Functional connectivity MRI tracks memory networks after maze learning in rodents.

Learning and memory employs a series of cognitive processes which require the coordination of multiple areas across the brain. However in vivo imaging of cognitive function has been challenging in rodents. Since these processes involve synchronous firing among different brain loci we explored functional connectivity imaging with resting-state fMRI. After 5-day training on a hidden platform watermaze task, notable signal correlations were seen between the hippocampal CA3 and other structures, including thalamus, septum and cingulate cortex, compared to swim control or naïve animals. The connectivity sustained 7 days after training and was reorganized toward the cortex, consistent with views of memory trace distribution leading to memory consolidation. These data demonstrates that, after a cognitive task, altered functional connectivity can be detected in the subsequently sedated rodent using in vivo imaging. This approach paves the way to understand dynamics of area-dependent distribution processes in animal models of cognition.

Spatial memory training induces morphological changes detected by manganese-enhanced MRI in the hippocampal CA3 mossy fiber terminal zone.

Hippocampal mossy fibers (MFs) can show plasticity of their axon terminal arbor consequent to learning a spatial memory task. Such plasticity is seen as translaminar sprouting from the stratum lucidum (SL) of CA3 into the stratum pyramidale (SP) and the stratum oriens (SO). However, the functional role of this presynaptic remodeling is still obscure. In vivo imaging that allows longitudinal observation of such remodeling could provide a deeper understanding of this presynaptic growth phenomenon as it occurs over time. Here we used manganese-enhanced magnetic resonance imaging (MEMRI), which shows a high-contrast area that co-localizes with the MFs. This technique was applied in the detection of learning-induced MF plasticity in two strains of rats. Quantitative analysis of a series of sections in the rostral dorsal hippocampus showed increases in the CA3a' area in MEMRI of trained Wistar rats consistent with the increased SO+SP area seen in the Timm's staining. MF plasticity was not seen in the trained Lister-Hooded rats in either MEMRI or in Timm's staining. This indicates the potential of MEMRI for revealing neuro-architectures and plasticity of the hippocampal MF system in vivo in longitudinal studies.

Is the place cell a "supple" engram?

This short note, which honors Nobelists O'Keefe and the Mosers, asks how the patterning of inputs to a single place cell regulates its firing. Because the combination of inputs to a single CA1 place cell is very large, the generally accepted view is rejected that inputs to a place cell are relatively restricted, near identical repetition upon re-presentation of the environment. The alternative proposed here is that when any 100 excitatory inputs are fired activating a subset combination, which is a large number, selected from the 30,000 synapses, this leads to CA1 cell firing. The selection of the combination of inputs is a very large number it nonetheless leads to the conclusion that even though the same cell dutifully fires when the animal is in an identical location, the inputs that fire the place cell are nonetheless obligatorily non-identical. This CA1 input combinatorial proposal may help us understand the physiological underpinnings of the memory mechanism arising from supple synapses (Routtenberg (2013), Hippocampus 23:202-206).

Activity-dependent Wnt 7 dendritic targeting in hippocampal neurons: plasticity- and tagging-related retrograde signaling mechanism?

Wnt proteins have emerged as transmembrane signaling molecules that regulate learning and memory as well as synaptic plasticity at central synapses (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86; Maguschak and Ressler (2011) J Neurosci 31:13057-13067; Tabatadze et al. (2012) Hippocampus 22: 1228-1241; Fortress et al. (2013) J Neurosci 33:12619-12626). For example, there is both a training-selective and Wnt isoform-specific increase in Wnt 7 levels in hippocampus seven days after spatial learning in rats (Tabatadze et al. (2012) Hippocampus 22: 1228-1241). Despite growing interest in Wnt signaling pathways in the adult brain, intracellular distribution and release of Wnt molecules from synaptic compartments as well as their influence on synaptic strength and connectivity remain less well understood. As a first step in such an analysis, we show here that Wnt 7 levels in primary hippocampal cells are elevated by potassium or glutamate activation in a time-dependent manner. Subsequent Wnt 7 elevation in dendrites suggests selective somato-dendritic trafficking followed by transport from dendrites to their spines. Wnt 7 elevation is also TTX-reversible, establishing that its elevation is indeed an activity-dependent process. A second stimulation given 6 h after the first significantly reduces Wnt 7 levels in dendrites 3 h later as compared to non-stimulated controls suggesting activity-dependent Wnt 7 release from dendrites and spines. In a related experiment designed to mimic the release of Wnt 7, exogenous recombinant Wnt 7 increased the number of active zones in presynaptic terminals as indexed by bassoon. This suggests the formation of new presynaptic release sites and/or presynaptic terminals. Wnt signaling inhibitor sFRP-1 completely blocked this Wnt 7-induced elevation of bassoon cluster number and cluster area. We suggest that Wnt 7 is a plasticity-related protein involved in the regulation of presynaptic plasticity via a retrograde signaling mechanism as previously proposed (Routtenberg (1999) Trends in Neuroscience 22:255-256). These findings provide support for this proposal, which offers a new perspective on the synaptic tagging mechanism (Redondo and Morris (2011) Nat Rev Neurosci 12:17-30).

Lifetime memories from persistently supple synapses.

It is here proposed that the evanescent network derived from malleable or supple synapses is the substrate for long-lasting memory. The subjective sense of memory permanence is not derived, as suggested by Bain and others, from the stabilization of synaptic structure which gives rise to consolidated distributed networks. This generally held wisdom that synapses are activated and ultimately stabilized to reflect the long-lasting substrate of memory is reinforced by increased interest in the importance of sparse coding in memory consolidation. One line of evidence for sparse coding derives from studies on the lateral nucleus neurons of the amygdala (for review, see Josselyn, 2010). These findings lead to the conclusion that a small number of neurons are both necessary and sufficient to retrieve the fear engram. Recently, it has been shown that sparse coding in the dentate gyrus of the hippocampus is sufficient for retrieving the fear conditioning engram (Liu et al., 2012). One implication of these findings is that memory is stored in selected cells and that this restricted storage is more or less permanent, as the authors note: "… Defined cell populations can form the cellular basis for fear memory engrams (Liu et al., 2012, p. 89)." But the problem with this model is that because new learning is incorporated into existing networks, stabilization would work against this integration. For this reason and because of obligatory processes of metabolic protein turnover and ongoing synaptic malleability, the "supple synapse" model is proposed (Routtenberg, A., Rekart, 2005; Routtenberg, 2008a,b). Specifically, long-lasting memory is derived from the instructive, use-dependent sampling of neural networks selected from a very large universe of networks that are evanescent because they are linked together by supple synapses. Importantly, if such suppleness did not exist, the stabilization of synapses would then prevent the physiological malleability of brain circuitry that is essential both for proper integrated information storage and for flexible information retrieval. A corollary of this proposal is that it suggests an alternative view of consolidation: the same agent which is disruptive immediately after learning is no longer effective later because the network, over time, becomes widely distributed and evanescent. Thus, time-dependence is replaced with space-dependence.

Delayed yet persistent hippocampal molecular and structural alteration after learning: new players in the debate on time-dependent long-lasting memory processes.

Two articles in this issue concern the presynaptic structural remodeling and the molecular events that are important novel mechanisms underlying long-term memory storage processes.

Wnt transmembrane signaling and long-term spatial memory.

Transmembrane signaling mechanisms are critical for regulating the plasticity of neuronal connections underlying the establishment of long-lasting memory (e.g., Linden and Routtenberg (1989) Brain Res Rev 14:279-296; Sossin (1996) Trends Neurosci 19:215-218; Mayr and Montminy (2001) Nat Rev Mol Cell Biol 2:599-609; Chen et al. (2011) Nature 469:491-497). One signaling mechanism that has received surprisingly little attention in this regard is the well-known Wnt transmembrane signaling pathway even though this pathway in the adult plays a significant role, for example, in postsynaptic dendritic spine morphogenesis and presynaptic terminal neurotransmitter release (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86). The present report now provides the first evidence of Wnt signaling in spatial information storage processes. Importantly, this Wnt participation is specific and selective. Thus, spatial, but not cued, learning in a water maze selectively elevates the levels in hippocampus of Wnt 7 and Wnt 5a, but not the Wnt 3 isoform, indicating behavioral selectivity and isoform specificity. Wnt 7 elevation is subfield-specific: granule cells show an increase with no detectable change in CA3 neurons. Wnt 7 elevation is temporally specific: increased Wnt signaling is not observed during training, but is seen 7 days and, unexpectedly, 30 days later. If the Wnt elevation after learning is activity-dependent, then it may be possible to model this effect in primary hippocampal neurons in culture. Here, we evaluate the consequence of potassium or glutamate depolarization on Wnt signaling. This represents, to our knowledge, the first demonstration of an activation-dependent elevation of Wnt levels and surprisingly an increased number of Wnt-stained puncta in neurites suggestive of trafficking from the cell body to neuronal processes, probably dendrites. It is proposed that Wnt signaling pathways regulate long-term information storage in a behavioral-, cellular-, and isoform-specific manner.

Lidocaine injections targeting CA3 hippocampus impair long-term spatial memory and prevent learning-induced mossy fiber remodeling.

Learning a spatial location induces remodeling of the mossy fiber terminal field (MFTF) in the CA3 subfield of the dorsal hippocampus (Ramirez-Amaya et al. (2001) J Neurosci 21:7340-7348; Holahan et al. (2006) Hippocampus 16:560-570; Rekart et al. (2007a) Learn Mem 14:416-421). These fibers appear to grow from the stratum lucidum into distal stratum oriens. Is this axonal growth dependent on "repeated and persistent" neural activity in the CA3 region during training? To address this issue, we targeted local inactivation of the MFTF region in a post-training, consolidation paradigm. Male Wistar rats, bilaterally implanted with chronic indwelling cannulae aimed at the MFTF CA3 region, were trained on a hidden platform water maze task (10 trials per day for 5 days). Immediately after the 10th trial on each training day, rats were injected with lidocaine (4% w/v; 171 mM; n=7) or phosphate-buffered saline (PBS; n=7). Behavioral measures of latency, path length, and thigmotaxis were recorded, as was directional heading. A retention test (probe trial) was given 7 days after the last training day, and brains were subsequently processed for MFTF distribution (Timm's stain) and cannula location. Lidocaine treatment was found to block the learning-associated structural remodeling of the MFTF that was reported previously and observed in the PBS-injected controls. During training, the lidocaine group showed elevated latencies and a misdirected heading to locate the platform on the first trial of each training day. On the 7-day retention probe trial, the lidocaine-injected group showed poor retention indicated by the absence of a search bias in the area where the platform had been located during training. These data suggest that the reduction of neuronal activity in the CA3 region impairs long-term storage of spatial information. As this was associated with reduced MFTF structural remodeling, it provides initial anatomical and behavioral evidence for an activity-dependent, presynaptic growth model of memory.

Adult learning and remodeling of hippocampal mossy fibers: unheralded participant in circuitry for long-lasting spatial memory.

Two articles in this issue concerning the overexpression of GAP-43 on mossy fiber growth are related to the plasticity of these axons in relation to learning and memory.

Overexpression of GAP-43 reveals unexpected properties of hippocampal mossy fibers.

The mossy fiber (MF) system targets the apical dendrites of CA3 pyramidal cells in the stratum lucidum (SL). In mice overexpressing the growth-associated protein GAP-43 there is an apparent ectopic growth of these MFs into the stratum oriens (SO) targeting the basal dendrites of these same pyramidal cells (Aigner et al. (1995) Cell 83:269-278). This is the first evidence to our knowledge that links increased GAP-43 expression with growth of central axons. Here we studied the Aigner et al. transgenic mice but were unable to confirm such growth into SO. However, using quantitative methods we did observe enhanced growth within the regions normally targeted by MFs, for example, the SL in the CA3a region. These contrasting results led us to study MFs with double-immunostaining using an immunohistochemical marker for MFs, the zinc transporter, ZnT3, to visualize the colocalization of transgenic GAP-43 within MFs. Unexpectedly, using both fluorescence and confocal microscopy, we were unable to detect colocalization of GAP-43-positive axons with ZnT3-positive MF axons within the MF pathways, either in the region of the MF axons or in the SL, where MF terminals are abundant. In contrast, the plasma membrane-associated presynaptic marker SNAP-25 did colocalize with transgenic GAP-43-positive terminals in the SL. Synaptophysin, the vesicle-associated presynaptic terminal marker, colocalized with ZnT3 but did not appear to colocalize with GAP-43. The present findings raise important questions about the properties of granule cells and the MF mechanisms that differentially regulate axonal remodeling in the adult hippocampus: (1) Because there appears to be at least two populations of granule cells defined by their differential protein expression, this points to the existence of an intrinsic heterogeneity of granule cell expression beyond that contributed by adult neurogenesis; (2) Giventhe present evidence that growth is induced in mice overexpressing GAP-43 in adjacent non-GAP-43 containing MFs, the potential exists for a heretofore unexplored interaxonal communication mechanism.

Ectopic growth of hippocampal mossy fibers in a mutated GAP-43 transgenic mouse with impaired spatial memory retention.

In a previous study, it was shown that transgenic mice, designated G-NonP, forget the location of a water maze hidden platform when tested 7 days after the last training day (Holahan and Routtenberg (2008) Hippocampus 18:1099-1102). The memory loss in G-NonP mice might be related to altered hippocampal architecture suggested by the fact that in the rat, 7 days after water maze training, there is discernible mossy fiber (MF) growth (Holahan et al. (2006) Hippocampus 16:560-570; Rekart et al. (2007) Learn Mem 14:416-421). In the present report, we studied the distribution of the MF system within the hippocampus of naïve, untrained, G-NonP mouse. In WT mice, the MF projection was restricted to the stratum lucidum of CA3 with no detectable MF innervation in distal stratum oriens (dSO). In G-NonP mice, in contrast, there was an ectopic projection terminating in the CA3 dSO. Unexpectedly, there was nearly a complete loss of immunostaining for the axonal marker Tau1 in the G-NonP transgenic mice in the MF terminal fields indicating that transgenesis itself leads to off-target consequences (Routtenberg (1996) Trends Neurosci 19:471-472). Because transgenic mice overexpressing nonmutated, wild type GAP-43 do not show this ectopic growth (Rekart et al., in press) and the G-NonP mice overexpress a mutated form of GAP-43 precluding its phosphorylation by protein kinase C (PKC), the possibility exists that permanently dephosphorylated GAP-43 disrupts normal axonal fasciculation which gives rise to the ectopic growth into dSO.

The protein kinase C phosphorylation site on GAP-43 differentially regulates information storage.

Protein kinase C (PKC) is known to regulate phosphorylation of substrates such as MARCKS, GAP-43, and the NMDA receptor, all of which have been linked to synaptic plasticity underlying information storage processes. Here we report on three transgenic mice isoforms differentiated both by mutation of the PKC site on GAP-43 as well as by their performance in three learning situations: (1) a radial arm maze task, which evaluates spatial memory and its retention, (2) fear conditioning which assesses contextual memory, and (3) the water maze which also evaluates spatial memory and its retention. The present results show, for the first time to our knowledge, that the phosphorylation state of a single site on an identified brain growth- and plasticity-associated protein differentially regulates performance of three different memory-associated tasks.

The substrate for long-lasting memory: if not protein synthesis, then what?

The prevailing textbook view that de novo protein synthesis is required for memory (e.g., [Bear, M. F., Connors, B., & Paradiso, M. 2006. Neuroscience. Lippincott, New York]) is seriously flawed and an alternative hypothesis has been proposed in which post-translational modification (PTM) of proteins already synthesized and already present within the synapse is 'the' substrate for long-lasting memory. Protein synthesis serves a replenishment role. The first part of this review discusses how long-lasting memory can be achieved with 'only' PTM of existing synaptic proteins. The second part critically reviews a recent report published in Neuron 2007 that exemplifies the current view of protein synthesis and memory while also illustrating how these results can be understood within this new PTM framework. A necessary yet unexpected conclusion to emerge from consideration of the consequences of a PTM mechanism as the necessary, sufficient and exclusive substrate for long-lasting memory, is that the central Hebbian dogma that cells that 'fire together, wire together' is an unlikely mechanism for long-lasting memory. Thus, a unique feature of the PTM model is that longevity of information storage is achieved not by stability of the synaptic mechanism, but by impermanent pseudoredundant circuits. This is so because PTM is a reversible process and thus any permanent connection, any 'lasting effect' cannot be in the form of stable synapse formation. We have therefore proposed a solution in which network level processes regulate cellular mechanisms, even as such mechanisms regulate the network. Thus, synapses are 'meta-stabilized' by regulated feedback mediated by the circuit in which the synapse is embedded. For example, spontaneous activity is proposed to be a substrate feedback mechanism we term 'cryptic rehearsal' to sustain for some period of time after learning an approximation to the state initially created by input. Additionally, because the duplication of these traces is ongoing, this provides a degenerate code for the engram. Stability is thus achieved, not by stabilizing the synapse, but by implementing a pseudo-redundant yet malleable circuitry so that memory can be protected in the face of small catastrophes in network representation.

Long-lasting memory from evanescent networks.

Current models of memory typically require a protein synthetic step leading to a more or less permanent structural change in synapses of the network that represent the stored information. This instructive role of protein synthesis has recently been called into question [Routtenberg, A., Rekart, J.L. 2005. Post-translational modification of synaptic proteins as the substrate for long-lasting memory. Trends Neurosci. 28, 12-19]. In its place a new theory is proposed in which post-translational modifications (PTMs) of proteins already synthesized and present within the synapse calibrate synaptic strength. PTM is thus the only mechanism required to sustain long-lasting memories. Activity-induced, PTM-dependent structural modifications within brain synapses then define network formation which is thus a product of the concatenation of cascaded PTMs. This leads to a formulation different from current protein synthesis models in which neural networks initially formed from these individual synaptic PTM-dependent changes is maintained by regulated positive feedback maintains. One such positive feedback mechanism is 'cryptic rehearsal' typically referred to as 'noise' or 'spontaneous' activity. This activity is in fact not random or spontaneous but determined in a stochastic sense by the past history of activation of the nerve cell. To prevent promiscuous network formation, the regulated positive feedback maintains the altered state given specific decay kinetics for the PTM. The up or down state of individual synapses actually exists in an infinite number of intermediate states, never fully 'up', nor fully 'down.' The networks formed from these uncertain synapses are therefore metastable. A particular memory is also multiply represented by a 'degenerate code' so that should loss of a subset of representations occur, erasure can be protected against. This mechanism also solves the flexibility-stability problem by positing that the brain eschews synaptic stability having its own uncertainty principle that allows retrieval from a probabilistic network, so that a retrieved memory can be represented by a selection of components from an essentially infinite number of networks. The network so formed, that is the retrieval, thus emerges from a hierarchy of connectionistic probabilities. The relation of this new theory of memory network formation to current and potential computational implementations will benefit by its unusual point of initiation: deep concerns about the molecular substrates of information storage.

GAP-43 gene expression regulates information storage.

Previous reports have shown that overexpression of the growth- and plasticity-associated protein GAP-43 improves memory. However, the relation between the levels of this protein to memory enhancement remains unknown. Here, we studied this issue in transgenic mice (G-Phos) overexpressing native, chick GAP-43. These G-Phos mice could be divided at the behavioral level into "spatial bright" and "spatial dull" groups based on their performance on two hidden platform water maze tasks. G-Phos dull mice showed both acquisition and retention deficits on the fixed hidden platform task, but were able to learn a visible platform task. G-Phos bright mice showed memory enhancement relative to wild type on the more difficult movable hidden platform spatial memory task. In the hippocampus, the G-Phos dull group showed a 50% greater transgenic GAP-43 protein level and a twofold elevated transgenic GAP-43 mRNA level than that measured in the G-Phos bright group. Unexpectedly, the dull group also showed an 80% reduction in hippocampal Tau1 staining. The high levels of GAP-43 seen here leading to memory impairment find its histochemical and behavioral parallel in the observation of Rekart et al. (Neuroscience 126: 579-584) who described elevated levels of GAP-43 protein in the hippocampus of Alzheimer's patients. The present data suggest that moderate overexpression of a phosphorylatable plasticity-related protein can enhance memory, while excessive overexpression may produce a "neuroplasticity burden" leading to degenerative and hypertrophic events culminating in memory dysfunction.

Remodeling of hippocampal mossy fibers is selectively induced seven days after the acquisition of a spatial but not a cued reference memory task.

Relating storage of specific information to a particular neuromorphological change is difficult because behavioral performance factors are not readily disambiguated from underlying cognitive processes. This issue is addressed here by demonstrating robust reorganization of the hippocampal mossy fiber terminal field (MFTF) when adult rats learn the location of a hidden platform but not when rats learn to locate a visible platform. Because the latter task requires essentially the same behavioral performance as the former, the observed MFTF growth is seen as the consequence of specific input-dependent hippocampal activity patterns selectively generated by processing of extramaze but not intramaze cues. Successful performance on the hidden platform task requires formation of spatial memory. Increased MFTFs in hidden platform-trained rats are observed 7 d but not 2 d after training nor in swim controls. These results suggest that structural plasticity of the mossy fiber:CA3 circuit may contribute to the maintenance of long-lasting memory but not to the initial storage of the spatial context.

Post-translational protein modification as the substrate for long-lasting memory.

Prevailing models of memory identify mRNA translation as necessary for long-lasting information storage. However, there are enough instances of memory storage in the virtual absence of protein synthesis to prompt consideration of alternative models. A comprehensive review of the protein synthesis literature leads us to conclude that the translational mechanism is exclusively a permissive, replenishment step. Therefore, we propose that post-translational modification (PTM) of proteins already at the synapse is the crucial instructive mechanism underlying long-lasting memory. A novel feature of this model is that non-random spontaneous (or endogenous) brain activity operates as a regulated positive-feedback rehearsal mechanism, updating network configurations by fine-tuning the PTM state of previously modified proteins. Synapses participating in memory storage are therefore supple, a feature required for networks to alter complexity and update continuously. In analogy with codons for amino acids, a long-lasting memory is represented by a 'degenerate code' - a set of pseudo-redundant networks that can ensure its longevity.

Resting state functional connectivity data supports detection of cognition in the rodent brain.

Learning is a process which induces plastic changes in the synapses and connections across different regions of the brain. It is hypothesized that these new connections can be tracked with resting state functional connectivity MRI. While most of the evidence of learning-induced plasticity arises from previous human data, data from sedated rats that had undergone training for either 1 day or 5 days in a Morris Watermaze is presented. Seed points were taken from the somatosensory and visual cortices, and the hippocampal CA3 to detect connectivity changes. The data demonstrates that 5-day trained rats showed increased correlations between the hippocampal CA3 and thalamus, septum and cingulate cortex, compared to swim control or naïve animals. Seven days after the training, persistent but reorganized networks toward the cortex were observed. Data from the 1-day trained rats, on the contrary, showed connectivity similar to the swim control and less persistent. The connectivity in several regions was highly correlated with the behavioral performance in these animals. The data demonstrates that longitudinal changes following learning-induced plasticity can be detected and tracked with resting state connectivity.

Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy.

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (µg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240±9µM in wet weight level (135±5µg/g dry weight) in the stratum lucidum (SL) compared to 144±6µM in wet weight level (81±3µg/g dry weight) in the stratum pyramidale (SP) and 78±10µM in wet weight level (44±5µg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

Post-translational synaptic protein modification as substrate for long-lasting, remote memory: an initial test.

The current view of the molecular basis for information storage is that post-translational modification (PTM) of brain proteins is important for the early stages of memory storage and that protein synthesis is necessary for long-lasting memory. This view has been challenged by the proposal that PTM of synaptic proteins is the critical instructive mechanism underlying both recent as well as long-lasting memories (Routtenberg and Rekart, 2005). As an initial test, a broad spectrum serine/threonine kinase inhibitor (H-7) was delivered bilaterally to rat anterior cingulate cortex 1 h before a 3 week retention test of contextual fear conditioning. This significantly blocked 21-Day retention. In the second experiment evaluating extinction of a 21-Day remote memory, H-7 injected into mouse medial prefrontal cortex blocked fear extinction. As the H-7-induced impairment in 21-Day retention was indexed by a decrease in freezing, while the extinction blockade by no decrease in freezing, the results could not be ascribed to a direct effect of the drug on behavioral performance. This represents the first demonstration, to our knowledge, that PTM inhibition, here serine/threonine kinase activity, interferes with long-lasting memory, providing initial support for the PTM model.