1. Selective impairment of learning and blockade of long-term potentiantion by an NMDA receptor antagonist, AP5 Presentation by: David Alexanian Ellen Almirol Kate Beider Kunal Agrawal R.G.M. Morris, E. Anderson, G.S. Lynch, & M. Baundry

2. Presentation Breakdown PART 1: Introduction & Background PART 2: Three parts to the experiment 1. Spatial learning 2. Visual discrimination task 3. In vivo Long-term potentiation (LTP) recordings PART 3: Conclusions, Critics and Improvements

3. PART 1 Background & Introduction

4. Long Term Potentiation STRONG stimulation High [Glu] LTP results in:  Higher concentration of AMPA- and NMDA-Rs on the postsyn. membrane.  Higher conductance of the postsyn. AMPA-Rs.  Retrograde signals that enhance Glutamate release are sent to the presyn. neuron.  Ultimately, the postsyn. neuron is more sensitive to presyn. stimulation. NMDA-Rs are both ligand-gated and voltage-dependant! So, they are only activated when postsyn. neuron is depolarized above a certain threshold! NMDA-Rs are ESSENTIAL to LTP! Mg2+

5. PLACE CELLS in the Hippocampus  Place cells within the hippocampus  Neurons that fire action potentials only when an animal is in certain locations  These place cells encode spatial memories  If damaged, it would be difficult for animals to learn in spatial learning tasks

6. Experimental Question What is the effect of the AP5 drug on NMDA receptors and what role do these receptors play in spatial and visual discrimination learning in the hippocampus?

7. 1. SPATIAL LEARNING

8. Experiment Background Morris Water Maze –Used for spatial learning tasks –A circular tank filled with opaque water –Opaque water conceals a platform that the rats are trying to find –Rats can also use extra-maze cues (lamps, desks, etc)

9. Morris Water Maze ?

10. Experimental Methods: Setup Male Lister rats Implanted with minipumps connected to a cannula that was lowered in the right lateral ventricle –Group 1: D, L-AP5 (40mM in 0.9% saline) –Group 2: L-AP5 (20mM) –Group 3: Control (Saline only) –Group 4: Unoperated (No minipumps)

11. Experimental Methods: Training Platform was either in SW or NE quadrant Total of 15 trials given (3 trials/day at 4 hr intervals between trials) for 5 days Measure the time it took for the rats to find the platform Given maximum 120 seconds to find platform, and 30 seconds given to stay on platform

12. Results: Acquisition Training Minipump implantation Latency (s) AcquisitionFurther trainingReversal Transfer test = Control = D,L-AP5 = L-AP5

13. Experimental Methods: Retention  Transfer test: to determine how much had been learned of the platform location  Platform was removed from maze  Rats given 60 seconds to swim freely with no escape opportunity  Measured time it took for rats to be in the same quadrant that the platform was in  Further training  Eight trials given in rapid succession (30 sec. between trials) with platform back in original location

14. Results: Transfer Test Minipump implantation Latency (s) AcquisitionFurther trainingReversal Transfer test = Control = D,L-AP5 = L-AP5

15. Experimental Methods: Reversal Training  Reversal Trials  Rats trained to learn a new platform location  Platform located in the opposite quadrant  Only one trial per day for four days

16. Reversal Results Minipump implantation Latency (s) AcquisitionFurther trainingReversal Transfer test = Control = D,L-AP5 = L-AP5

17. Overall Results: Comparison Minipump implantation Latency (s) AcquisitionFurther trainingReversal Transfer test = Control = D,L-AP5 = L-AP5

18. Experimental Results: Summary Initial acquisition training: –Control and L-AP5 groups escaped rapidly within 15 trials –D, L-AP5 learned more slowly, but not significantly Memory Retention (Transfer Test): –Control and L-AP5 groups escaped with minimum latencies –D, L-AP5 took longer, but stabilized their latency times Reversal Training: –Control and L-AP5 groups showed a strong decline in latency times across the 4 trials –D, L-AP5 group failed to learn the new platform location

19. 2. VISUAL DISCRIMINATION

20. Visual Discrimination Tests: Why & How? This experiment follows upon the transfer tests done in day 9. The goal of this test is to track the motion of the rats and see if there was a preference for the trained quadrant when there was no platform present. For each animal a computerized tracking system calculated the time spent in the four main quadrants of the pool during 60s transfer tests. This data was organized into: Tr- time in trained quadrant Opp – time in opposite quadrant Adj/L, Adj/R – time spent in two adjacent quadrants

21. Visual Discriminations Test: Results Control (saline) D,L-AP5 L-AP5 Time per quadrant (s)

22. Visual Discrimination: Summary Further analysis of the paths taken during the transfer test showed even more convincingly that D,L-AP5 rats had not learned the location of the platform and were wandering around aimlessly. L-AP5 also showed a somewhat effected movement pattern as compared to the control. The bar graphs quantify the preference for each quadrant. As can be seen, the D,L-AP5 rats had almost no preference for one quadrant or another. D,L-AP5 Control (saline) L-AP5

23. Critical Issue: How do you know that you haven’t damaged other areas of the brain that might be hindering performance?

24. Sensory-motor/motivational damage test  Experiment focused on visual cues to see if secondary sensory systems were damaged.  Used Morris Maze to keep motivation the same.  Two colored and clearly visible platforms were used.  One was rigid and could support the rats weight, the other would sink when the rat attempted to get on.  A black curtain was drawn around the maze to prevent any extra-maze cues.  The location of the platforms was also rotated during training.  The task was to approach the rigid platform irrespective of its location.

25. Test Results No significant difference in learning curve for all of the groups. Thus showed that there was no significant performance detriment from secondary brain damage.

26. 3. In vivo RECORDINGS of LTP

27. In vivo LTP recordings This final test was to determine the effect of AP5 on induction of LTP in vivo. A bipolar stimulating electrode electrode was placed into the perforant path A recording electrode was then placed in the dentate gyrus. After a stable baseline voltage was recorded, low frequency bursts were given for 100 min. At 20min and 40 min a high frequency tetanus was applied and the voltages were recorded

28. In vivo LTP recordings: Results % of baseline slope Time (min) L-AP5D,L-AP5Control D,L-AP5 had no difference in the low frequency stimulation, but showed no LTP excitation during 20/40 min tetanus.

29. PART 3 Conclusion, Critiques, & Improvements

30. Part I: Morris Maze Results Purpose: To prove that blocking hippocampus NMDA receptors with an NMDA antagonist impairs place learning in rats. Therefore, this data strongly implies that NMDA-Rs are involved in LTP and place learning. Initial Acquisition Training Memory Retention (Transfer Test) Reversal Training Control and L- AP5 Escaped rapidly within 15 trials Escaped with minimum latencies Showed a strong decline in latency trails across 4 trials D, L-AP5Escaped relatively slow compared to the control and L-AP5 Escaped with longer latencies but stabilized their latency times Failed to learn the new platform location

31. Critiques 1) Small sample size and unequal distribution Solution: more quantity of rats, even distribution among trials, age/sex of rats 2) Function of L-AP5 unknown Solution: use more focused tests to separate the differences between D,L-AP5 and L-AP5 3)Modern techniques to see if there is an actual structural change of synapse at molecular level. 4)Could AP5 be having a downstream effect that is difficult to distinguish with the tests used? 5)During secondary damage test, rats still had to learn and remember that a specific pattern meant the rigid platform.

32. Questions?