Selective impairment of learning and blockade of

long-term potentiantion by an NMDA receptor

antagonist, AP5

Presentation by:David Alexanian

Ellen AlmirolKate Beider

Kunal Agrawal

R.G.M. Morris, E. Anderson, G.S. Lynch, & M. Baundry

Presentation Breakdown

PART 1: Introduction & BackgroundPART 2: Three parts to the experiment

1. Spatial learning2. Visual discrimination task3. In vivo Long-term potentiation (LTP)

recordings

PART 3: Conclusions, Critics and Improvements

PART 1

Background & Introduction

Long Term PotentiationSTRONG 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+

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

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?

1. SPATIAL LEARNING

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)

Morris Water Maze

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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)

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

Results: Acquisition TrainingM

inip

um

p im

pla

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tio

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Lat

ency

(s)

Acquisition Further training Reversal

Tra

ns

fer

tes

t

= Control

= D,L-AP5

= L-AP5

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

Results: Transfer TestM

inip

um

p im

pla

nta

tio

n

Lat

ency

(s)

Acquisition Further training Reversal

Tra

nsf

er t

est

= Control

= D,L-AP5

= L-AP5

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

Reversal ResultsM

inip

um

p im

pla

nta

tio

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Lat

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(s)

Acquisition Further training Reversal

Tra

nsf

er t

est

= Control

= D,L-AP5

= L-AP5

Overall Results: Comparison

Min

ipu

mp

imp

lan

tati

on

Lat

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(s)

Acquisition Further training Reversal

Tra

nsf

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est

= Control

= D,L-AP5

= L-AP5

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

2. VISUAL DISCRIMINATION

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 quadrantOpp – time in opposite quadrantAdj/L, Adj/R – time spent in two adjacent

quadrants

Visual Discriminations Test:

Results Control (saline) D,L-AP5 L-AP5

Tim

e p

er q

uad

ran

t (s

)

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

Critical Issue:

How do you know that you haven’t damaged other areas

of the brain that might be hindering performance?

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.

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.

3. In vivo RECORDINGS of LTP

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

In vivo LTP recordings: Results

% o

f b

asel

ine

slo

pe

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.

PART 3

Conclusion, Critiques, & Improvements

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-AP5 Escaped 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

Critiques1) 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.

Questions?