J. Kauer, R. Malenka, and R. Nicoll A Persistent Postsynaptic Modification Mediates Long-Term Potentiation in the Hippocampus J. Kauer, R. Malenka, and R. Nicoll Neuron vol 1, p 911-917 December 1988

Background Why is LTP important? Long Term Potentiation (LTP) - a long-lasting enhancement of synaptic transmission that can be induced by brief and repetitive stimulation of excitatory pathways in the hippocampus. Why is LTP important? LTP has been intensively studied as a model for information storage in the vertebrate brain and shares properties with associative learning.

Background (cont.) NMDA Receptors: Long lasting Glutamate released by the CA3 region of the hippocampus activates 2 subtypes of receptors: NMDA Receptors: Long lasting Sensitive to APV antagonist Non-NMDA Receptors: Shorter time course Sensitive to CNQX antagonist

The Big Question Where does the process causing increased synaptic transmission associated with LTP take place…Pre- or post-synaptically? At the time of this experiment, there were 3 suggested possibilities: Increased neurotransmitter release from presynaptic terminals A morphological change in pre- or post-synaptic structure A post-synaptic change in sensitivity to neurotransmitters

Methods In this series of experiments, the LTP is induced at the Schaffer collateral-commissural synapse between the CA3 and CA1 pyramidal cells. Study is done on the sliced hippocampus of a rat. Two methods used to induce LTP Tetanic Stimulation Pairing Protocol

Tetanus vs. Pairing Protocol A tetanus is a brief, high-frequency stimulation of pre-synaptic neuron, which, in addition to inducing LTP, causes a 1-2 min. “Post-tetanic potentiation” (PTP) resulting from increased glutamate release from pre-synaptic terminals. Pairing protocol refers to a low-frequency (0.1 Hz) stimulation of the pre-synaptic neuron along with artificial intracellular depolarization of the post-synaptic neuron. This provides just enough to induce LTP, but without such strong stimulation and neurotransmitter release associated with tetanic stimulation.

Pairing & Tetanus LTP induction by pairing EPSPs with Depolarization or by Tetanic Stimulation of the afferent pathway.

The Premise After LTP induction, an increase in neurotransmitter release by the pre-synaptic neuron would result in a simultaneous increase in both EPSP components. It is already known that the non-NMDA mediated current increases after LTP, but to prove that more glutamate is released, it would have to be shown that the NMDA mediated current increases after LTP as well.

Experiment 1 PROCEDURE: LTP induced using either Tetanic stimulation Pairing protocol Chemical antagonist CNQX was applied to block the non-NMDA receptors. The NMDA component of the EPSP was examined in isolation from the non-NMDA component before and after LTP using CNQX. Cells were monitored for 40 min. following LTP inducing stimuli.

Experiment 1 RESULTS: After addition of CNQX: Pairing protocol produced no lasting changes in the NMDA component of the EPSP. Tetanic stimulation induced PTP Caused a transient increase in NMDA component, but quickly returned to baseline

Experiment 1 INTERPRETATION: The NMDA component of the EPSP is not enhanced following LTP inducing stimuli. Since NMDA mediated response did not change after pairing and tetanus, an increase in glutamate release probably does not explain the changes in LTP.

Experiment 2 BACKGROUND AND PROCEDURE: Previous work has shown that activation of NMDA receptors is required to induce LTP. It is possible that non-NMDA are also required to induce LTP, and that CNQX simply prevents the induction of LTP. In the presence of CNQX: record EPSP from 2 independent stimulating inputs using 2 extracellular microelectrodes: One pathway was tetanized The other received low frequency stimulation To rule out excess neurotransmitter release from tetanus-induced PTP, recordings were repeated using the pairing protocol.

Experiment 2 RESULTS: As CNQX washed away – 30-60 minutes The tetanized pathway exhibited LTP The pathway which received low frequency stimulation returned to baseline. As CNQX washed away, the post-synaptic cell exhibited LTP.

Experiment 2B PROCEDURE : OBSERVATIONS: Intracellular and Extracellular recording electrodes recorded EPSPs before and after the pairing protocol in the presence of CNQX. OBSERVATIONS: -Intracellular EPSP became potentiated, while the extracellular EPSP was unaffected. -CNQX does not interfere with the induction of LTP.

Experiment 2 & 2B INTERPRETATION: This experiment demonstrates the following: CNQX does not interfere with the induction of LTP. LTP can be induced, even when the activity of non-NMDA receptors has been abolished.

Experiment 3 Goal: - Show that LTP results from a selective increase in the non-NMDA component of the EPSP. Procedure: Reversed EPSPs were recorded Isolate early and late components of the EPSP by using chemical antagonist APV that blocks the NMDA receptors. Compare EPSPs before and after the induction of LTP, and in the presence and absence of APV.

Experiment 3 RESULTS: INTERPRETATION: Large increase in the amplitude of the non-NMDA (early component), after a tetanus or pairing. Shows that the NMDA component (late component) is not altered after tetanus or pairing. INTERPRETATION: The early non-NMDA component is selectively increased following LTP inducing stimuli.

Summary Late component of EPSP enhanced only during post tetanic potentiation (PTP) After pairing and tetanus only the early component of the EPSP was increased. After a few minutes after tetanization the late component of the EPSP returned back to control level.

Conclusions What does this mean?? Results from these experiments are best explained by a modification of the postsynaptic neuron such that the non-NMDA component of the EPSP is selectively increased. LTP is not a result of an increase in glutamate release between the CA3 and CA1 regions.

Further thoughts It is possible that at single post-synaptic dendritic spines, pre-synaptic fiber stimulation, plus activation of NMDA receptors modifies neighboring non-NMDA receptors. The calcium dependent biochemical events are localized to the postsynaptic cell, and may either increase the number or modify the gating properties of the non-NMDA class of glutamate receptor/ion channels.

Critiques During Experiment 2: Adding APV and CNQX simultaneously would make it possible to conclude that induction of LTP was from NMDA activation.

J. Kauer, R. Malenka, and R. Nicoll™ Credits Made Possible By: Andy Chiu Stephanie Chow Aaron Cheung Kimberlyn Dang Cassie Coleman Alice Hue Chen Romerson Dimla Zackary Craddock Jeff Diamond Giang Dinh Isaac Cho Deepak Dhaliwal Michael Douzjian Michael Dearinger Susan Cheng Krijin Dijkstra Synthia Dabiri Produced by BIPN 148 Group 2™ Animation by Kim Dang, Inc. ™ Research by J. Kauer, R. Malenka, and R. Nicoll™  2007, Group 2 Productions ™