each well was loaded with 3 μg of crude synaptosomes. C, D: Graphs of protein expression of the ζ1, ε1, and ε2 subunits of the NMDA receptor and syntaxin in 3 and 26 month old mice, expressed as μg caudal cortex equivalents/μg protein loaded, in prefrontal/frontal cortex (C) and hippocampus (D). * p < .05 for difference from 3 month old mice (analysis of variance and Fisher's protected least significant difference post-hoc analysis). n = 8 for 3 month olds and n = 6 for 26 month old mice for prefrontal/frontal cortex. n = 6 for both ages for hippocampus. Error bars represent SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Effects of age on protein expression of NMDA receptor subunits in crude synaptosomes. A: Representative bands from crude synaptosomes prepared from cortex and labelled with an antibody specific for the ε2 subunit. To the right are bands from different μg loads of caudal cortex used to obtain a standard curve. Five different μg amounts of caudal cortex were loaded as standards for each blot. To the left are representative bands from the prefrontal/frontal (P/F) cortex of a 3 (3 mo) and 26 (26 mo) month old mouse. B: Representative bands for all other proteins analyzed in crude synaptosomes from prefrontal/frontal cortex and hippocampus. Columns show labelling of different proteins from the same animal and well. Each well was loaded with 3 μg of crude synaptosomes. C, D: Graphs of protein expression of the ζ1, ε1, and ε2 subunits of the NMDA receptor and syntaxin in 3 and 26 month old mice, expressed as μg caudal cortex equivalents/μg protein loaded, in prefrontal/frontal cortex (C) and hippocampus (D). * p < .05 for difference from 3 month old mice (analysis of variance and Fisher's protected least significant difference post-hoc analysis). n = 8 for 3 month olds and n = 6 for 26 month old mice for prefrontal/frontal cortex. n = 6 for both ages for hippocampus. Error bars represent SEM.
Mentions: 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 ζ1 and ε2 subunits of the NMDA receptor (p = .0014 and .004, respectively) in crude synaptosomes from the prefrontal/frontal cortex of 26-month-old mice as compared to young (Figure 4A–C). There was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ε1 (p = .10) subunit of the NMDA receptor in the prefrontal/frontal cortex (Figure 4B, C). Syntaxin also showed a significant decrease in expression between 3 and 26 months of age in the prefrontal/frontal cortex (p = .0221; Figure 4B, C). There was no significant effect of age on caudal cortex equivalents per μg of protein loaded for the ζ1 (p = .5441), ε1 (p = .0715), or ε2 (p = .2568) subunits of the NMDA receptor in the hippocampus (Figure 4B, D). Syntaxin showed a near-significant increase in expression between 3 and 26 months of age in the hippocampus (p = .0603; Figure 4D).

Bottom Line: NMDA receptor subunit and syntaxin proteins were analyzed with Western blotting.A significant decrease in performance was seen between 3 and 26 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 epsilon2 and zeta1 subunits of the NMDA receptor and syntaxin in prefrontal/frontal cortex.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA. Kathy.Magnusson@oregonstate.edu

ABSTRACT

Background: C57BL/6 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.

Results: 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 3 and 26 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 epsilon2 and zeta1 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.

Conclusion: 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.

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