Neuronal activity determines the protein

synthesis dependence of long-term potentiation Fonseca R, Nagerl UV, Bonhoeffer T.

Group 8 : Seaton Tai, Kristie Tanaka, Hanz Tao, Cindy Tsau, Vincent Tse, Christine Tran, Victor Tung, Christian Villarosa, Courtney Warren, Jared Whittier, Tim Wong, Abel Wu

OutlineI. Introduction

a) Definitions

b) Background and Findings

II. Experiment 1: Protein Synthesis Blockade on L-LTP at 0.017Hz

III. Experiment 2: Protein Synthesis Blockade on L-LTP at 0.100Hz

IV. Experiment 3: Protein Synthesis, Test Pulse Stimulations, and NMDA Receptors

V. Conclusions

VI. Critiques

VII.Questions

IntroductionThis paper looks at how protein synthesis inhibition affects long term potentiation, both early and late stage.

Definitions:Long term potentiation (LTP) is divided into two phases:

• E-LTP = Early Phase Long Term Potentiation. - Increased synaptic sensitivity that occurs up to one hour

following LTP induction.

• L-LTP = Late Phase Long Term Potentiation. - Potentiation that occurs one hour and beyond.

Background and Findings• It is generally accepted that only L-LTP was

dependent on protein synthesis. – This experiment finds that E-LTP may also be dependent on protein synthesis

• It is also generally accepted that L-LTP maintenance is dependent on translation in the early induction phase.– This experiment shows that L-LTP maintenance depends on synaptic stimulation

• The TAKE HOME POINTS:– Neuronal Activity is crucial in determining the role of protein synthesis in E and L-LTP– Protein synthesis occurs at the dendrites

MethodsSlice Preparations

• Male Wistar rats• 3-4 weeks old• Hippocampi were

isolated and cut into 400 μm transverse slices.

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Methods Electrophysiological Recordings

• Schaffer collaterals were stimulated by pulses lasting 0.2 ms (unless otherwise noted) at varying frequencies. – Test Pulse stimulation

• Field excitatory postsynaptic potentials (fEPSP) were recorded extracellulary in the stratum radiatum of the CA1 region.

MethodsInduction of LTP

• Two stimulating

electrodes were used,

positioned in the

stratum radiatum.

• This allowed for the activation of two sets of Schaffer collaterals which were independent of each other.

MethodsInduction of LTP

• Experimental pathway– Received a tetanus at a frequency of 100 Hz for 1

second

• Control pathway. – Test pulse frequency set at 0.1 Hz.

MethodsProtein Synthesis Inhibitor

• 25 μM of Anisomycin in 0.01% DMSO

• Anisomycin inhibits protein synthesis– Blocks translation of mRNA

Experiment 1 Protein Synthesis Blockade on L-LTP

• Tests the effect of protein synthesis blockade on L-LTP

• Anisomycin was bath applied for 100 minutes. At 40 minutes, LTP was induced by tetanic stimulation.– Test pulse frequency was 0.017 Hz (roughly 1/min)

• Results:– E-LTP = unaffected.

– L-LTP = affected

Experiment 1Figure 1a and 1b

Experiment 1c and 1d

• The other part of experiment 1 in this paper shows that when protein synthesis inhibitors are added after LTP induction, there is no change to E-LTP or L-LTP.

Experiment 1Figure 1c and 1d

Experiment 1Conclusions

• Anisomycin must be present during the induction of LTP in order to affect L-LTP.

• L-LTP is crucially dependent on protein synthesis during early induction of LTP

• E-LTP is seemingly unaffected (0.017 Hz).

Experiment 2

• Experiment 1 was repeated using different test pulse frequencies.– 0.017 Hz vs 0.1 Hz

• Test to see if LTP maintenance and protein synthesis increases with increased levels of synaptic activity

Experiment 2Figure 2a and 2b

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Experiment 2Figure 1a vs Figure 2a

Figure 1a = 0.017 Hz Figure 2a = 0.1 Hz

Experiment 2Figure 2c and 2d

Experiment 2Conclusions

• At higher frequencies, E-LTP is affected.

• The stimulation frequency during inhibition does not effect the final amount of LTP reduction

• Higher levels of synaptic activity require more protein synthesis for LTP maintenance

Experiment 3

• To change the protocol of previous experiments and observe LTP decay– Anisomycin applied for 100 mins after tetanus

• 1 expt: anisomycin applied during period of no test pulses

• 2nd expt: anisomycin applied 100 minutes with 20 minutes of test pulses in the middle of application

• 3rd expt: repeat 2nd expt with concurrent AP5 (NMDA antagonist) treatment and removal

• Help prove hypothesis that elevated synaptic activation may decrease availability of proteins important for LTP

Experiment 3Figure 3a and 3b

• Inhibitor applied with no test pulse stimulation– No decrease in L-LTP

Experiment 3Figure 3c and 3d

• Inhibitor applied with test pulse stimulation– L-LTP decreases

Experiment 3Figure 3d and 3f

• Inhibitor applied with AP-5 and test pulse stimulation– L-LTP is saved, no decrease

Experiment 3Conclusions

• Test pulse stimulation must occur during protein synthesis inhibitor application in order to have any effect on L-LTP.– LTP decays as a supply of synthesized proteins for

maintenance is inhibited

• If AP-5 is applied with the inhibitor (with test pulse stimulation), LTP decrease is prevented.– Implicates translation is happening at the potentiated

synapses and dendritic area– Implicates Ca2+ as a modulator of translational activity

Paper Conclusions• Experiment 1

– L-LTP is crucially dependent on protein synthesis during early induction of LTP

• Experiment 2– Increase in synaptic activity (test pulse frequency) reveals

that E-LTP may also require protein synthesis.• Higher frequency leads to accelerated decay of LTP

• Experiment 3– Stimulation of potentiated synapses recruits protein

synthesis for LTP maintenance

– Protein synthesis is modulated by NMDA R/Ca2+ activity

– Protein synthesis very likely occurs at dendritic area and the potentiated synapse

Critiques• 30% of the data was rejected and not included

in the paper– Fix: Include the data in the write up

• Reasoning behind why the experiments worked was not discussed– No explanation as to why a change in frequency

affects LTP– No explanation about the interaction between

protein synthesis and AP-5