Le Good JA, et al. showed more brain PKC immunoreactivity than males without a difference between genotypes (modulates anxiety-like behavior, particularly in male mice. Open in a separate window Figure 2 Reduced anxiety-like behavior PD 0332991 Isethionate in kinase assay using purified PKM, PKC and PKC (Table 1, Supplementary Fig. 3). We found that both ZIP and scrambled ZIP inhibited PKM in the micromolar range, which is less potent than recently reported21. There was only a 7.3-fold difference in inhibitory activity of compounds against purified atypical PKC isozymes (studies suggest that the current pharmacological reagents commonly used to inhibit PKM are not specific for this kinase. More importantly, our studies with mice indicate that PKM is not required for long-term memory and that ZIP can impair memory through mechanisms that do not involve PKM. These findings cast doubt on the importance of PKM in the Copper PeptideGHK-Cu GHK-Copper maintenance of long-term memory. Methods Summary Generation and testing of Prkcz?/? mice A targeting construct containing a 1.1 kb floxed region of exon 9 was used to generate chimeric mice that were crossed to produce wild-type and test. The relationship between machine and hand scoring of fear conditioning was analyzed by calculating a Pearson product-moment correlation coefficient. In all tests of learning and memory we did not detect a sex difference; to increase power and the possibility of detecting a genotype difference, we combined results from male and female mice and analyzed these data by ANOVA, and wild-type mice based on the protocol in Li, et al.3, but using a two-chamber apparatus (Med Associates Inc.) and 20-min conditioning sessions. Injections of saline or 10 mg/kg cocaine (Sigma-Aldrich) were counterbalanced across groups. Cocaine preference index was calculated as the time (seconds) spent in the cocaine-paired chamber on test day minus the time spent in the same chamber before conditioning. Three mice in the non-cannulated group (1 wild-type and 2 mice) and three mice (2 saline-treated and 1 ZIP-treated) in the cannulated group did not develop CPP and were therefore excluded PD 0332991 Isethionate from analysis. One outlier in the cannulated group that was identified by a Grubbs test was also removed from the analysis. Surgery and microinjection Mice were anaesthetized with ketamine (100 mg/kg kinase assays. J.D. produced the constructs for the kinase assays and for generation of the mutant mice, and genotyped the mice. R.O.M. designed experiments, analyzed data, and coauthored the manuscript. The authors declare no competing financial interest. REFERENCES PD 0332991 Isethionate 1. Hardt O, Migues PV, Hastings M, Wong J, Nader K. PKMzeta maintains 1-day- and 6-day-old long-term object location but not object identity memory in dorsal hippocampus. Hippocampus. 2010;20:691C695. [PubMed] [Google Scholar] 2. He YY, et al. PKMzeta maintains drug reward and aversion memory in the basolateral amygdala and extinction memory in the infralimbic cortex. Neuropsychopharmacology. 2011;36:1972C1981. [PMC free article] [PubMed] [Google Scholar] 3. Li YQ, et al. Inhibition of PKMzeta in nucleus accumbens core abolishes long-term drug reward memory. J Neurosci. 2011;31:5436C5446. [PMC free article] [PubMed] [Google Scholar] 4. Migues PV, et al. PKMzeta maintains memories by regulating GluR2-dependent AMPA receptor trafficking. Nat Neurosci. 2010;13:630C634. [PubMed] [Google Scholar] 5. Parsons RG, Davis M. Temporary disruption of fear-potentiated startle following PKMzeta inhibition in the amygdala. Nat Neurosci. 2011 [PMC free article] [PubMed] [Google Scholar] 6. Pastalkova E, et al. Storage of spatial information by the maintenance mechanism of LTP. Science. 2006;313:1141C1144. [PubMed] [Google Scholar] 7. Serrano P, et al. PKMzeta maintains spatial, instrumental, and classically conditioned long-term memories. PLoS Biol. 2008;6:2698C2706. [PMC free article] [PubMed] [Google Scholar] 8. Shabashov D, Shohami E, Yaka R. Inactivation of PKMzeta in the NAc Shell Abolished Cocaine-Conditioned Reward. J Mol Neurosci. 2011 [PubMed] [Google Scholar] 9. Shema R, Sacktor TC, Dudai Y. Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta. Science. 2007;317:951C953. [PubMed] [Google Scholar] 10. Hernandez AI, et al. Protein kinase M zeta synthesis from a brain mRNA encoding an independent protein kinase C zeta catalytic domain. Implications for the molecular mechanism of memory. J Biol Chem. 2003;278:40305C40316. [PubMed] [Google Scholar] 11. Chou MM, et al. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr.Proc Natl Acad Sci U S A. protein expression of PKC and PKM by western blot analysis. We detected an immunoreactive band at 52 kDa corresponding to PKM in brain samples from C57BL/6, wild-type and heterozygous samples at ~72 kDa. His-tagged human PKC (PKC std) was run as a positive control. c, Females showed more brain PKC immunoreactivity than males without a difference between genotypes (modulates anxiety-like behavior, particularly in male mice. Open in a separate window Figure 2 Reduced anxiety-like behavior in kinase assay using purified PKM, PKC and PKC (Table 1, Supplementary Fig. 3). We found that both ZIP and scrambled ZIP inhibited PKM in the micromolar range, which is less potent than recently reported21. There was only a 7.3-fold difference in inhibitory activity of compounds against purified atypical PKC isozymes (studies suggest that the current pharmacological reagents commonly used to inhibit PKM are not specific for this kinase. More importantly, our studies with mice indicate that PKM is not required for long-term memory and that ZIP can impair memory through mechanisms that do not involve PKM. These findings cast doubt on the importance of PKM in the maintenance of long-term memory. Methods Summary Generation and testing of Prkcz?/? mice A targeting construct containing a 1.1 kb floxed region of exon 9 was used to generate chimeric PD 0332991 Isethionate mice that were crossed to produce wild-type and test. The relationship between machine and hand scoring of fear conditioning was analyzed by calculating a Pearson product-moment correlation coefficient. In all tests of learning and memory we did not detect a sex difference; to increase power and the possibility of detecting a genotype difference, we combined results from male and female mice and analyzed these data by ANOVA, and wild-type mice based on the protocol in Li, et al.3, but using a two-chamber apparatus (Med Associates PD 0332991 Isethionate Inc.) and 20-min conditioning sessions. Injections of saline or 10 mg/kg cocaine (Sigma-Aldrich) were counterbalanced across groups. Cocaine preference index was calculated as the time (seconds) spent in the cocaine-paired chamber on test day minus the time spent in the same chamber before conditioning. Three mice in the non-cannulated group (1 wild-type and 2 mice) and three mice (2 saline-treated and 1 ZIP-treated) in the cannulated group did not develop CPP and were therefore excluded from analysis. One outlier in the cannulated group that was identified by a Grubbs test was also removed from the analysis. Surgery treatment and microinjection Mice were anaesthetized with ketamine (100 mg/kg kinase assays. J.D. produced the constructs for the kinase assays and for generation of the mutant mice, and genotyped the mice. R.O.M. designed experiments, analyzed data, and coauthored the manuscript. The authors declare no competing financial interest. Recommendations 1. Hardt O, Migues PV, Hastings M, Wong J, Nader K. PKMzeta maintains 1-day time- and 6-day-old long-term object location but not object identity memory space in dorsal hippocampus. Hippocampus. 2010;20:691C695. [PubMed] [Google Scholar] 2. He YY, et al. PKMzeta maintains drug incentive and aversion memory space in the basolateral amygdala and extinction memory space in the infralimbic cortex. Neuropsychopharmacology. 2011;36:1972C1981. [PMC free article] [PubMed] [Google Scholar] 3. Li YQ, et al. Inhibition of PKMzeta in nucleus accumbens core abolishes long-term drug reward memory space. J Neurosci. 2011;31:5436C5446. [PMC free article] [PubMed] [Google Scholar] 4. Migues PV, et al. PKMzeta maintains remembrances by regulating GluR2-dependent AMPA receptor trafficking. Nat Neurosci. 2010;13:630C634. [PubMed] [Google Scholar] 5. Parsons RG, Davis M. Short term disruption of fear-potentiated startle following PKMzeta inhibition in the amygdala. Nat Neurosci. 2011 [PMC free article] [PubMed] [Google Scholar] 6. Pastalkova E, et al. Storage of spatial info from the maintenance mechanism of LTP. Technology. 2006;313:1141C1144. [PubMed] [Google Scholar] 7. Serrano P, et al. PKMzeta maintains spatial, instrumental, and classically conditioned long-term remembrances. PLoS Biol. 2008;6:2698C2706. [PMC free article] [PubMed] [Google Scholar] 8. Shabashov D, Shohami E, Yaka R. Inactivation of PKMzeta in the NAc Shell Abolished Cocaine-Conditioned Incentive. J Mol Neurosci. 2011 [PubMed] [Google Scholar] 9. Shema R, Sacktor TC, Dudai Y. Quick erasure of long-term memory space associations in the cortex by an inhibitor of PKM zeta. Technology. 2007;317:951C953. [PubMed] [Google Scholar] 10. Hernandez AI, et al. Protein kinase M zeta synthesis from a.
