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[…] For LTP experiments, acute mouse hippocampal slices (450 µm) were prepared as previously described (). Hippocampi were dissected, bathed in ice-cold dissection buffer, and sliced with a McIlwain tissue slicer in a cold room (4˚C). The dissection buffer contained (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 11 glucose, 10 MgCl2, and 0.5 CaCl2, and was bubbled with 95% O2/5% CO2 to maintain the pH at 7.4. The slices were immediately transferred into an interface recording chamber (31.5 ± 1°C) (). The recording superfusate consisted of (in mM): 118 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 24 NaHCO3, and 15 glucose, bubbled with 95% O2/5% CO2, with a flow rate of 0.5 ml/min. In oligodeoxynucleotide experiments, the bath level was increased to fully submerge the slices, and the superfusate containing the oligodeoxynucleotide was recirculated (5 ml at 5 ml/min for 30 min), using a custom-made recirculation system employing piezoelectric pumps (Bartels Mikrotechnik GmbH, Dortmund, Germany). Thereafter, the bath containing the oligodeoxynucleotide was lowered again to interface level, and the flow rate was returned to 0.5 ml/min for the remainder of the experiment.Field EPSPs were recorded with a glass extracellular recording electrode (2–5 MΩ) placed in the CA1 stratum radiatum, and concentric bipolar stimulating electrodes were placed on either side within CA3 or CA1. Hippocampal slices were excluded from study if initial analysis showed fEPSP spike threshold was <2 mV. Pathway independence was confirmed by the absence of paired-pulse facilitation between the two pathways. The high-frequency stimulation consisted of standard two 100 Hz-1 s tetanic trains, at 25% of spike threshold, spaced 20 s apart, which is optimized to produce a relatively rapid onset of protein synthesis-dependent late-LTP (). The maximum slope of the rise of the fEPSP is analyzed on a PC using the WinLTP data acquisition program ().For postsynaptic dialysis of PKMζ and PKCι/λ and activation of conventional/novel PKCs by bath applications of phorbol esters, hippocampal slices (400 µm) were prepared from 19- to 30-day-old Sprague-Dawley rats, using a Vibratome tissue sectioner, as previously described (). The slices were placed in an incubation chamber at 31-33°C in oxygenated (95% O2, 5% CO2) physiological saline consisting of (in mM): 124 NaCl, 5 KCl, 26 NaHCO3, 1.6 MgCl2, 4 CaCl2, 10 glucose for a minimum of 1.5 hr. Single slices were then transferred to a recording chamber (1.5 ml) placed on the stage of an upright microscope (Zeiss Axioskop 2; Carl Zeiss, Oberkochen, Germany) and perfused with warm (31–33°C) saline at ~4.5 ml/min. The recording pipettes had tip resistance of 2–4 MΩ and contained (in mM): 130 Cs-MeSO4, 10 NaCl, 2 EGTA, 10 HEPES, 1 CaCl2, 2 Na-ATP, 0.5 Na-GTP. Purified PKMζ (final concentration in the pipette, 7–20 nM, 0.5–0.9 pmol·min-1·μl-1 phosphotransferase activity []) or PKCι/λ (final concentration, 7.4 ng/ml, 0.8 pmol·min-1·μl-1 [ProQinase GmbH, Breisgau, Germany]) was added to the pipette solution prior to whole-cell patch. Whole-cell recordings were obtained from visualized CA1 pyramidal cells, and synaptic events were evoked by extracellular stimulation (pulse width 0.1 ms) every 15 s with bipolar electrodes placed in stratum radiatum. The cells were held at –75 mV, and EPSC was recorded under the voltage-clamp mode with a Warner Instruments PC-501A amplifier (Hamden, CT) and filtered at 2 kHz (-3 dB, four-pole Bessel). Brief voltage steps (−5 mV, 5 ms) from holding potential were applied during the course of recording to monitor cell access resistance, input resistance, and capacitance. Only recordings with an initial input resistance of >100 MΩ and an initial access resistance of <10 MΩ with insignificant change (<20%) during the course of recordings were accepted for study. Signals were digitized with Digidata 1322A and acquired and analyzed with pClamp software (Molecular Devices, Sunnyvale, CA) running on a PC. The peak amplitude of EPSCs was further analyzed with Excel (Microsoft, Redmond, WA). The means ± SEMs of 1 min bins of responses were plotted in the figures. [...] Sample sizes vary for the different experimental approaches (biochemistry, in vitro intracellular current and extracellular field potential physiology, and behavior). The PKMζ is unnecessary and the PKMζ is compensated hypotheses predict all-or-none effects in the experiments, and this provided a basis for sample size estimates. Power analyses were performed using G*Power Version 3.0.6 with α = 0.05 and β = 0.8 and large effect sizes of 1.5–2.0. The effect size estimates were based on prior studies that demonstrated essentially all-or-none effects of PKMζ inhibition on the biochemical, physiological, and behavioral assays used here (; ; ; ; ). Two-population Student’s t tests were performed to compare protein levels in the PKMζ-null and wild-type mice. For LTP experiments the responses to test stimuli were averaged across 5 min for statistical comparisons. Paired Student’s t tests were used to compare the change in the potentiated response at time points at the beginning and end of drug application. Multi-factor comparisons were performed using ANOVA with repeated measures, as appropriate. The degrees of freedom for the critical t values of the t tests and the F values of the ANOVAs are reported as subscripts. Post-hoc multiple comparisons were performed by Newman-Keuls tests as appropriate. Statistical significance was accepted at p<0.05. Effect sizes for binary comparisons and one-way ANOVAs are reported as Cohen's d and as η2 for two-way ANOVA effects. […]

Pipeline specifications

Software tools pCLAMP, G*Power
Applications Miscellaneous, Patch-clamp
Organisms Mus musculus