Appropriate application of ZIP for PKMζ inhibition, LTP reversal, and memory erasure

We thank Dr. Lisman for his interest in our experiments on PKMf, long-term potentiation (LTP), and long-term memory. PKMf is the leading candidate molecule for maintaining long-term memory because, uniquely, its activation enhances and its inhibition disrupts previously stored memories (Pastalkova et al., 2006; Sacktor, 2011; Shema et al., 2011). Three structurally unrelated PKMf inhibitors disrupt established long-term memory: (1) the zeta inhibitory peptide (ZIP) (Pastalkova et al., 2006; Serrano et al., 2008), (2) the small molecule protein kinase C (PKC) inhibitor chelerythrine (Serrano et al., 2008; Li et al., 2011), and (3) the dominant negative mutant of PKMf (Shema et al., 2011). PKMf inhibition disrupts long-term memory in the hippocampus, insular cortex, sensorimotor cortex, association cortex, cingulate cortex, basolateral amygdala, and nucleus accumbens, as assayed using a variety of behavioral paradigms for spatial, aversive, appetitive, skilled sensorimotor, drug reward, pain sensitization, trace and classical conditioning in rodents, and longterm sensitization in Aplysia californica (Pastalkova et al., 2006; Shema et al., 2007; Serrano et al., 2008; Kwapis et al., 2009; Shema et al., 2009; Hardt et al., 2010; Li et al., 2010; Madronal et al., 2010; Migues et al., 2010; Sacco and Sacchetti, 2010; von Kraus et al., 2010; Cai et al., 2011; Li et al., 2011). In his letter, Lisman makes two claims, one questioning whether ZIP’s amnestic effect on memory is due to its inhibition of PKMf, and the other concerning its effect on LTP of the population spike response. As shown in detail in this letter, neither claim is justified. The first concern is that the dose of ZIP is ‘‘three orders of magnitude greater than the IC-50 of PKM-zeta and two orders of magnitude higher than the concentration required to reverse LTP in hippocampal slices. ’’ The appropriate target concentration of ZIP is that which fully blocks the function of PKMf in neurons and its effect on LTP maintenance. We determined this concentration of ZIP by introducing PKMf postsynaptically into pyramidal cells in hippocampal slices to induce potentiation of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated postsynaptic responses, and adding various concentrations of the drug to the bath (Serrano et al., 2005). We found the lowest concentration of ZIP required to fully block the action of PKMf on the functionally relevant physiological substrates that cause synaptic potentiation was 5 lM (Serrano et al., 2005). Five-micromolar ZIP was also found to be the lowest that effectively reversed late-LTP that had been maintained for several hours [not 1 lM, as stated in Lisman’s letter, which is the dose effective at a time window of an hour after tetanization, but not later (Serrano et al., 2005)]. In Pastalkova et al., we wished to examine the role of PKMf and the persistence of LTP maintenance in long-term spatial memory storage beginning a day after training; thus, our target dose in the hippocampus was 5 lM. If the ZIP injected from a point source was distributed equally in the hippocampus, as Dr. Lisman had calculated, the final concentration would be 100 lM– 203 higher than the target dose. Because synaptic changes thought to encode spatial information are believed to be widely distributed in the dorsal hippocampus, however, we did not wish to assume that a membrane-permeant peptide like ZIP distributes equally throughout the hippocampus. Indeed, using a biotinylated ZIP, we found a significant gradient of 1–2 mm, in part due to the spread of the agent from a point source (Pastalkova et al., 2006). In Dr. Lisman’s comment, the concentration levels of this standing gradient are overestimated, because an assumption was made that the cannula was left in place for the duration of the experiment causing ‘‘continuous leakage from the cannula,’’ whereas, in fact, in our experiments, the cannula was removed 2–3 min after the bolus injection, and the image was obtained 2 h after the injection. Similar millimeter gradients of ZIP at 2-h postinjection were seen by Sacco and Sacchetti (2010), who injected the same dose of ZIP in neocortex and also removed the cannula after 2–3 min (B. Sacchetti, personal communication, Fig. 1). These data show that the spread of the drug is limiting. Indeed, other data show that injecting one-third of this dose of ZIP decreases its amnestic effect (Shema et al., 2009). Thus, to assure a sufficient spatial extent of ZIP spread in the hippocampus, we used an array of electrodes from 0.5 to 2 mm from the injection site to determine that LTP is reversed at distal sites (Pastalkova Department of Physiology and Pharmacology, State University of New York Downstate Medical Center, Brooklyn, New York 11203; Department of Neurology, State University of New York Downstate Medical Center, Brooklyn, New York 11203; Center for Neural Science, New York University, New York, New York 10003 This article was first published online 04 Oct 2011. An error was subsequently identified. A copy editing error mistated the unit of measure in lines 3 and 4 of column 2 as micrometer. It has been corrected to micromolar as originally intended by the author on DD MMM 2011. The publisher regrets the error *Correspondence to: Todd C. Sacktor, Departments of Physiology, Pharmacology, and Neurology, State University of New York Downstate Medical Center. Telephone: 718-270-3933, Fax: 718-270-8974. E-mail: tsacktor@downstate.edu Accepted for publication 12 August 2011 DOI 10.1002/hipo.20978 Published online 4 October 2011 in Wiley Online Library (wileyonlinelibrary.com). HIPPOCAMPUS 22:645–647 (2012)