Zeta Inhibitory Peptide Research Articles

Maintenance of PKMζ-modulated synaptic efficacies despite protein turnover

How can synaptic efficacies be maintained despite the fast turnover of proteins at synapses? Partially, we know that the synthesis of new proteins is essential for the induction of the late, long-lasting phase of long-term potentiation (L-LTP). Recent experiments suggest that the concentration of protein kinase Mζ (PKMζ) is increased during L-LTP and that inhibiting the PKMζ activity during the maintenance phase can effectively reverse L-LTP. Experiments have also shown that phosphorylation is necessary for the activation of PKMζ. However, it is not clear what mechanism maintains the level and activity of PKMζ despite protein turnover and phosphatase activity. Using a mathematical modeling framework, I examine the hypothesis that the activity of PKMζ is sustained through a local switching mechanism. The model for the switching mechanism is motivated by several experimental observations: 1) PKMζ has two phosphorylation sites; one is mediated by another constitutively active kinase, Phosphoinositide-dependent kinase 1 PDK1 (T410) and is essential for its activity, and another is an autophosphorylation site, T560. 2) The phosphorylation of PKMζ increases its stability and the doubly phosphorylated PKMζ has a significantly longer lifetime than the unphosphorylated and singly phosphorylated states of PKMζ. 3) The doubly phosphorylated PKMζ also regulates the new synthesis of PKMζ through a translation feedback loop. The present study implemented a mass action model consistent with these observations. The results show that such a model can be bistable and that L-LTP induction produces an increase in the total amount of PKMζ at active synapses. The increase in PKMζ concentration was maintained through the regulation of new protein synthesis by PKMζ. The results also show that blocking the activity of PKMζ in a dose-dependent manner can effectively abolish the increase in the total amount of PKMζ, which is consistent with the effect that the PKMζ inhibitor zeta inhibitory peptide (ZIP) has experimentally demonstrated. The model is consistent with available experimental results regarding the phosphorylation levels of PKMζ and the temporal aspects of blocking experiments and produces a new prediction.

Infusing zeta inhibitory peptide into the perirhinal cortex of rats abolishes long-term object recognition memory without affecting novel object location recognition.

Infusing the amnesic agent zeta inhibitory peptide (ZIP) into the dorsal hippocampus disrupts established long-term object location recognition memory without affecting object identity recognition, which likely depends on the perirhinal cortex. Here, we tested whether infusing ZIP into the perirhinal cortex can abolish long-term memory supporting object identity recognition, leaving long-term object location recognition memory intact. We infused ZIP into the perirhinal cortex of rats either 1 day or 6 days after exposing them to two identical objects in an open field arena. One day after ZIP infusion, that is, 2 or 7 days after object exposure, we either assessed whether the animals recognized that now one of the two objects was novel or whether they recognized that one of the two familiar objects was at a new location. Our results show for both retention intervals, infusions of ZIP into the perirhinal cortex impaired novel object recognition but spared novel object location recognition. Rats that received a scrambled version of ZIP had no deficit in either test at both retention intervals and expressed stronger novel object recognition compared to rats infused with ZIP. These findings support the view that object recognition depends on dissociable memory representations distributed across different brain areas, with perirhinal cortex maintaining long-term memory for what objects had been encountered, and hippocampus supporting memory for where these objects had been placed.

Impact of peptide permeation enhancer on tight junctions opening cellular mechanisms

The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any in vitro cytotoxicity, a proteomics study revealed that L-R5 interferes with other regulatory pathways, e.g., apoptosis and immune response. We suggest that structural modification of the peptide may increase the specificity TJ protein-peptide interaction.

The role of PKMζ in the maintenance of long-term memory: a review.

Long-term memories are thought to be stored in neurones and synapses that undergo physical changes, such as long-term potentiation (LTP), and these changes can be maintained for long periods of time. A candidate enzyme for the maintenance of LTP is protein kinase M zeta (PKMζ), a constitutively active protein kinase C isoform that is elevated during LTP and long-term memory maintenance. This paper reviews the evidence and controversies surrounding the role of PKMζ in the maintenance of long-term memory. PKMζ maintains synaptic potentiation by preventing AMPA receptor endocytosis and promoting stabilisation of dendritic spine growth. Inhibition of PKMζ, with zeta-inhibitory peptide (ZIP), can reverse LTP and impair established long-term memories. However, a deficit of memory retrieval cannot be ruled out. Furthermore, ZIP, and in high enough doses the control peptide scrambled ZIP, was recently shown to be neurotoxic, which may explain some of the effects of ZIP on memory impairment. PKMζ knockout mice show normal learning and memory. However, this is likely due to compensation by protein-kinase C iota/lambda (PKCι/λ), which is normally responsible for induction of LTP. It is not clear how, or if, this compensatory mechanism is activated under normal conditions. Future research should utilise inducible PKMζ knockdown in adult rodents to investigate whether PKMζ maintains memory in specific parts of the brain, or if it represents a global memory maintenance molecule. These insights may inform future therapeutic targets for disorders of memory loss.

Overexpression of protein kinase Mζ in the hippocampal dentate gyrus rescues amyloid-β-induced synaptic dysfunction within entorhinal-hippocampal circuit

Entorhinal cortex (EC) is one of the first cerebral regions affected in the early phase of Alzheimer's disease (AD). Soluble forms of amyloid beta (Aβ) impair synaptic transmission in experimental AD models. Protein kinase Mζ (PKMζ) is an atypical persistently active protein kinase C, known to maintain long term synaptic plasticity and memory, but its role in AD has not yet been described. We examined effect of PKMζ overexpression on the late long-term potentiation (L-LTP) in the dentate gyrus (DG) following EC amyloidopathy. Oligomeric Aβ 1-42 (oAβ) or vehicle was bilaterally microinjected into the EC of the male Wistar rats. After 1 week, 2 µL of lentiviral vector (~108 TU/mL) encoding PKMζ genome was injected into the DG. One week later, synaptic responses and the LTP persistence were assessed in DG of freely moving animals during 90 minutes to 7 days period. Novel object recognition, passive avoidance and spatial memories were also tested. In rats with EC amyloidopathy, LTP was induced with less amplitude compared to the control group, and extinguished after 24 h. PKMζ overexpression in DG augmented synaptic responses (PS-LTP amplitudes) and maintained LTP over 1 week. PKMζ ameliorated recognition and memory deficits in rats with EC amyloidopathy. Microinjection of PKMζ inhibitor, zeta inhibitory peptide, into the DG abolished the boosting effect of PKMζ on synaptic activity and memory performance. PKMζ-dependent pathway could be a potential therapeutic target to combat synaptic failure and memory deficit in the early phase of AD.

Scabronine G Methyl Ester Improves Memory-Related Behavior and Enhances Hippocampal Cell Proliferation and Long-Term Potentiation via the BDNF-CREB Pathway in Olfactory Bulbectomized Mice.

A previous study reported that scabronine G methyl ester (SG-ME) potentially enhances the in vitro secretion of neurotrophic factors such as nerve growth factor via the protein kinase C (PKC)-ζ pathway. However, it remains unknown whether SG-ME can improve cognitive dysfunctions in olfactory bulbectomized (OBX) mice. To address this question, we evaluated SG-ME-treated and untreated OBX mice in a passive avoidance test. We also investigated potential effects of SG-ME on several parameters: cell proliferation and cAMP response element-binding protein (CREB) phosphorylation in the hippocampal dentate gyrus by immunohistochemistry, brain-derived neurotrophic factor (BDNF) levels in the hippocampus by Western blotting, p-CREB levels in the hippocampus by MapAnalyzer, and long-term potentiation (LTP) by electrophysiology. On the 14th day after surgery OBX mice showed altered passive avoidance and decreases in both cell proliferation and long-term potentiation in the hippocampus, while these changes were reversed by SG-ME (20 μg/mouse) 24 h after the treatment. The improvement in memory deficits was prevented when SG-ME was co-administeredwith either zeta inhibitory peptide (PKC-ζ inhibitor), anti-BDNF antibody, ANA-12 (TrkB antagonist), U0126 (MEK inhibitor), H-89 (PKA inhibitor), LY294002 (PI3K inhibitor) or KN-93 (CaMKII inhibitor). We found that SG-ME enhanced brain-derived neurotrophic factor and p-CREB levels in the hippocampus while p-CREB was localized in neurons, but not in astrocytes nor microglial cells. These findings revealed the potential of SG-ME in improving memory impairments by enhancing cell proliferation and LTP via activation of the BDNF/CREB signaling pathway in neurons.

GSK-3\u03b2 activation is required for ZIP-induced disruption of learned fear

The myristoylated zeta inhibitory peptide (ZIP), which was originally developed as a protein kinase C/Mζ (PKCζ/PKMζ) inhibitor, is known to produce the loss of different forms of memories. However, ZIP induces memory loss even in the absence of PKMζ, and its mechanism of action, therefore, remains elusive. Here, through a kinome-wide screen, we found that glycogen synthase kinase 3 beta (GSK-3β) was robustly activated by ZIP in vitro. ZIP induced depotentiation (a cellular substrate of memory erasure) of conditioning-induced potentiation at LA synapses, and the ZIP-induced depotentiation was prevented by a GSK-3β inhibitor, 6-bromoindirubin-3-acetoxime (BIO-acetoxime). Consistently, GSK-3β inhibition by BIO-acetoxime infusion or GSK-3β knockdown by GSK-3β shRNA in the LA attenuated ZIP-induced disruption of learned fear. Furthermore, conditioned fear was decreased by expression of a non-inhibitable form of GSK-3β in the LA. Our findings suggest that GSK-3β activation is a critical step for ZIP-induced disruption of memory.

Zeta Inhibitory Peptide attenuates learning and memory by inducing NO-mediated downregulation of AMPA receptors

Zeta inhibitory peptide (ZIP), a PKMζ inhibitor, is widely used to interfere with the maintenance of acquired memories. ZIP is able to erase memory even in the absence of PKMζ, via an unknown mechanism. We found that ZIP induces redistribution of the AMPARGluA1 in HEK293 cells and primary cortical neurons, and decreases AMPAR-mediated currents in the nucleus accumbens (NAc). These effects were mimicked by free arginine or by a modified ZIP in which all but the arginine residues were replaced by alanine. Redistribution was blocked by a peptidase-resistant version of ZIP and by treatment with the nitric oxide (NO)-synthase inhibitor L-NAME. ZIP increased GluA1-S831 phosphorylation and ZIP-induced redistribution was blocked by nitrosyl-mutant GluA1-C875S or serine-mutant GluA1-S831A. Introducing the cleavable arginine-alanine peptide into the NAc attenuated expression of cocaine-conditioned reward. Together, these results suggest that ZIP may act as an arginine donor, facilitating NO-dependent downregulation of AMPARs, thereby attenuating learning and memory.

Histone deacetylase inhibitors rescue the impaired memory in terrestrial snails.

It is becoming increasingly clear that the long-term plasticity can be regulated via histone modifications. Many studies demonstrated the role of histone acetylation in acquisition, maintenance, and extinction of long-term memory. Nonetheless, the role of histone acetylation in memory reinstatement following its disruption by antimnemonic treatments was not studied in details. In terrestrial snails, we examined effects of the histone deacetylases inhibitors (HDACi) sodium butyrate (NaB) and trichostatin A (TSA) on reinstatement of the context fear memory impaired by antimnemonic agents such as protein synthesis blocker anisomycin (ANI) + reminding or a specific inhibitor of protein-kinase Mζ, zeta inhibitory peptide (ZIP). It was observed that both NaB and TSA applications restored the ANI-impaired context memory regardless of memory reactivation, while a combination of NaB or TSA plus memory reactivation (or additional training) was necessary for the effective reinstatement of the ZIP-impaired memory. Additionally, NaB injections significantly facilitated development of long-term memory in animals with weak memory, while no effect was observed in animals with strong memory. The data obtained confirmed the assumption that histone acetylation is a critical regulatory component of memory development and reinstatement.

Intra-amygdala infusion of zeta inhibitory peptide attenuates neuropathic pain but not inflammatory pain in adult rats.

Protein kinase Mζ (PKMζ), a typical brain-specific PKC isoform, has been shown to be critical in the maintenance of long-term potentiation and memory storage. Zeta inhibitory peptide (ZIP), a peptide with selective inhibition of PKMζ, has been used in relieving experimental neuropathic pain and disrupting memory. The aim of this study was to investigate the effects of intra-amygdalar infusion of ZIP on neuropathic pain induced by chronic constriction injury (CCI), and inflammatory pain induced by complete Freund's adjuvant (CFA) in adult rats. (I) ZIP was infused into the amygdala 30 minutes (min) before CCI was performed. Mechanical withdrawal threshold (MWT) was determined prior to the infusion of ZIP, and 30, 60, 90, 120, 150, 180 min after CCI (n=8 per group). (II) ZIP was infused into the amygdala 3, 7, 14 days after CCI (n=8 per group). MWT was measured 30 min before the infusion and 2, 12, 24 h after the infusion. (III) Three days after CCI, ZIP was infused into the amygdala repeatedly once a day for 2 days. MWT was measured before each infusion and 2 or 24 h after each infusion (n=8 per group). (IV) ZIP was infused into the amygdala 24 h after the establishment of inflammatory pain induced by complete Freund's adjuvant (CFA). MWT was determined 30 min before the infusion and 30, 60, 90, 120 and 150 min after the infusion (n=8 per group). As shown in figures, (I) amygdalar infusion of ZIP prior to the CCI produced no effect on CCI-induced hyperalgesia when compared to scr-ZIP or saline infusion [n=8, interaction effect, F (7.312, 76.776) =1.237, P>0.05; time as main factor, F (3.656, 76.776) =115.346, P<0.001; group as main factor, F (2, 21) =0.648, P>0.05]. (II) BLA infusion of ZIP significantly increased mechanical withdrawal threshold 7 days after CCI [n=8, interaction effect, F (5.476, 57.500) =15.279, P<0.001; time as main factor, F (2.738, 57.500) = 242.357, P<0.001; group as main factor, F (2,21) =4.786, P<0.05], just the same as 3 and 14 days after CCI (data not shown). (III) Bilateral injection of ZIP into the BLA significantly reduced mechanical hyperalgesia 2 h after the administration [n=8, paired t-test, t (0.05,7) =-5.561, P<0.05; n=8, paired t-test, t (0.05,7) =-4.745, P<0.05], and returned to baseline 24 h after the administration [n=8, paired t-test, t (0.05,7) =1.039, P>0.05; n=8, paired t-test, t (0.05,7) =-1.173, P>0.05]. (IV) ZIP produced no effect on mechanical withdrawal thresholds at all time points examined after the infusion, compared with scr-ZIP or saline control groups (n=8, interaction effect, F (6.135, 126) =1.724, P>0.05; group as main factor, F (2,21) =0.608, P>0.05). Intra-amygdala infusion of ZIP attenuates mechanical hyperalgesia induced by CCI but has no effect on inflammatory pain induced by CFA in rats, suggesting that amygdala PKMζ may be a therapeutic target in the treatment of neuropathic pain.

Reversing Cocaine-Induced Plasticity with Zeta Inhibitory Peptide.

Cocaine-induced plasticity persists during abstinence and is thought to underlie cue-evoked craving. Reversing this plasticity could provide an opportunity for therapeutic intervention. Converging evidence suggest that zeta inhibitory peptide (ZIP) eliminates memories for experience-dependent behaviors, including conditioned drug associations. However, the effect of ZIP on reward seeking and drug-induced plasticity is unknown. The current study examined the effect of ZIP administration in the nucleus accumbens on reinstatement (RI) of cocaine seeking, a rodent model of relapse. We demonstrate that intra-accumbal ZIP administration blocks cocaine-primed RI in rats when administered 24 h or 1 week before testing. These effects of ZIP on drug seeking are specific, as we did not see any effect of ZIP on RI of sucrose seeking. ZIP is a synthetic compound designed to inhibit the atypical PKC, PKMζ, a protein implicated in learning and memory. However, recent evidence from PKMζ-knock-out (KO) mice suggests that ZIP may function through alternative mechanisms. In support of this, we found that ZIP was able to block cue-induced RI in PKMζ-KO mice. One possible mechanism underlying addictive phenotypes is the ability of cocaine to block further plasticity. We hypothesized that ZIP may be working to reverse this anaplasticity. Although ZIP has no effect on accumbal LTD in slices from naive or yoked saline mice, it is able to restore both NMDA-dependent and mGluR5-dependent LTD in animals after cocaine self-administration and withdrawal. These findings demonstrate that intra-accumbal ZIP persistently reverses cocaine-induced behavioral and synaptic plasticity in male and female rodents.SIGNIFICANCE STATEMENT Zeta-inhibitory peptide (ZIP) has been shown to disrupt memory maintenance for experience-dependent behaviors. We examined the effect of ZIP infused into the nucleus accumbens on the reinstatement (RI) of cocaine seeking. We found that intra-accumbal ZIP blocked RI of cocaine seeking 24 h and 1 week later. This effect was specific to RI of cocaine seeking as ZIP did not disrupt RI of food seeking. In conjunction with these behavioral studies we examined the ability of ZIP to reverse cocaine-induced deficits in LTD. We found that ZIP was able to rescue two forms of LTD in cocaine-experienced mice. These studies demonstrate that ZIP is able to reverse cocaine-induced behavioral and synaptic plasticity in a persistent manner.

Cortical zeta-inhibitory peptide injection reduces local sleep need.

Local sleep need within cortical circuits exhibits extensive interregional variability and appears to increase following learning during preceding waking. Although the biological mechanisms responsible for generating sleep need are unclear, this local variability could arise as a consequence of wake-dependent synaptic plasticity. To test whether cortical synaptic strength is a proximate driver of sleep homeostasis, we developed a novel experimental approach to alter local sleep need. One hour prior to light onset, we injected zeta-inhibitory peptide (ZIP), a pharmacological antagonist of protein kinase Mζ, which can produce pronounced synaptic depotentiation, into the right motor cortex of freely behaving rats. When compared with saline control, ZIP selectively reduced slow-wave activity (SWA; the best electrophysiological marker of sleep need) within the injected motor cortex without affecting SWA in a distal cortical site. This local reduction in SWA was associated with a significant reduction in the slope and amplitude of individual slow waves. Local ZIP injection did not significantly alter the amount of time spent in each behavioral state, locomotor activity, or EEG/LFP power during waking or REM sleep. Thus, local ZIP injection selectively produced a local reduction in sleep need; synaptic strength, therefore, may play a causal role in generating local homeostatic sleep need within the cortex.

Antisense Oligodeoxynucleotide Perfusion Blocks Gene Expression of Synaptic Plasticity-related Proteins without Inducing Compensation in Hippocampal Slices.

The elucidation of the molecular mechanisms of long-term synaptic plasticity has been hindered by both the compensation that can occur after chronic loss of the core plasticity molecules and by ex vivo conditions that may not reproduce in vivo plasticity. Here we describe a novel method to rapidly suppress gene expression by antisense oligodeoxynucleotides (ODNs) applied to rodent brain slices in an "Oslo-type" interface chamber. The method has three advantageous features: 1) rapid blockade of new synthesis of the targeted proteins that avoids genetic compensation, 2) efficient oxygenation of the brain slice, which is critical for reproducing in vivo conditions of long-term synaptic plasticity, and 3) a recirculation system that uses only small volumes of bath solution (< 5 ml), reducing the amount of reagents required for long-term experiments lasting many hours. The method employs a custom-made recirculation system involving piezoelectric micropumps and was first used for the acute translational blockade of protein kinase Mζ (PKMζ) synthesis during long-term potentiation (LTP) by Tsokas et al., 2016. In that study, applying antisense-ODN rapidly prevents the synthesis of PKMζ and blocks late-LTP without inducing the compensation by other protein kinase C (PKC) isoforms that occurs in PKCζ/PKMζ knockout mice. In addition, we show that in a low-oxygenation submersion-type chamber, applications of the atypical PKC inhibitor, zeta inhibitory peptide (ZIP), can result in unstable baseline synaptic transmission, but in the high-oxygenation, "Oslo-type" interface electrophysiology chamber, the drug reverses late-LTP without affecting baseline synaptic transmission. This comparison reveals that the interface chamber, but not the submersion chamber, reproduces the effects of ZIP in vivo. Therefore, the protocol combines the ability to acutely block new synthesis of specific proteins for the study of long-term synaptic plasticity, while maintaining properties of synaptic transmission that reproduce in vivo conditions relevant for long-term memory.

Male-Specific Conditioned Pain Hypersensitivity in Mice and Humans

Pain memories are hypothesized to be critically involved in the transition of pain from an acute to a chronic state. To help elucidate the underlying neurobiological mechanisms of pain memory, we developed novel paradigms to study context-dependent pain hypersensitivity in mouse and human subjects, respectively. We find that both mice and people become hypersensitive to acute, thermal nociception when tested in an environment previously associated with an aversive tonic pain experience. This sensitization persisted for at least 24hr and was only present in males of both species. In mice,context-dependent pain hypersensitivity was abolished by castrating male mice, pharmacological blockade of the hypothalamic-pituitary-adrenal axis, or intracerebral or intrathecal injections of zeta inhibitory peptide (ZIP) known to block atypical protein kinase C (including the protein kinase Mζ isoform). In humans, men, but not women, self-reported higher levels of stress when tested in a room previously associated with tonic pain. These models provide a new, completely translatable means for studying the relationship between memory, pain, and stress.

Developmental transitions in amygdala PKC isoforms and AMPA receptor expression associated with threat memory in infant rats

Although infants learn and remember, they rapidly forget, a phenomenon known as infantile amnesia. While myriad mechanisms impact this rapid forgetting, the molecular events supporting memory maintenance have yet to be explored. To explore memory mechanisms across development, we used amygdala-dependent odor-shock conditioning and focused on mechanisms important in adult memory, the AMPA receptor subunits GluA1/2 and upstream protein kinases important for trafficking AMPAR, protein kinase M zeta (PKMζ) and iota/lambda (PKCι/λ). We use odor-shock conditioning in infant rats because it is late-developing (postnatal day, PN10) and can be modulated by corticosterone during a sensitive period in early life. Our results show that memory-related molecules did not change in pups too young to learn threat (PN8) but were activated in pups old enough to learn (PN12), with increased PKMζ-PKCι/λ and GluA2 similar to that observed in adult memory, but with an uncharacteristic decrease in GluA1. This molecular signature and behavioral avoidance of the conditioned odor was recapitulated in PN8 pups injected with CORT before conditioning to precociously induce learning. Blocking learning via CORT inhibition in older pups (PN12) blocked the expression of these molecules. PN16 pups showed a more adult-like molecular cascade of increased PKMζ-PKCι/λ and GluA1–2. Finally, at all ages, zeta inhibitory peptide (ZIP) infusions into the amygdala 24 hr after conditioning blocked memory. Together, these results identify unique features of memory processes across early development: AMPAR subunits GluA1/2 and PKC isoform expression are differentially used, which may contribute to mechanisms of early life forgetting.

Persistent Increases of PKMζ in Sensorimotor Cortex Maintain Procedural Long-Term Memory Storage

SummaryProcedural motor learning and memory are accompanied by changes in synaptic plasticity, neural dynamics, and synaptogenesis. Missing is information on the spatiotemporal dynamics of the molecular machinery maintaining these changes. Here we examine whether persistent increases in PKMζ, an atypical protein kinase C (PKC) isoform, store long-term memory for a reaching task in rat sensorimotor cortex that could reveal the sites of procedural memory storage. Specifically, perturbing PKMζ synthesis (via antisense oligodeoxynucleotides) and blocking atypical PKC activity (via zeta inhibitory peptide [ZIP]) in S1/M1 disrupts and erases long-term motor memory maintenance, indicating atypical PKCs and specifically PKMζ store consolidated long-term procedural memories. Immunostaining reveals that PKMζ increases in S1/M1 layers II/III and V as performance improved to an asymptote. After storage for 1 month without reinforcement, the increase in M1 layer V persists without decrement. Thus, the persistent increases in PKMζ that store long-term procedural memory are localized to the descending output layer of the primary motor cortex.

Persistent Increases of PKMM in Sensorimotor Cortex Maintain Procedural long-Term Memory Storage

Procedural memories, such as for riding a bicycle, can be maintained without practice for long periods of time and are thought to be supported by the persistent reorganization of sensorimotor cortices (S1/M1). Whereas enhanced synaptic strength and structural changes accompany the learning of motor tasks, the persistent molecular modifications that store long-term procedural memories within specific layers of sensorimotor cortex have not been identified. The persistent increase in the autonomously active, atypical PKC isoform, PKMζ, is a putative molecular mechanism for maintaining enhanced synaptic strength during long-term potentiation (LTP) and several forms of long-term memory. Here we examine whether persistent increases in PKMζ store long-term memory for a reaching task in rat sensorimotor cortex that could reveal the sites of procedural memory storage. Perturbing PKMζ synthesis with PKMζ – antisense oligodeoxynucleotides or blocking atypical PKC activity with zeta inhibitory peptide (ZIP) in S1/M1 disrupts and erases the maintenance of long-term motor memories. Only memories that are maintained without daily reinforcement are affected, indicating atypical PKCs (via ZIP) and PKMζ specifically (via antisense) stores consolidated long-term procedural memories. Analysis of changes in the amount of PKMζ in S1/M1 reveals PKMζ increases in layers II/III and V of both S1 and M1 cortices as performance improves to an asymptote during training. After storage for 1 month without reinforcement, the increase in M1 layer V but not other layers persists without decrement. Thus, the sustained increases in PKMζ reveal that the persistent molecular changes storing long-term procedural memory are localized to the descending output layer of primary motor cortex.

Transcription-independent expression of PKMζ in the anterior cingulate cortex contributes to chronically maintained neuropathic pain.

Protein kinase M ζ is well known for its role in maintaining memory and pain. Previously, we revealed that the activation of protein kinase M ζ in the anterior cingulate cortex plays a role in sustaining neuropathic pain. However, the mechanism by which protein kinase M ζ is expressed in the anterior cingulate cortex by peripheral nerve injury, and whether blocking of protein kinase M ζ using its inhibitor, zeta inhibitory peptide, produces analgesic effects in neuropathic pain maintained chronically after injury, have not previously been resolved. In this study, we show that protein kinase M ζ expression in the anterior cingulate cortex is enhanced by peripheral nerve injury in a transcription-independent manner. We also reveal that the inhibition of protein kinase M ζ through zeta inhibitory peptide treatment is enough to reduce mechanical allodynia responses in mice with one-month-old nerve injuries. However, the zeta inhibitory peptide treatment was only effective for a limited time.

Neuroprotection by Propofol Post-Conditioning: Focus on PKMζ/KCC2 Pathway Activity.

Critical and major operations are often accompanied by brain ischemic complications. Previous studies found that propofol post-conditioning provided neuroprotective functions through upregulating the expression of potassium chloride cotransporter 2 (KCC2) in gamma-aminobutyric acid (GABA) interneurons. Membrane expression and phosphorylation represents KCC2 activity, which were modulated by a protein kinase C (PKC)-dependent mechanism. However, the role of propofol in increasing KCC2 phosphorylation and the involvement of protein kinase Mζ (PKMζ), a major subtype of PKC, in the KCC2 pathway remained unclear. In this study, we established middle cerebral artery occlusion model in rats to evaluate the long-term recovery of brain functions using behavioral experiments. KCC2 and PKMζ were assessed via western blot. We used the selective inhibitor, zeta inhibitory peptide (ZIP), to investigate the relationship between KCC2 and PKMζ. Intracellular chloride concentration in the hippocampal CA1 area was measured to determine KCC2 activity. We found that propofol, infused at a speed of 20mgkg-1h-1 for 2h at the onset of reperfusion, improved neurological deficits and cognitive dysfunction following ischemia/reperfusion injury. PKMζ expression was significantly upregulated, which improved KCC2 membrane expression and phosphorylation in the ischemic hippocampal CA1 area, and these effects could last up to 28days. But ZIP inhibited this process. Ultimately, we showed that propofol increased KCC2 phosphorylation and PKMζ was the upstream of KCC2. Propofol led to long-term recovery of brain functions by upregulating the activity of the PKMζ/KCC2 pathway.

Try to Remember: Interplay between Memory and Substance Use Disorder.

Memories associated with substance use disorders, or substance-associated cues increase the likelihood of craving and relapse during abstinence. There is a growing consensus that manipulation of synaptic plasticity may reduce the strength of substance abuse-related memories. On the biological front, there are new insights that suggest memories associated with substance use disorder may follow unique neurobiological pathways that render them more accessible to pharmacological intervention. In parallel to this, research in neurochemistry has identified several potential candidate molecules that could influence the formation and maintenance of long-term memory. Drugs that target these molecules (blebbistatin, isradipine and zeta inhibitory peptide) have shown promise at the preclinical stage. In this review, we shall provide an overview of the evolving understanding on the biochemical mechanisms involved in memory formation and expound on the premise that substance use disorder is a learning disorder.