Memory Consolidation: Molecular and Cellular Processes

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Memory is a complex biological process involving multiple brain systems, each with a specialized function, and many molecular and cellular mechanisms that process and consolidate information in the brain. Although studies in recent years have made considerable inroads into the molecular and cellular mechanisms required for triggering the intraneuronal synaptic processes underlying the initial stages of memory, little is known about the mechanisms that consolidate memories. Among the processes most intensively studied, mechanisms that regulate transcription seem to have a clear role in memory consolidation. Also, many studies have demonstrated that memory consolidation involves multiple brain systems. For example, while the hippocampus has a critical role in the initial stages of memory consolidation, remote memories seem to be dependent on cortical storage sites.

Memory and Protein Synthesis

Evidence from a variety of systems and organisms demonstrates that protein synthesis—during or shortly after training—is essential for the formation of long-term memory (LTM) (Davis and Squire, 1984). For example, the protein synthesis inhibitor anisomycin, given systemically before or immediately after training, blocks LTM (typically tested twenty-four hours after training) but not short-term memory (STM; 30-120 minutes after training) tested in a wide spectrum of behavioral tasks. Anisomycin can also block the later phases of long-term potentiation (LTP) without affecting its early stages (Frey et al., 1993). LTP is an experimental model of the synaptic plasticity mechanisms underlying learning and memory.

Unfortunately, most of the protein-synthesis inhibitor drugs have a variety of side effects that complicate the interpretation of the findings. Protein-synthesis inhibitors can make animals sick, and some of these inhibitors have nonspecific effects on the levels of "housekeeping" proteins necessary for cell health. Thus, the dramatic effects of protein-synthesis inhibitors on memory triggered a search for the transcription factors that direct the gene expression required for memory. Transcription factors bind to specific sequences in the promoters of genes and serve as attachment sites for the machinery that transcribes genes. Alterations of these transcription factors have far more specific biological effects than the transcription and translation inhibitors used to study memory.

CREB and Memory

Several lines of evidence from studies with multiple organisms and brain systems demonstrate that the cAMP Responsive Element Binding Protein (CREB) is one of the transcription factors regulating the synthesis of proteins necessary for the formation of LTM.

Learning activates several signaling pathways, including cAMP, CaMKIV and MAPK signaling cascades. Activation of these signaling cascades seems to lead to the phosphorylation and activation of CREB. Phosphorylated CREB bound to cAMP Responsive Elements (CRE sites) in the promoter of specific genes, such as 14-3-3 eta, can then bind to a complex of proteins that transcribes those genes. Insights into the biochemistry of CREB directed the design of experiments that tested the impact on memory of both increases and decreases in CREB function.

In a wide range of species, including Aplysia, Drosophila, song birds, mice, and rats, CREB-dependent transcription has been demonstrated to be crucial for the formation of LTM. Furthermore, the levels of active CREB may also be an important determinant of the amount and schedule of training required for LTM. Higher levels of CREB facilitate LTM, while LTM formation in animals with lower CREB levels requires more training with longer intervals between trials. The involvement of CREB in memory formation does not seem to be restricted to certain forms of memory. Tests as diverse as olfactory conditioning in flies, fear conditioning, spatial memory, conditioned taste aversion, social recognition, and social transmission of food preferences in rodents demonstrate the involvement of CREB in memory formation. Similarly, CREB has a role in the stability of synaptic changes in a variety of species, including Aplysia, rats, and mice. Furthermore, CREB also has a role in other forms of plasticity, including topographical cortical reorganization and circadian rhythms (Silva et al., 1998)).

There is compelling evidence that cAMP-dependent signaling can activate CREB (Silva et al., 1998). Deleting both calcium/calmodulin induced adenyl cyclases (1 and 8), enzymes that generates cAMP, block the maintenance but not the induction of LTP in the CA1 region. Similarly, these genetic manipulations also cause deficits in hippocampal long-term memory. Pharmacological activation of adenyl cyclases turns on CREB and rescues the LTP and memory deficits of this mutant (Wong et al., 1999). Another kinase that can phosphorylate and activate CREB is calcium/calmodulin kinase IV (CaMKIV). Experiments with a dominant-negative form of CaMKIV (dnCaMKIV) expressed in the postnatal forebrain showed that this kinase has a role in the later stages of LTP. Similarly, behavioral tests indicated that this kinase modulates long-term memory formation: the dnCaMKIV mice showed specific long-term memory deficits in both spatial learning and contextual fear conditioning tasks. CREB activation was impaired by the dnCaMKIV transgene, confirming that this kinase activates CREB during learning (Kang et al., 2001).

Downstream Targets of CREB and Their Role on Memory

CREB controls the expression of other proteins that seem to regulate the complex molecular and cellular events underlying long-term memory. For example, multiple pulses of serotonin activate CREB in Aplysia and induce a set of genes, many of which may regulate synaptic plasticity and memory. Researchers have identified several synaptic plasticity candidate genes, including the CCAAT enhancer-binding protein, ApC/EBP. ApC/EBP contains CRE sites in the promoter region, is rapidly induced by cAMP, and exhibits properties consistent with an immediate early gene. Low levels of ApC/EBP are present in unstimulated sensory neurons, but there are higher levels during the initial phase of long-term facilitation (LTF), a synaptic model of LTM in Aplysia. Decreasing the expression of ApC/EBP selectively blocks the formation of LTF but not short term facilitation or STF (Alberini et al., 1994). Remarkably, inhibitory avoidance training in rats induces two homologs of ApC/EBP (C/EBP β and C/EBP α), and down-regulation of C/EBP β with antisense oligonucleotides leads to deficits in long-but not in short-term memory in rats tested in this fear conditioning task (Taubenfeld et al., 2001).

C/EBP may regulate another transcription factor, ApAF (Aplysia Activating Factor) (Bartsch et al., 2000), which shares homology with the mammalian PAR family of transcription factors. Injection of recombinant ApAF into a cocultured Aplysia neuronal preparation converts the STF normally produced by one pulse of serotonin into LTF. Additionally, injection of antibodies against ApAF or injection of a dominant negative form of ApAF that contains only the bZIP domain blocks the LTF produced by five pulses of serotonin.

Zif268 is another transcription factor whose expression seems to be regulated by CREB. The promoter region of Zif268 contains two CRE sites and six SRE sites. The levels of Zif268 are upregulated shortly following LTP-inducing stimulation. Furthermore, mutant mice lacking Zif268 show specific deficits in the late phases of LTP. Similarly, LTM, but not STM tested with a variety of tasks, is abnormal in these mutants (Jones et al., 2001). Like the CREBαδ-/- mutant mice, the Zif268 mutants show deficits in spatial learning in the water maze following massed training, and these deficits are rescued by spaced training.

Hippocampal/Neocortical Interactions During Memory Consolidation

Memory consolidation involves the molecular cascades described above, but it also requires multiple brain regions. A number of studies have indicated that whereas memory is initially stored in the hippocampus, eventual storage takes place in cortical networks. Studies examining patients with hippocampal damage have found a loss of memory called temporally-graded retrograde amnesia (Squire, 1992): Patients with hippocampal damage suffer severe amnesia for memories that are a few years old at the time of injury (recent memory) but not for memories that are many years old (remote memory). Similar findings have been made in rodents. For example, contextual fear conditioning is severely disrupted by hippocampal lesions made one day, but not several weeks, after training. These studies suggest that the hippocampus processes new memories but that they are eventually stored elsewhere, presumably the cortex.

A study examining patients with cortical brain damage showed that they have recent but not remote memory, a result that is consistent with the theory that memory is only temporarily stored in the hippocampus and that it is eventually consolidated in the cortex. Interestingly, imaging brain function with the deoxyglucose technique showed that metabolic activity was high in the hippocampus when maze learning was recent, but relatively high in the cortex when maze learning was remote (Bontempi et al., 1999). Performance on the maze correlated only with hippocampal metabolism for recent memory and only with cortical metabolism for remote memory.

A study using molecular genetic techniques came to a strikingly similar conclusion (Frankland et al., 2001). In the mutants studied, LTP is normal in the hippocampus but deficient in the cortical regions thought to store memory. Remarkably, these mice showed normal memory for up to three days after training but dramatic forgetting over longer retention intervals (ten to fifty days). These data suggest that the abnormal LTP in the cortex interfered with the storage of memory there and that the normal LTP in the hippocampus may explain why memory was normal for up to three days after training. It is striking that a wide range of studies, ranging from genetic and imaging studies in mice to neuroanatomical lesions in rats, monkeys, and humans all demonstrate this striking interaction between the hippocampus and the neocortex in memory consolidation.

Editing Memory During Recall

Thus, memory goes through several stages where information is consolidated with different molecular mechanisms and in different brain structures. However, it would be misleading to think of animal memory as computer memory, a permanent etching of events and experiences. Instead, there is a overwhelming amount of data that show that memory is a constructive and dynamic process, designed to fine tune behavioral responses and not to faithfully record experiences. The very usefulness of memory has to do with this ability to extract and generalize from experience adaptive responses and rules that improve survival and fitness. It is not useful for the mouse to remember that a particular cat is dangerous. It is far more useful for it to learn that cats in general are dangerous. Moreover, a number of studies show that the very process of recall can bring memories to a labile state in which they can be edited or reconsolidated (Sara, 2000). Some of the very molecular mechanisms that lay down memories in the first place may be called upon during recall to edit these memory traces. Thus, blocking protein synthesis during recall interferes with memory just as dramatically as blocking it during training (Nader et al., 2000). Similarly, blocking CREB function during recall interferes with memory just as blocking it during training (Kida et al., 2002), an eloquent demonstration of the constructive essence of recall and of the fragile nature of memory.



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