PKMζ Inhibition Reverses Learning-Induced Increases in Hippocampal Synaptic Strength and Memory during Trace Eyeblink Conditioning

A leading candidate in the process of memory formation is hippocampal long-term potentiation (LTP), a persistent enhancement in synaptic strength evoked by the repetitive activation of excitatory synapses, either by experimental high-frequency stimulation (HFS) or, as recently shown, during actual learning. But are the molecular mechanisms for maintaining synaptic potentiation induced by HFS and by experience the same? Protein kinase Mzeta (PKMζ), an autonomously active atypical protein kinase C isoform, plays a key role in the maintenance of LTP induced by tetanic stimulation and the storage of long-term memory. To test whether the persistent action of PKMζ is necessary for the maintenance of synaptic potentiation induced after learning, the effects of ZIP (zeta inhibitory peptide), a PKMζ inhibitor, on eyeblink-conditioned mice were studied. PKMζ inhibition in the hippocampus disrupted both the correct retrieval of conditioned responses (CRs) and the experience-dependent persistent increase in synaptic strength observed at CA3-CA1 synapses. In addition, the effects of ZIP on the same associative test were examined when tetanic LTP was induced at the hippocampal CA3-CA1 synapse before conditioning. In this case, PKMζ inhibition both reversed tetanic LTP and prevented the expected LTP-mediated deleterious effects on eyeblink conditioning. Thus, PKMζ inhibition in the CA1 area is able to reverse both the expression of trace eyeblink conditioned memories and the underlying changes in CA3-CA1 synaptic strength, as well as the anterograde effects of LTP on associative learning

PKMζ is essential for spinal plasticity underlying the maintenance of persistent pain

<p>Abstract</p> <p>Background</p> <p>Chronic pain occurs when normally protective acute pain becomes pathologically persistent. We examined here whether an isoform of protein kinase C (PKC), PKMζ, that underlies long-term memory storage in various brain regions, also sustains nociceptive plasticity in spinal cord dorsal horn (SCDH) mediating persistent pain.</p> <p>Results</p> <p>Cutaneous injury or spinal stimulation produced persistent increases of PKMζ, but not other atypical PKCs in SCDH. Inhibiting spinal PKMζ, but not full-length PKCs, reversed plasticity-dependent persistent painful responses to hind paw formalin and secondary mechanical hypersensitivity and SCDH neuron sensitization after hind paw capsaicin, without affecting peripheral sensitization-dependent primary heat hypersensitivity after hind paw capsaicin. Inhibiting spinal PKMζ, but not full-length PKCs, also reversed mechanical hypersensitivity in the rat hind paw induced by spinal stimulation with intrathecal dihydroxyphenylglycine. Spinal PKMζ inhibition also alleviated allodynia 3 weeks after ischemic injury in rats with chronic post-ischemia pain (CPIP), at a point when allodynia depends on spinal changes. In contrast, spinal PKMζ inhibition did not affect allodynia in rats with chronic contriction injury (CCI) of the sciatic nerve, or CPIP rats early after ischemic injury, when allodynia depends on ongoing peripheral inputs.</p> <p>Conclusions</p> <p>These results suggest spinal PKMζ is essential for the maintenance of persistent pain by sustaining spinal nociceptive plasticity.</p

BDNF Facilitates L-LTP Maintenance in the Absence of Protein Synthesis through PKMζ

Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase ζ (PKMζ) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMζ reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMζ, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMζ level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMζ at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMζ in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMζ

Boundary conditions for the maintenance of memory by PKMζ in neocortex

We report here that ZIP, a selective inhibitor of the atypical protein kinase C isoform PKMζ, abolishes very long-term conditioned taste aversion (CTA) associations in the insular cortex of the behaving rat, at least 3 mo after encoding. The effect of ZIP is not replicated by a general serine/threonine protein kinase inhibitor that is relatively ineffective toward PKMζ, is independent of the intensity of training and the perceptual quality of the taste saccharin (conditioned stimulus, CS), and does not affect the ability of the insular cortex to re-encode the same specific CTA association again. The memory trace is, however, insensitive to ZIP during or immediately after training. This implies that the experience-dependent cellular plasticity mechanism targeted by ZIP is established following a brief time window after encoding, consistent with the standard period of cellular consolidation, but then, once established, does not consolidate further to gain immunity to the amnesic agent. Hence, we conclude that PKMζ is not involved in short-term CTA memory, but is a critical component of the cortical machinery that stores long- and very long-term CTA memories

Characteristics of LTP evoked repetitively at the CA3-CA1 synapse.

<p>LTP was evoked in a group of control mice (n = 10) by the presentation of two successive HFS sessions. Evoked fEPSPs reached values significantly larger than baseline recordings for the indicated days [asterisk, <i>P</i>≤0.05; F_(24,96) = 3.950]. Subsequent HFS sessions were presented on days 13 and 14, i.e., after the first LTP has decayed to baseline values. Note that in this case, LTP was evoked again reaching values non-significantly different (<i>P</i> = 0.674) from those collected following the first two HFS sessions.</p

Effects on hippocampal EEG and on fEPSPs evoked at the CA3-CA1 synapse of ZIP and scr-ZIP injections in the CA1 area.

<p>(A) Examples of EEG recordings carried out in representative control (C), ZIP-, and scr-ZIP-injected animals. (B) Spectral power analysis of EEG recordings collected from the three experimental groups indicated no significant differences (<i>P</i> = 0.809). (C) Input/output curves of the CA3-CA1 synapse collected from the three experimental groups (n = 5 animals per group). No significant differences (<i>P</i> = 0.874) were observed in the data collected from the three groups. (D) Results collected from the paired-pulse test applied to the three groups of animals. No significant differences (<i>P</i> = 0.978) between groups were observed. Drug infusions were carried out as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010400#s4" target="_blank">Methods</a> section. Each bar in B and each point in C and D represents the mean value collected from 5 animals ± s.e.m.</p

fEPSP and CR evolution for ZIP and scr-ZIP groups.

<p>(A, B) fEPSP slopes (A, white triangles) and percentage of CRs (B, white circles) for ZIP-injected animals (n = 10). For comparison, data (A, fEPSP, black triangles; B, percentage of CRs, black circles) corresponding to the scr-ZIP-injected group (n = 10) are also illustrated. In both groups, the injection took place 2 h before the 8th conditioning session (arrow). Illustrated fEPSP recordings (A, inset) were collected from the 1st and the 10th conditioning sessions of representative ZIP and scr-ZIP animals. Data are indicated as mean ± s.e.m. Asterisks indicate significant differences observed between the two groups for both fEPSP slopes (<i>P</i> = 0.021) and the percentage of CRs across training (<i>P</i> = 0.004) following ZIP injection.</p

Experimental design and analysis of eyeblink data.

<p>(A) Animals were implanted with EMG recording electrodes in the orbicularis oculi (O.O.) muscle and with stimulating electrodes on the supraorbital nerve. For trace eyeblink conditioning, a tone was used as CS and an electric shock at the trigeminal nerve as US. The location of hippocampal stimulating (St.) and recording (Rec.) electrodes and of the injection cannula is illustrated in the top diagram. Abbreviations: DG, dentate gyrus; D, L, M, V, dorsal, lateral, medial, and ventral; Sch., Schaffer collaterals; Sub., subiculum. (B) Photomicrographs illustrating the location (white arrows) of the injection cannula and of the stimulating and recording sites. Calibration bar is 200 µm. (C) Schematic representation of the trace conditioning paradigm, illustrating CS and US stimuli, and the moment when a single electrical pulse (100 µs, square, biphasic) was presented to Schaffer collaterals (St. Hipp.). Examples of EMG and hippocampal extracellular records obtained from the 8th conditioning session of a representative animal are shown. Note the fEPSP evoked by the single pulse (St.) presented to Schaffer collaterals. (D) Three superimposed EMG traces recorded from the orbicularis oculi muscle of control animal following electrical stimulation (a single, 500-µs, cathodic pulse, 2 × threshold) of the supraorbital nerve. Note the characteristic R1 and R2 components of the evoked blink response <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010400#pone.0010400-Gruart1" target="_blank">[2]</a>. (E) No significant differences (<i>P</i> = 0.575) in the latency to the R1 component between the three experimental groups were observed: controls (C), and ZIP- and scr-ZIP-injected mice. (F) Quantitative analysis of the area (expressed in µV × s) of the rectified EMG response corresponding to the R1 component of the evoked blink response. No significant differences (<i>P</i> = 0.302) between groups were observed. Drug infusions were carried out as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010400#s4" target="_blank">Methods</a> section. Each bar in B and C represents the mean value collected from 3 animals ± s.e.m.</p