Morris Water Maze — S Davis | ReplicateScience

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Morris Water Maze — S Davis | ReplicateScience

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This series of experiments investigated whether the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) could induce impairments of spatial learning across a dose range comparable to its impairment of hippocampal long-term potentiation (LTP) in vivo. Estimations of the extracellular concentration of D-AP5 in hippocampus using microdialysis were also made to compare whether these impairments occur at concentrations similar to those required to impair LTP in the in vitro hippocampal slice. Rats were chronically infused with D-AP5 into the lateral ventricle at a range of concentrations (0–50 mM) via osmotic minipumps. They were first trained to find and escape onto a hidden platform in an open-field water maze task. After the behavioral learning, they were anesthetized with urethane and an attempt was made to evoke and monitor hippocampal LTP. Extracellular samples of D-AP5 in hippocampus were then taken using microdialysis, and finally, the animals were killed and tissue samples dissected. The microdialysis and tissue samples were analyzed for D-AP5 content using HPLC with fluorescence detection. The results established, first, that D-AP5 impairs spatial learning in a linear dose-dependent manner, highly correlated with its corresponding impairment of hippocampal LTP in vivo. No concentration of D-AP5 was observed to block LTP without affecting learning. Second, the microdialysis estimates indicated that, subject to certain assumptions, D-AP5 causes these impairments at extracellular concentrations comparable to those that impair LTP in vitro. Third, comparison of the whole tissue and microdialysis samples revealed a concentration ratio of approximately 30:1, indicating that 97% of the intracerebral D-AP5 is inaccessible to the dialysis probes. Infusion of 20 mM EGTA was found to cause a sevenfold increase in D-AP5 in the dialysis perfusates, suggesting that at least part of the inaccessible D-AP5 is trapped by a calcium-dependent mechanism. Two further behavioral control studies indicated that the D-AP5-induced impairment of spatial learning is unlikely to be secondary to a drug- induced motor disturbance, and that the performance of the D-AP5 group whose concentration was just sufficient to block hippocampal LTP completely was statistically indistinguishable from that of a group of rats with bilateral hippocampal lesions induced by ibotenic acid. Taken together, these findings offer support for the hypothesis that activation of NMDA receptors is necessary for certain kinds of learning.

Protocol Steps

1

Chronic D-AP5 infusion setup

Rats were chronically infused with D-AP5 into the lateral ventricle using osmotic minipumps at a range of concentrations from 0 to 50 mM

Not specifiedNot specified

Note: Multiple concentration groups were tested to establish dose-response relationship

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“Rats were chronically infused with D-AP5 into the lateral ventricle at a range of concentrations (0–50 mM) via osmotic minipumps”

2

Morris Water Maze training

Rats were trained to find and escape onto a hidden platform in an open-field water maze task to assess spatial learning

Not specifiedNot specified

Morris Water Maze

Note: This behavioral learning task was performed while D-AP5 was being infused

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“They were first trained to find and escape onto a hidden platform in an open-field water maze task”

3

Anesthesia and LTP recording preparation

After behavioral learning, rats were anesthetized with urethane in preparation for hippocampal LTP recording

Not specifiedNot specified

Note: Anesthesia was necessary for invasive LTP recording procedures

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“After the behavioral learning, they were anesthetized with urethane and an attempt was made to evoke and monitor hippocampal LTP”

4

Hippocampal LTP evocation and monitoring

Hippocampal LTP was evoked and monitored in anesthetized rats to assess the electrophysiological effects of D-AP5

Not specifiedNot specified

Note: LTP measurements were compared across different D-AP5 concentration groups

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“an attempt was made to evoke and monitor hippocampal LTP”

5

Microdialysis sampling

Extracellular samples of D-AP5 in hippocampus were collected using microdialysis to determine the concentration of drug accessible to neural tissue

Not specifiedNot specified

Note: Microdialysis was performed after LTP recording while animals were still anesthetized

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“Extracellular samples of D-AP5 in hippocampus were then taken using microdialysis”

6

Tissue sample collection

Animals were killed and tissue samples were dissected from hippocampus for analysis of D-AP5 content

Not specifiedNot specified

Note: Tissue samples were collected after microdialysis sampling

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“finally, the animals were killed and tissue samples dissected”

7

HPLC analysis of D-AP5 content

Microdialysis and tissue samples were analyzed for D-AP5 content using HPLC with fluorescence detection

Not specifiedNot specified

Note: This analysis allowed comparison of extracellular versus total tissue D-AP5 concentrations

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“The microdialysis and tissue samples were analyzed for D-AP5 content using HPLC with fluorescence detection”

8

EGTA infusion control experiment

In a separate experiment, 20 mM EGTA was infused to test whether calcium-dependent mechanisms sequester D-AP5

Not specifiedNot specified

Note: EGTA infusion resulted in a sevenfold increase in D-AP5 in dialysis perfusates, suggesting calcium-dependent sequestration

View evidence from paper

“Infusion of 20 mM EGTA was found to cause a sevenfold increase in D-AP5 in the dialysis perfusates”

9

Behavioral control studies

Two additional behavioral control studies were conducted to rule out motor disturbances and to compare D-AP5 effects with hippocampal lesion effects

Not specifiedNot specified

Note: One control group received bilateral hippocampal lesions induced by ibotenic acid for comparison

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“Two further behavioral control studies indicated that the D-AP5-induced impairment of spatial learning is unlikely to be secondary to a drug-induced motor disturbance”