Activation of postsynaptically silent synapses during pairing-induced

1. Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice Dezhi Liao, Neal A. Hessier & Roberto Malinow

2. Abstract • The authors of this paper propose two main points. These are: • The existence of silent synapses in hippocampal area CA1 • The effective desilencing of a synapse occurs in response to a LTP inducing protocol which is thought to be mediated by AMPA insertion into the postsynaptic cell.

3. Background info • AMPA and NMDA receptors are found in synapses of the hippocampus • Both are necessary components for LTP • In order for the NMDA receptor to be activated two conditions must be met: • Presynaptic release of glutamate to its receptor site • Postynaptic depolarization.

4. Background info • The glutamate binding activates the receptor. • Calcium (Ca2+) cannot pass through the ion channel unless depolarization occurs, which will expel the magnesium (Mg2+) ion blocking the channel. • Depolarization occurs when the AMPA receptor has been activated by glutamate binding and Na+ influx depolarizes the membrane sufficiently. • Ca2+ influx signals the induction of LTP through activation of 2nd messenger systems.

5. Background info AMPA receptor NMDA receptor

6. Background info • LTP (long term potentiation) is an enhancement of synaptic strength that can be produced by pairing presynaptic activity with postsynaptic depolarization. (taken verbatim from Lomo) • Silent Synapse • A silent synapse is one whose postsynaptic membrane contains NMDA receptors but no AMPA receptors. Normal AMPA receptor-mediated depolarization does not occur, rendering the synapse "silent" or unresponsive to stimulation at normal resting potentials.

7. Background info Electrophysiology -The whole cell patch clamp technique was used to manipulate the postsynaptic cell potential. In addition, the composition of solutes present to the cell membrane was manipulated. --- -Stimulation was applied by injecting current into an afferent pathway.

8. Methods 1st experiment • Hippocampal slices from the CA1 subiculum, were taken from adult rats (10-18 days old) •  Cells were clamped at approximately -60 mV • Cells were dialyzed with a Ca2+ chelator (10mM EGTA or 10mM BAPTA) to prevent calcium dependent plasticity • The stimulus was adjusted to yield a 50% trial failure rate in transmission to find an adequate (weak) stimulation level

9. Methods • These trials were taken within two holding potential categories:       -  Negative holding potential category (-55 mV to -65 mV).       -  Positive holding potential category (40 mV to 60 mV). • These trials yielded a relatively higher failure rate at negative potentials than positive • When depolarized, cells are more likely to exhibit an EPSC

10. Methods 2nd Experiment • 100 micro-M D,L-APV was added to the solution bath, under a negative holding potential. • 100 micro-M D,L-APV was added to the solution bath, under a positive holding potential. • With the addition of APV (NMDA receptor antagonist), an equivalent failure rate at both positive and negative holding potentials was observed

11. Methods 3rd Experiment • Synapses only containing NMDA receptors were isolated in whole-cell recordings. • Cells were held at -65 mV • A stimulus was provided through an afferent pathway. • The level of stimulus was decreased by 0.05 V until a minimal intensity produced 100 consecutive failures. • Depolarizing the cells to 55 mV resulted in the presence of NMDA mediated EPSCs upon stimulation

12. Methods More fun experiments…. • 39 experiments were conducted under the following guidelines and were split into one of two possible experimental method groups: • One option involved depolarizing the postsynaptic membrane to -10 mV with subminimal stimulus for 100 trials, followed by returning the cell to -65 mV. • The second experimental method left the postsynaptic membrane potential at -65 mV, while the subminimal stimulus continued. • Out of the 39 experiments, 22 utilized a pairing protocol (option1) and 17 were completed without a pairing protocol (option 2).

13. Results Hyperpolarized vs Depolarized • EPSC amplitudes at positive and negative potentials. Notice the increased frequency of zero amplitude events at negative potentials • When exposed to a weak stimulus, neurons held at a negative potential had a higher failure rate percentage than those held at a positive potential (p<0.002)

14. Results Antagonism of NMDA Receptors by APV • When APV was added, there was no significant difference between failure rates in the hyperpolarized and depolarized cells. • APV antagonizes NMDA, which causes depolarized cells to respond like hyperpolarized cells, in which NMDA normally has poor or no function.

15. Results Pairing produces AMPA responses at previously silent synapses • After the pairing protcol, the failure rate of postsynaptic activation decreased drastically.

16. Results Whole lotta failin’ going on!!! (don’t like the sound of that) • Compared to cells that did not undergo the pairing procedure, those that were subjected to it had a significantly lower failure rate when hyperpolarized (p<0.02).

17. Results LTP is expressed by AMPA • Decreased failure rates after pairing are due to changes in the AMPA component (increase 56%± 9%) of transmission, not the NMDA component (increase 1%± 1%) of transmission. After LTP induction, the responses at negative holding potentials increased significantly in size, while there was no increase at the positive holding potential

18. Results LTP increases EPSC strength in hyperpolarized neurons But no increase?? • There was a significant increase (p<0.01) in the AMPA component of transmission. } increase due to AMPA

19. Discussion Weakness? -The model proposed in this paper predicts that following LTP decreased failure rates and changes in synaptic response strength at negative potentials are due to the incorporation of AMPA receptors at the postsynaptic site. -A logical prediction from this is that the size of responses at positive potentials should increase (as well as at negative potentials) after LTP induction because these are actually a mix of AMPA and NMDA mediated current. -HOWEVER, there is not evidence for this in the results of this paper!

20. Discussion -They observed little enhancement of the early component at depolarized potentials -Review: In EPSC kinetics the AMPA receptor accounts for the early component and the NMDA receptor for the slow component. So again, why would they not see enhancement of AMPA mediated current at depolarized potentials? Why didn’t response sizes increase after LTP at depolarized potentials?

21. The authors explain the problem with 2 potential hypotheses: • The AMPA receptors inserted are very inwardly rectifying and therefore would not contribute substantially to the depolarized response. BUT…If this is the case, the paper would have been improved by adding an IV curves examining the isolated AMPA responses to demonstrate such a rectification. • It might be due to the fact that the NMDA currents dominate the response HOWEVER, it is fairly well accepted that mature central synapses are dominated by AMPA mediated responses.

22. Conclusions -Silent Synapses?? Strong evidence is provided that synapses exist in which only NMDA responses can be detected, yet this is dependent upon the stimulation methods used here.Thus, one must be careful in declaring the presence of silent synapses in the mature CA1 region of the hippocampus under normal physiological conditions. -Activation of these silent synapses following LTP induction due to AMPA addition? Following induction of LTP using a pairing protocol, postsynaptic responses were seen at hyperpolarized potentials in synapses that had previously been silent at these potentials. Again there is the caveat about physiological relevance.

23. Conclusions -Evidence that LTP induction and expression are postsynaptic? -These experiments are important because provide evidence for a potential mechanism underlying LTP that is independent (although not necessarily mutually exclusive) of presynaptic changes.

24. Future considerations To name a few…. -Are their results replicable in other brain areas? -They did this work in adult brains, how might this apply to development? -What are other methods that could tell us about silent synapses? (immunostaining?) -What are the (calcium specific) mechanisms underlying postsynaptic modification? -How is AMPA added to the synapse?

25. References http://www.ux1.eiu.edu/~cfbpn/ZOO_4950/Chapter_23.htm http://www.bbsonline.org/Preprints/OldArchive/bbs.shors.html http://en.wikipedia.org/wiki/Silent_synapse http://www.abdn.ac.uk/sms/ugradteaching/PY4302/PY4302_09122004_22.htm http://www.gsu.edu/~biodhe/LTP%20Notes.doc http://www.bris.ac.uk/Depts/Synaptic/info/glutamate.html Mechanisms of memory, J. David Sweatt Activation of postsynaptically silent synapses during paring-induced LTP in CA1 region of hippocampal silce: Dezhi Liao, neal A. Hessier & Roberto Mallnow

26. Presenters: Karen Randel Issa Rammal Adam Qwan Colleen Roy Payton Quintel Natalie Shanks Erica Smith Babak Shirdel