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

Outline I.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

Introduction This 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

Methods Slice Preparations Male Wistar rats 3-4 weeks old Hippocampi were isolated and cut into 400 μm transverse slices. hello

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.

Methods Induction 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.

Methods Induction 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.

Methods Protein 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 Hz (roughly 1/min) Results: –E-LTP = unaffected. –L-LTP = affected

Experiment 1 Figure 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 1 Figure 1c and 1d

Experiment 1 Conclusions 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 2 Figure 2a and 2b hello

Experiment 2 Figure 1a vs Figure 2a Figure 1a = Hz Figure 2a = 0.1 Hz

Experiment 2 Figure 2c and 2d

Experiment 2 Conclusions 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 2 nd expt: anisomycin applied 100 minutes with 20 minutes of test pulses in the middle of application 3 rd expt: repeat 2 nd 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 3 Figure 3a and 3b Inhibitor applied with no test pulse stimulation –No decrease in L-LTP

Experiment 3 Figure 3c and 3d Inhibitor applied with test pulse stimulation –L-LTP decreases

Experiment 3 Figure 3d and 3f Inhibitor applied with AP-5 and test pulse stimulation –L-LTP is saved, no decrease

Experiment 3 Conclusions 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