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On the problematic use of intracranial ZIP
Submit responseWe appreciate the attention Drs. Fenton and Sacktor have given to our work and their concern at reconciling their previous results using Zeta inhibitory peptide (ZIP) with ours. We need to highlight that the main point of our paper was to specifically demonstrate a troubling confound of the use of intracranial ZIP, namely, a severe disruption of neural activity, which we believe has been clearly documented in our work (LeBlancq et al., 2016). From our standpoint, the demonstration that this agent can suppress spontaneous network interactions and also generate seizure activity is troubling for prior interpretations that suggested a more specific and benign action on PKMzeta.
More generally, we would like to take this opportunity to caution against the common approach taken in neuroscience which involves jumping directly from the molecular/biochemical level of investigation to the whole behaving animal without carefully taking into consideration the intervening levels of cellular, network, and systems neuroscience. This is a not a focused criticism of the PKMzeta/ZIP story per se, but rather a more widespread criticism of the entire field.
Below we address the specific points made by Drs. Fenton and Sacktor in their comment:
1) Pastalkova et al., (2006) reported a lack of effect of ZIP on baseline evoked field potentials, specifically fEPSP slope. However, a rapid and nearly complete (i.e. lower than baseline) depression of population spikes was reported and shown in Figure 1E. Moreover, Volk et al., (2013) reported a ZIP-induced decrease in baseline evoked potential amplitude in the hippocampal slice using far less ZIP than that used in any in vivo study. In our present study, we did not test the effects of ZIP on evoked potentials so we cannot comment further. However, based on our previous work that studied the effects of intrahippocampal infusions of the translational inhibitor anisomycin (another amnestic substance that was strongly suggested to have little to no influence on neural activity - i.e., Davis and Squire, 1984), we observed a similar catastrophic loss of both spontaneous AND evoked potentials (Sharma et al, 2012). We consider it likely that we would have observed a parallel finding using ZIP in our current study had we examined evoked responses as well.
2) Although Barry et al. (2012) reported no changes in single unit hippocampal electrophysiology (other than spatial remapping) post-ZIP, we note that no data were explicitly shown for the period of time reported in our present study, i.e., within a 2 hour time window post infusion. We agree that an explicit demonstration of a behavioural effect of ZIP in the complete absence of any detrimental electrophysiological effect (most importantly including changes in local field ensemble dynamics), would go a long way in demonstrating a more specific effect of ZIP. However, in the absence of this, and in the face of our present demonstration, we still feel that our results point out a serious confound of ZIP.
3) Although it might seem difficult to reconcile the effects we report as an explanation for some prior behavioural results, notably the demonstration of ZIP-induced amnesia at longer latencies from initial training (typically one day or more), we note that very few studies of ZIP have gone beyond the one or two day limit (Kwapis and Helmstetter, 2014), which is well within the window of retrograde amnesia reported for the majority of hippocampal lesion studies (Sutherland et al, 2010). Given the recent demonstrations of the excitotoxic nature of ZIP (Hanif et al., 2012; Sadeh et al. 2015 - both studies that we unfortunately neglected to cite in our original article) we feel that the confounding problems of intracranial ZIP are compounded. Although we reported no lesions within the time frame of our experiment, we did not use specific markers of cell death to evaluate this possibility. Interestingly, and in confirmation of our own work, Sadeh et al. (2015) showed that ZIP can also induce epileptiform activity. Although the detrimental influence of this aberrant activity is disputed in their comment, we would trust that Drs. Fenton and Sacktor would agree that it still constitutes a serious confound for ZIP, since it is well known that amnestic effects can be promoted by both spontaneous and induced epileptic events. Lastly, we know of no study that explicitly pits the effects of alterations or suppression of neural activity against a more specific biochemical effect of ZIP as the primary explanation of amnesia induced by this agent. In the absence of this evidence, our findings of profound suppressive and seizure-inducing effects of ZIP are still troubling issues in the interpretation of the prior behavioural effects of ZIP. We agree with Drs. Fenton and Sacktor that this still requires definitive resolution.
4) The assertion that ZIP does not affect short-term memory does not really apply to the findings of Pastalkova et al, (2006) in which retraining was conducted outside of the 2-hour window of time reported in our study. Furthermore, this same problem applies to Shema et al., (2007), in which training was likewise conducted 2 hours post infusion. Shema et al, (2009), however, did show that ZIP infused into the insular cortex 20 minutes prior to training did not prevent a conditioned taste aversion (CTA) from being acquired as well as demonstrating that a similar ZIP infusion 1 hour following CTA training did not prevent consolidation of this memory. As are Drs. Fenton and Sacktor, we are puzzled by these results, given our clear and profound documentation of neural suppression using ZIP. Again, this is an interesting question requiring further exploration.
5) Drs. Fenton and Sacktor reflect that the (buffered) vehicle we used might be a cause of the detrimental effects on activity we report. Although we certainly agree that unbuffered acid would have (rapid) detrimental effects on neurophysiological measures (similar to those we reported for the sodium channel blocker lidocaine which were more rapid than those we found for ZIP), had this indeed been the case, we would have expected similar results for the influences of scrambled ZIP in addition to the presence of noticeable histological lesions for both ZIP and scrambled ZIP; neither of which were observed. We believe that our interpretation of a detrimental effect of ZIP on activity is a more probable and substantially less convoluted idea than the notion that PKMzeta might have a "neuroprotective effect on the potentially toxic action of intracranial injections of acetic acid".
We and many other groups are compelled by the prior behavioural results shown with ZIP. However, the demonstration of continued amnestic effects of ZIP in PKMzeta knock outs (Volk et al., 2013; Lee et al., 2013), multiple reports of the neurotoxicity of ZIP (Hanif et al., 2012, Sadeh et al., 2015), in addition to our demonstration of detrimental influences on network activity (LeBlancq et al., 2016) shake the clear interpretation of these prior data.
In conclusion, we believe that the ramifications of the suppressive and disruptive influences of ZIP as demonstrated here (LeBlancq et al., 2016) and elsewhere (Hanif et al., 2012, Sadeh et al., 2015) for prior behavioural results remains to be further explored and clarified.
References
Barry JM, Rivard B, Fox SE, Fenton AA, Sacktor TC, Muller RU (2012) Inhibition of protein kinase Mzeta disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. J Neurosci 32:13753- 13762
Davis HP, Squire LR (1984) Protein synthesis and memory: a review. Psychol Bull 96:518 -559.
Hanif W, Wang M, Sutherland RJ (2012) zeta-Pseudosubstrate Inhibitory Peptide (ZIP) is neurotoxic when infused into the hippocampus in anesthetized rat. Society for Neuroscience Conference Abstract 44.03. Society for Neuroscience.
Kwapis JL, Helmstetter FJ (2014) Does PKM(zeta) maintain memory? Brain Res Bull 105:36-45.
LeBlancq MJ, McKinney TL, and Dickson CT (2016) ZIP It: Neural Silencing Is an Additional Effect of the PKM-Zeta Inhibitor Zeta- Inhibitory Peptide J Neurosci 36:6193-6198.
Pastalkova E, Serrano P, Pinkhasova D, Wallace E, Fenton AA, Sacktor TC (2006) Storage of spatial information by the maintenance mechanism of LTP. Science 313:1141-1144.
Sadeh N, Verbitsky S, Dudai Y, and Segal M (2015) Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase M?, Is Excitotoxic to Cultured Hippocampal Neurons. J Neurosci, 35: 12404-12411.
Sharma AV, Nargang FE, Dickson CT (2012) Neurosilence: Profound Suppression of Neural Activity following Intracerebral Administration of the Protein Synthesis Inhibitor Anisomycin The Journal of Neuroscience 32:2377-2387.
Shema, R., Sacktor, T. C., Dudai, Y. (2007). Rapid Erasure of Long- Term Memory Associations in Cortex by an Inhibitor of PKMzeta, Science, 317:951-953.
Shema R., Hazvi S., Sacktor T. C., Dudai Y. (2009). Boundary conditions for the maintenance of memory by PKMzeta in neocortex. Learn Mem: 16:122-128.
Sutherland RJ, Sparks FT, Lehmann H (2010) Hippocampus and retrograde amnesia in the rat model: a modest proposal for the situation of systems consolidation. Neuropsychologia 48:2357-2369.
Conflict of Interest:
None declared
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On ZIP, neuronal silencing, and the differences between the results of LeBlancq et al. and other reports examining effects of ZIP on memory
Submit responseIt is difficult to reconcile the data reported in LeBlancq et al. with a large number of published observations.
1) In the urethane-anesthetized rat, Pastalkova et al. (2006) observed that ZIP has no effect on baseline population postsynaptic responses (Fig. 1D, and supplemental Fig. S2). After inducing LTP, ZIP depotentiated postsynaptic responses, which returned to baseline values and stabilized. This was measured by both the field excitatory postsynaptic potential (fEPSP; Fig. 1C) and population (action potential) spike (PS; supplemental Fig. S1). After infusing ZIP, stable fEPSPs and PS responses depotentiated to baseline could not have been observed if ZIP had caused neuronal silencing or seizures.
2) In freely-behaving rats, Barry et al. (2012) observed that ZIP had no effects on action potential waveforms and local field potentials 2 h afterwards. Unpublished recordings were made 10-20 min after ZIP infusion, and we observed no evidence of seizure or silencing due to ZIP.
3) The LeBlancq et al. authors assert that seizure and/or temporary neuronal silencing by ZIP is sufficient to explain the amnesia for various forms of long-term memory that have been observed following ZIP infusion. But this assertion is contrary to a large body of evidence. In experiments to test its effect on established long-term memory, the peptide is infused at least 1 day after training. Seizures, the neuronal silencing induced by short-lasting lidocaine (~30 min), the longer-lasting AMPA/kainate receptor antagonist LY326325 (> 6 h), or tetrodotoxin (~12 h), are all known to effectively impair learning, the initial cellular consolidation of memory lasting a few hours, and memory retrieval. But in contrast to ZIP, these treatments do not impair the storage of memories that have had a period of 24 h or longer to consolidate after memory activation. This is demonstrated by intact retrieval of hippocampus-dependent memory, so long as the retention test is sufficiently long (> 5 h) after the seizure or neural inactivation (Fenton et al., 1994; 1995; Jezek et al., 2002; Riedel et al., 1999). Furthermore, place cell firing and spatial performance are only altered after days of repeated seizures (Lin et al., 2009; 2012). Thus, even if ZIP caused seizures or neuronal silencing (neither of which we have observed during our in vivo recordings), experiments that investigate the effect of ZIP on memory persistence infuse ZIP 1 to 30 days after training, when seizure and neuronal silencing are ineffective on memory persistence.
4) In contrast to effects on long-term memory persistence, ZIP does not affect short-term memories or initial consolidation of short-term into long-term memory (Pastalkova et al, 2006; Shema et al., 2007, 2009), precisely the forms of memory that are disrupted by neuronal silencing (Riedel, et al., 1999).
In conclusion, we do not know the source of differences between experiments that show no neuronal silencing and those of LeBlancq et al. that do, but we and others typically inject ZIP in phosphate buffered saline (PBS). LeBlancq et al. injected ZIP, PBS, and acetic acid up to 14% (v:v in PBS). It would be useful to determine whether PKMzeta has a neuroprotective effect on the potentially toxic action of intracranial injections of acetic acid.
References
Fenton, A.A., and Bures, J. (1994). Interhippocampal transfer of place navigation monocularly acquired by rats during unilateral functional ablation of the dorsal hippocampus and visual cortex with lidocaine. Neuroscience 58, 481-491.
Fenton, A.A., Arolfo, M.P., Nerad, L., and Bures, J. (1995). Interhippocampal synthesis of lateralized place navigation engrams. Hippocampus 5, 16-24.
Jezek, K., M. Wesierska, et al. (2002). "Hippocampus-dependent retrieval and hippocampus-independent extinction of place avoidance navigation, and stress-induced out-of-context activation of a memory revealed by reversible lesion experiments in rats." Physiol Res 51 Suppl 1: S35-47.
Lin, H., Holmes, G. L., Kubie, J. L., Muller, R. U. (2009). Recurrent seizures induce a reversible impairment in a spatial hidden goal task. Hippocampus 19: 817-827.
Lin, H., Hangya, B., Fox, S. E., Muller, R. U. (2012). Repetitive convulsant-induced seizures reduce the number but not precision of hippocampal place cells. J Neurosci 32: 4163-4178.
Riedel, G.Riedel, G., Micheau, J., Lam, A. G., Roloff, E. L., Martin, S. J., Bridge, H., de Hoz, L., Poeschel, B., McCulloch, J., Morris, R. G.(1999). Reversible neural inactivation reveals hippocampal participation in several memory processes. Nat Neurosci 2: 898-905.
Shema, R., Sacktor, T. C., Dudai, Y. (2007). Rapid Erasure of Long- Term Memory Associations in Cortex by an Inhibitor of PKMzeta, Science, 317:951-953
Shema R., Hazvi S., Sacktor T. C., Dudai Y. (2009). Boundary conditions for the maintenance of memory by PKMzeta in neocortex. Learn. Mem.: 16:122-128.
Tsokas et al. (2016). "Compensation for PKMzeta in long-term potentiation and spatial long-term memory in mutant mice." eLife 2016;5:e14846
Conflict of Interest:
None declared
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