Analogous to learning and memory storage, long-term potentiation (LTP) is divided into induction and maintenance phases. Testing the hypothesis that the mechanism of LTP maintenance stores information requires reversing this mechanism in vivo and finding out whether long-term stored information is lost. This was not previously possible. Recently however, persistent phosphorylation by the atypical protein kinase C isoform, protein kinase Mzeta (PKMz), has been found to maintain late LTP in hippocampal slices. Here we show that a cell-permeable PKMz inhibitor, injected in the rat hippocampus, both reverses LTP maintenance in vivo and produces persistent loss of 1-day-old spatial information. Thus, the mechanism maintaining LTP sustains spatial memory.

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Protein kinase M (PKM) is a newly described form of PKC that is necessary and sufficient for the maintenance of hippocampal long term potentiation (LTP) and the persistence of memory in Drosophila. PKM is the independent catalytic domain of the atypical PKC isoform and produces long term effects at synapses because it is persistently active, lacking autoinhibition from the regulatory domain of PKC. PKM has been thought of as a proteolytic fragment of PKC. Here we report that brain PKM is a new PKC isoform, synthesized from a PKM mRNA encoding a PKC catalytic domain without a regulatory domain. Multiple -specific antisera show that PKM is expressed in rat forebrain as the major form of in the near absence of full-length PKC. A PKC knockout mouse, in which the regulatory domain was disrupted and catalytic domain spared, still expresses brain PKM, indicating that this form of PKM is not a PKC proteolytic fragment. Furthermore, the distribution of brain PKM does not correlate with PKC mRNA but instead with an alternate RNA transcript thought incapable of producing protein. In vitro translation of this RNA, however, generates PKM of the same molecular weight as that in brain. Metabolic labeling of hippocampal slices shows increased de novo synthesis of PKM in LTP. Because PKM is a kinase synthesized in an autonomously active form and is necessary and sufficient for maintaining LTP, it serves as an example of a link coupling gene expression directly to synaptic plasticity. LTP1 is a persistent enhancement of synaptic transmission widely studied as a physiological model of memory (1). LTP can be divided into two phases: induction, which triggers the potentiation, and maintenance, which sustains it over time. Many molecules have been implicated in LTP induction, which is initiated by the activation of N-methyl-D-aspartate (NMDA) receptors and involves several protein kinases (2). In contrast, very little is known about the molecular mechanism of maintenance. Recently, however, a specific, autonomously active form of the atypical PKC isozyme (3, 4), PKM, has been found both necessary and sufficient for maintaining LTP (5-7). Overexpression of PKM also prolongs memory in Drosophila melanogaster, suggesting it is part of an evolutionarily conserved molecular mechanism for memory storage (8).The unique role of PKM in LTP maintenance is due, in part, to its unusual structural and enzymatic properties as an autonomously active kinase. PKM consists of the independent catalytic domain of a PKC isoform (5). PKC isoforms are divided into three classes: conventional, novel, and atypical (reviewed in Refs. 9 -11). Each isoform is a single polypeptide consisting of an N-terminal regulatory domain and a C-terminal catalytic domain linked by a hinge (Fig. 1A, left). The regulatory domain contains binding sites for second messengers and an autoinhibitory pseudosubstrate sequence, which interacts with and blocks the active site of the catalytic domain. Second messengers stimulate PKC by binding to the regulatory domain, translocating th...

Although the maintenance mechanism of late long-term potentiation (LTP) is critical for the storage of long-term memory, the expression mechanism of synaptic enhancement during late-LTP is unknown. The autonomously active protein kinase C isoform, protein kinase M (PKM), is a core molecule maintaining late-LTP. Here we show that PKM maintains late-LTP through persistent N-ethylmaleimide-sensitive factor (NSF)/glutamate receptor subunit 2 (GluR2)-dependent trafficking of AMPA receptors (AMPARs) to the synapse. Intracellular perfusion of PKM into CA1 pyramidal cells causes potentiation of postsynaptic AMPAR responses; this synaptic enhancement is mediated through NSF/GluR2 interactions but not vesicle-associated membrane protein-dependent exocytosis. PKM may act through NSF to release GluR2-containing receptors from a reserve pool held at extrasynaptic sites by protein interacting with C-kinase 1 (PICK1), because disrupting GluR2/PICK1 interactions mimic and occlude PKM-mediated AMPAR potentiation. During LTP maintenance, PKM directs AMPAR trafficking, as measured by NSF/GluR2-dependent increases of GluR2/3-containing receptors in synaptosomal fractions from tetanized slices. Blocking this trafficking mechanism reverses established late-LTP and persistent potentiation at synapses that have undergone synaptic tagging and capture. Thus, PKM maintains late-LTP by persistently modifying NSF/GluR2-dependent AMPAR trafficking to favor receptor insertion into postsynaptic sites.

How long-term memories are stored is a fundamental question in neuroscience. The first molecular mechanism for long-term memory storage in the brain was recently identified as the persistent action of protein kinase Mzeta (PKMζ), an autonomously active atypical protein kinase C (PKC) isoform critical for the maintenance of long-term potentiation (LTP). PKMζ maintains aversively conditioned associations, but what general form of information the kinase encodes in the brain is unknown. We first confirmed the specificity of the action of zeta inhibitory peptide (ZIP) by disrupting long-term memory for active place avoidance with chelerythrine, a second inhibitor of PKMζ activity. We then examined, using ZIP, the effect of PKMζ inhibition in dorsal hippocampus (DH) and basolateral amygdala (BLA) on retention of 1-d-old information acquired in the radial arm maze, water maze, inhibitory avoidance, and contextual and cued fear conditioning paradigms. In the DH, PKMζ inhibition selectively disrupted retention of information for spatial reference, but not spatial working memory in the radial arm maze, and precise, but not coarse spatial information in the water maze. Thus retention of accurate spatial, but not procedural and contextual information required PKMζ activity. Similarly, PKMζ inhibition in the hippocampus did not affect contextual information after fear conditioning. In contrast, PKMζ inhibition in the BLA impaired retention of classical conditioned stimulus–unconditioned stimulus (CS-US) associations for both contextual and auditory fear, as well as instrumentally conditioned inhibitory avoidance. PKMζ inhibition had no effect on postshock freezing, indicating fear expression mediated by the BLA remained intact. Thus, persistent PKMζ activity is a general mechanism for both appetitively and aversively motivated retention of specific, accurate learned information, but is not required for processing contextual, imprecise, or procedural information.

Protein kinase M (PKM), an autonomously active atypical PKC isoform, is both necessary and sufficient for enhanced synaptic transmission during long-term potentiation (LTP) maintenance. LTP, however, evolves through several temporal phases, which may be mediated by distinct molecular mechanisms of potentiation. Here, we determined the specific phase of LTP maintained by PKM. Using a selective, cell-permeable -pseudosubstrate inhibitor at concentrations that block potentiation produced by postsynaptic perfusion of PKM, we inhibited PKM activity at various times after tetanization of Schaffer collateral/commissural-CA1 synapses. Inhibition of PKM did not affect baseline AMPA receptor-mediated synaptic transmission or an early phase of LTP. In contrast, the inhibitor reversed established LTP when applied 1, 3, or 5 h after tetanic stimulation. Control nontetanized pathways within the hippocampal slices were unaffected. An inactive scrambled version of the peptide had no effect on LTP. Thus, persistent, increased phosphorylation by PKM specifically maintains the late phase of LTP.

Ramó n y Cajal proposed 100 years ago that memory formation requires the growth of nerve cell processes. One-half century later, Hebb suggested that growth of presynaptic axons and postsynaptic dendrites consequent to coactivity in these synaptic elements was essential for such information storage. In the past 25 years, candidate growth genes have been implicated in learning processes, but it has not been demonstrated that they in fact enhance them. Here, we show that genetic overexpression of the growthassociated protein GAP-43, the axonal protein kinase C substrate, dramatically enhanced learning and long-term potentiation in transgenic mice. If the overexpressed GAP-43 was mutated by a Ser 3 Ala substitution to preclude its phosphorylation by protein kinase C, then no learning enhancement was found. These findings provide evidence that a growth-related gene regulates learning and memory and suggest an unheralded target, the GAP-43 phosphorylation site, for enhancing cognitive ability. High-resolution imaging studies of altered nerve cell structure under the influence of synaptic input (1-3) provide a cellular basis for the view that learning involves structural modification of synapses (4, 5). One molecule that has been implicated in input-dependent alterations of synaptic morphology is the growth-associated GAP-43 protein (6), a protein kinase C (PKC) (7,8) substrate and an intrinsic determinant of structural change at the synapse. GAP-43, previously implicated in memory storage processes (9-16), binds to actin (17) and fodrin (18), and by such protein-protein interactions may affect morphological change.To determine whether the neuron-specific GAP-43 growth protein in fact regulates memory formation, we studied the effect on learning and synaptic potentiation of its overexpression in transgenic mice. The GAP-43-null mutation is lethal (19). Because evidence from this and other laboratories indicated that learning increases GAP-43 phosphorylation (9-16), one might expect that a transgenic mouse that overexpresses phosphorylatable GAP-43 would demonstrate enhanced learning. A critical corollary of this prediction is that such genetically enhanced learning would not occur if the PKC site of the overexpressed GAP-43 were mutated to prevent its phosphorylation. Materials and MethodsAnimals. Transgenic mice production has been described in detail elsewhere (20). Brief ly, to construct the expression cassette, an 8.2-kb EcoRI GAP-43 genomic fragment including the Thy-1.2 promoter was used. Germline-transmitting chimeras were obtained by standard injection into C57BL͞6 blastocysts, and the mutation was crossed into either C57BL͞6 or C2D2͞DBA genetic backgrounds. G-Phos is the S42wt line, G-NonP the S42A line, and G-Perm the S42D line. Nontransgenic, wild-type (WT) mice from the breeding program were used as controls. Transgenic animals were screened by slot blot hybridization.Slot Blot and in Situ Hybridization. Genomic DNA purified from mouse tail was used for slot blot hybridization by using a 32 P-labeled chick ...

Our data indicate that female mice exposed to ES display a behavioral and physiologic profile that mimics symptoms of depression in the clinical population. As such, this experimental model may be adopted to examine vicarious stress-induced mood-related disorders, as well as pharmacological antidepressant response, in a sex-specific manner.