Peer review of: Zeta Inhibitory Peptide attenuates learning and memory by inducing NO-mediated (2020)



Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors have addressed my concerns. only a few minor editing concerns remain: 
 
1) Line 77: change AMPAR to AMPARs (same on 82 –first occurance; 90; 107; 128; 228, 242) 
 
2) Sentence starting on line 107 is incomplete. 
 
3) Line 228: change ‘its’ to ‘their’ 
 
 
 
Reviewer #2: 
Remarks to the Author: 
I am highly impressed by the thorough and extensive revisions that these authors have taken with 
respect to both my and the other reviewers' comments. 
I am very satisfied with the answers and additions regarding my first comment but wanted to 
revisit my second since it appears to have been misunderstood. 
 
Previous work supporting the PKMzeta story with respect to ZIP had always evaluated data in a 
window of time at least 2hrs following infusions. Others have reported more rapid (and 
detrimental) effects less than or equivalent to this time window. My interest was not in time 
windows greater than this 2 hour period but less than or equal. Can the authors not comment 
using their data from either the cell culture or slice data? I note that the time frame for the 
electrophysiological studies goes up to 80 minutes but do they have data beyond? I realize that 
this may not be feasible due to the techniques and preparations used. 
 
As a perhaps related follow up, I note the section in the discussion as follows (p 13 lines 273-278): 
"Recently, it was shown that in anesthetized rats, ZIP profoundly reduced spontaneous 
hippocampal local field potentials, comparable in magnitude to infusions of lidocaine, but with a 
slower onset and longer duration (19). However, in the current study we found that ZIP had no 
effect on membrane potential during the first 50 min of bath application, whereas 
its inhibitory effect on AMPAR-EPSCs was apparent. This suggests that ZIP has no effect on 
membrane potential, at least not when it interferes with AMPAR activity." 
One thing that the authors should realize is that field potentials in vivo most likely represent 
synaptic potentials in dipolar arrangements of pyramidal neurons (see for example chapter 2 in 
Electroencephalography - Niedermeyer and Lopes da Silva). From the data presented here, it 
seems like the effects are highly consistent with this previous study in both the reduction of 
excitatory synaptic neurotransmission as well as the time frame. Perhaps the "However" is 
misplaced in their wording here? 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The authors addressed some of my questions but there remain significant concerns in their MS. 
1. Liberation of free arginines from ZIP is a critical factor for their hypothesis. To show this clearly, 
the authors need to measure the concentration of free arginines after ZIP treatment (i.e., 
measurement of free arginines in an extract from the cells incubated with ZIP using HPLC). It is 
known that the concentration of free arginines in cells is around 100 – 200 μM, indicating that a 
large amount of free arginines is already present in cells. To stimulate NOS as its substrate, ZIP 
should be proteolyzed very effectively within a short time (10 minutes). 
2. The authors need to show that S831 phosphorylation of GluA1 is required for AMPAR 

endocytosis in neurons (not in HEK cells). 
3. ZIP is known to act specifically on potentiated synapses but not non-potentiated, naïve 
synapses. Based on the ZIP effect on NAC synapses (usually larger than 50 % inhibition), ZIP is 
likely to have some effects on non-potentiated synapses. As shown in one figure in their revised 
MS, ZIP effects should be tested after LTP induction. In addition, the authors need to show that 
ZIP does not have any significant effects on naïve synapses as shown in previous studies.