Protein Synthesis I Pharmacology

Oliver Stork and Hans Welz! Abstract

The formation of long-lasting memory traces depends on the de novo synthesis of proteins. For more than 30 years substantial experimental evidence has been collected in species ranging from insects to mammals, in support of this hypothesis. A pharmacological approach to investigate the dependence of long-term memory formation on de novo protein synthesis is to administer drugs that prevent protein synthesis on the transcriptional or translational level. When injected during or after learning, these drugs block the development of long-term memory while leaving short-term memory unaffected. Recent research investigating the time course of protein synthesis following learning revealed the existence of two or probably more distinct time windows during which new proteins are synthesized in order to form an enduring memory trace. Another current topic addresses the question where in the neuron de novo protein synthesis takes place. Whereas a large part of the new proteins are synthesized in the soma, some of them are assembled specifically at those synapses whose modification of synaptic efficiency underlies memory formation.

Asking about the 'Where' and 'When' of Learning-Related Protein Synthesis

Over the past 30 years and more, a large body of evidence has been raised for an involvement of protein synthesis in the formation of long-term memory (LTM). The use of protein synthesis inhibitors (PSIs) has been fundamental for the development of this view. Short-term memory (STM), which is insensitive to protein synthesis inhibition, is now believed to be based on transient modifications of preexisting molecules, most importantly phosphorylation and dephosphorylation of enzymes, receptors and/or ion channels. For an alteration of synaptic efficiency beyond the scope of STM, however, the de novo synthesis of proteins appears to be indispensable. These proteins include transcriptional activators and regulators, neurotrophic factors, cytoskeletal and cell recognition molecules, to name but a few. In essence, it is believed that the protein synthesis during formation of LTM is required for structural changes of existing synapses or generation of new neuronal circuits through reactive sprouting and synaptogenesis.4,2 ,67,83 A strong similarity is apparent to the regulation of growth during development and regeneration in the nervous system. All these processes are activity-dependent, require retrograde messengers and recruit cellular programs of growth including de novo protein synthesis to evoke changes in synaptic structure and function.18,62,101

The fact that LTM in vertebrates and invertebrates can be blocked by PSIs has long been the sole evidence for an involvement of protein synthesis.29 In recent years, however, this hypothesis has obtained support from studies that employed molecular and genetic approaches, as the targeted disruption of transcription factors (e.g., c-fos, zif268, CREB) or specific effector genes (e.g., BDNF, NCAM, CamKII) disturbed the formation of LTM. It must be considered though that mutation of a gene may affect a variety of biological functions other than memory

Lumbar Spine Anatomy

Figurel. Sites ofPSI action within the cell. Actinomycin-D interferes with transcription, whereas 8-azaguanin is incorporated into the growing mRNA and subsequently disturbs its translation. Anisomycin, cyclohex-imide, emetine and puromycin disturb cytoplasmic translation throughout the cell. Puromycin and chloramphenicol interfere with mitochondrial translation. Rapamycin, by interfering with the function of TOR proteins, can be used to specifically disturb synaptic protein synthesis.

Figurel. Sites ofPSI action within the cell. Actinomycin-D interferes with transcription, whereas 8-azaguanin is incorporated into the growing mRNA and subsequently disturbs its translation. Anisomycin, cyclohex-imide, emetine and puromycin disturb cytoplasmic translation throughout the cell. Puromycin and chloramphenicol interfere with mitochondrial translation. Rapamycin, by interfering with the function of TOR proteins, can be used to specifically disturb synaptic protein synthesis.

formation, especially if the gene is active during development. Mutant phenotypes may often be related to compensatory up-regulation or cis-activation of other genes91 rather than the mutation itself, and are sensitive to variations in genetic background.2 ,46 Some of these problems are overcome with inducible transgenic or knock out mutants,110,118 with injection of anti-sense oligonucleotides or with virus-mediated gene-transfer.61,131 However, it may still be difficult to clearly distinguish roles of the mutated molecules in STM and LTM as experimentally induced changes in gene expression require time and can hardly be controlled to occur in an exact time window before, during or after training.

Thus, while the tools are now available to investigate protein synthesis with unprecedented molecular specificity, PSIs become increasingly important to investigate the temporal and spatial aspects, in other words the "When? and Where?" of learning-related protein synthesis. They appear to be particularly powerful when studied in combination with or comparison to specific molecular interventions. In the following we describe the function of the most commonly used PSIs and their effect on memory formation in various paradigms.

O NH

Actinomvcin-D

Anisomycin Cvcloheximide

Figure 2. Chemical structure of the most commonly used PSIs in memory research, actinomycin-D, anisomycin and cycloheximide.

Inhibitors of Protein Synthesis

Transcription Inhibitors

Actinomycin-D (dactinomycin) and 8-azaguanine can be used to interfere with protein synthesis at the transcriptional level. Actinomycin-D is one of the best known antibiotics and used as a cytostatic agent in, e.g., Hodgkin's disease. It is produced by Streptomyces antibioticus and intercalates between GC base pairs of the DNA double strand without binding to single stranded RNA, and thus at low concentrations can inhibit transcription without interfering with translation or DNA replication.99 Actinomycin-D does not cross the blood-brain barrier and, therefore, has to be injected intracranially when a blockade of transcription in the central nervous system is desired. 8-Azaguanine, which is identical to Pathocidin from Streptomyces spectabilis, is incorporated during transcription43 and subsequently disturbs the translation of the affected mRNA. It also exerts influence on a variety of enzymes in cell metabolism and inhibits 5-phosphoribosylpyrophospate-amidotransferase, the key enzyme in purine synthesis. Unfortunately, doses of 8-azaguanine and actinomycin-D that are sufficient to substantially suppress cerebral protein synthesis cause rapid and irreversible systemic toxicity as well as ne-crosis.21,121 Intracranial injections of very low doses (l^g) of actinomycin-D, on the other hand, have little effect on RNA synthesis but still attenuate retention even when injected as late as 24h after training.121 This suggests that retention deficits may be the result of drug-induced brain damage and abnormalities in electrical activity. Thus, experiments with these transcription inhibitors should include anatomical and electrophysiological controls and have to be interpreted with caution.

M BM CO

Figure 3. Gene expression during memory formation. (A) Induction of the immediate early gene c-fos in the amygdala, 30min after fear conditioning training. A number of strongly labeled cells can be found throughout the amygdala, whereas baseline c-fos expression levels (not shown) are hardly detectable. (B) Expression of a novel ubiquitination factor Prajal in the amygdala, 6h after fear conditioning training. (1) Fear conditioned, (2) pseudo-trained and (3) fear memory retrieving mice, (4) naive controls. The graded increase across groups indicates fear-, stress- and learning-related regulation of gene-expression.1 8 CE, central amygdala; L, lateral amygdala; BL, basolateral amygdala; MB, basomedial amygdala; M, medial amygdala and CO, cortical amydala.

Translation Inhibitors

Three classes of translation inhibitors have most commonly been used in memory experiments: puromycin (PURO), anisomycin (ANI), and various glutarimides (cycloheximide (CXM), emetine and acetoxycycloheximide). PURO is a nucleoside-antibiotic from streptomy-ces alboniger and inhibits protein synthesis at both the 70S and 80S, i.e., prokaryotic/mito-chondrial and eukaryotic, ribosomes. Through its structural analogy to aminoacyl-tRNA, PURO can be incorporated into the growing peptide chain at its carboxyl end, which results in premature release of peptidyl-PURO fragments from the ribosomal complex. Unfortunately, such peptidyl-PURO fragments may by themselves have a long-lasting effect on cell function that causes amnesia.29 In addition to inhibiting protein synthesis, PURO induces hippocampal seizures, swelling of mitochondria and disaggregation of ribosomes.36 Due to its numerous side effects, PURO cannot be recommended to investigate the effects of protein synthesis inhibition on memory formation (for a discussion see ref. 29).

ANI is a pyrolidine antibiotic from Streptomycesgriseolus. It inhibits the peptidyl transferase activity in eukaryotic ribosomes and thus interferes with peptide bond formation.94 ANI is, at doses that successfully block retention, a fairly nontoxic protein synthesis inhibitor. Successive injections of ANI permit an inhibition of variable duration in the range of 2-8h, with the inactive form deacetyl-ANI serving as control compound.36 However, it should be considered that ANI is a potent agonist of Jun-NH2 terminal kinase (JNK) as well as mitogen-activated kinase (MAPK), and can induce apoptosis.15,136 CXM, which is isolated from Streptomyces griseus, interferes with the function of peptidyl transferase at the large subunit of the eukaryotic ribosome. However, CXM not only disturbs translation (initiation, translocation, and steps of elongation processes), but also DNA, rRNA and tRNA synthesis,43,49 and in effective doses is far more toxic than ANI.35 Doses inhibiting protein synthesis by 80% or more can cause sickness and, under stressful training conditions, even death. On the behavioral level, changes

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Figure 4. A model for protein synthesis during LTM formation. An incoming signal can produce several cellular responses: (1) post-translational modifications (e.g., phosphorylation) of proteins at activated synapses, responsible for STM, (2) a translational induction of protein synthesis at preexisting mRNA, to support ITM and possibly the generation of synaptic "tags", and (3) an induction of gene-expression through the activation of transcription factors, which are indispensable for LTM. All these processes appear to be activated in parallel during learning.

Figure 4. A model for protein synthesis during LTM formation. An incoming signal can produce several cellular responses: (1) post-translational modifications (e.g., phosphorylation) of proteins at activated synapses, responsible for STM, (2) a translational induction of protein synthesis at preexisting mRNA, to support ITM and possibly the generation of synaptic "tags", and (3) an induction of gene-expression through the activation of transcription factors, which are indispensable for LTM. All these processes appear to be activated in parallel during learning.

in locomotor activity occur that are not seen after ANI injections. Both protein synthesis inhibitors can cause diarrhea, but the signs are hardly noticeable after ANI injections.36 Emetine is the main alkaloid component in Radix ipecacuanhae and inhibits protein synthesis through an interaction with the ribosomal protein S14 of the small eukaryotic ribosomal subunit. Emetine is very toxic and irritant and although considered one of the classical PSI antibiotics has only been used in few studies to interfere with memory formation.

Antibiotics that specifically interfere with the function of 70S ribosomes, such as the Strep-tomyces venezuela antibiotic chloramphenicol, may be used to inhibit prokaryotic and mito-chondrial protein synthesis. Chloramphenicol binds to the 50S mitochondrial ribosome subunit and disturbs the mitochondrial peptidyl transferase reaction without affecting protein synthesis of eukaryotic ribosomes. Indirectly, however, chloramphenicol also affects nuclear and cytoplasmic gene expression and leads to the generation of new mitochondrial RNA species.1 7 Moreover, the inhibition of mitochondrial protein synthesis may change energy provision or Ca2+ sequestration in the mitochondria. The effects of chloramphenicol treatment thus have to be interpreted with care, in particular as its blockade of LTM can be accompanied by profound changes in motor behavior.42

Rapamycin finally is a lipophilic macrolide, which has been employed to show synaptic protein synthesis in Aplysia californica.16 Rapamycin is isolated from a strain of Streptomyces hygroscopicus indigenous to Easter Island ("Rapa Nui"). It interacts with the ubiquitous protein FKBP12 to specifically interfere with the TOR protein (target of rapamycin ). TOR modulates the phosphorylation of proteins that are involved in translation initiation and elongation of the peptide chain, and controls the abundance of the translation machinery in response to mitogen and growth factor stimulation. TOR localizes to synaptic sites and interacts with gephrin,109 and is therefore thought to play a pivotal role in the control of synaptic protein synthesis.

In summary, a number of PSIs are available to interfere with different steps of protein synthesis during memory formation. However, possible unspecific effects have to be carefully controlled in experiments with PSIs. In fact, several hypotheses have been put forth to explain the amnesic effects of protein synthesis inhibitors by mechanisms other than their blockade of protein synthesis (e.g., by producing sickness and conditioned aversion, changes in locomotor activity, inhibition of steroidogenesis, and disturbance of catecholamine neurotransmission), but a number of experiments in different laboratories have provided results that make all these alternative explanations unlikely (for a review see ref. 29). Still, it should be considered that inhibitors of protein synthesis may affect behavioral performance not only through their interference with memory formation, but also by acting on learning-related processes involved in, e.g., attention or motivation. In addition to PSIs, the inhibitors of specific signal transduction pathways can be used to interfere with gene expression during memory formation. In fact, inhibition of the protein kinase A (PKA) and MAPK pathways, or blockade of glucocorticoid receptors have revealed striking similarity to the effects of PSIs.

Effects of Protein Synthesis Inhibitors on Memory Formation

Invertebrates

Learning in invertebrates has been extensively used to investigate cellular and molecular mechanisms of memory formation, including the role of protein synthesis in LTM. Invertebrates are capable of simple forms of learning that are controlled by less complicated and more accessible nervous systems than in vertebrates.

Aplysia californica

In the sea snail Aplysia californica, a simple gill-withdrawal reflex habituates with repeated stimulation (direct touch), but is sensitized by a noxious stimulation of the tail (for a review see ref. 32). The basic elements of this reflex can be simulated in vitro in a preparation consisting of sensory and motor neurons, and application of exogenous serotonin. Long-term facilitation has been shown to be synapse-specific and to require local protein synthesis, as well as RNA and protein synthesis in the cell body.16,74 Long-term - but not short-term — in vivo sensitization or in vitro facilitation are prevented when mRNA or protein synthesis is inhibited during or up to 30min following treatments that normally induce the formation of a memory trace.5,17,8 ANI, when applied around the time of such treatments (-1h to +2h) or 4-7h later, can also block structural changes that are associated with long-term sensitization and facilitation.90 In addition to STM and LTM, an intermediate-term memory (ITM), which requires transla-tional but not transcriptional activation, has been described.48,75,129,130 The translational activation during this ITM seems to be independent of the soma and based on mRNA already existing at the synapse.

Hermissenda crassicornis

In this sea snail classical conditioning of the foot contraction reflex is achieved by pairing a light stimulus (conditioned stimulus; CS) with a rotation (unconditioned stimulus; US; for a review see ref. 32). Several different paradigms have been used on different nervous system preparations from Hermissenda. Although this makes a direct comparison of experimental data difficult, a number of key features, resembling those obtained in Aplysia, emerged. First, extensive conditioning causes structural changes of the neurons involved.1 Second, three different stages of memory formation could be distinguished: STM,25,26,97 which is independent of protein and RNA synthesis, ITM, which is dependent on translational but not transcriptional processes,27 and LTM, which requires mRNA as well as protein synthesis for its formation.25,26 And third, the requirement for protein synthesis is probably not restricted to the immediate post-training period, but continues for several hours, since prolonged and selective changes in RNA expression have been observed after extensive training (maximal at 24h and still detectable after four days88).

Insects

The fruitfly Drosophila melanogaster has been a long-time favourite with behavioral geneticists, due to the ease with which mutations can be created and their consequences are investigated (for a review see ref. 32). In a simple odor discrimination task, an anesthesia-resistant and CXM-insensitive memory can be induced that decays within 4 days after training. With spaced training, however, a LTM for odors is established that lasts for at least 7 days and can be blocked by application of CXM during training.139 Few studies exist that investigated protein synthesis and memory formation in other insects. In honeybees (Apis mellifera), LTM for an odour-sucrose association is blocked by inhibition of either transcription or translation. The blocking effect can be detected 4 days after conditioning, if the drugs are injected 1h or 6h — but not 24h - after conditioning. Memory lasting for up to 2 days, however, is unaffected by the protein synthesis inhibition.80,142,144 Inhibition of protein synthesis also prevents passive avoidance learning in the praying mantis (Stagmatoptera biocellata59). When tested after 24h, animals that were injected with CXM immediately after training act like naive animals. CXM injection 2h after training has no such effect.

Birds

The analysis of memory formation in birds has been particularly helpful to identify critical temporal phases of protein synthesis and the biochemical processes that occur after training.

Imprinting

When newly hatched chicks are briefly exposed to a distinct moving object, they become imprinted to it and the time spent near this object will be increased during future exposures. This imprinting process can be attenuated by immediate post trial injection of CXM.47 Imprinting is accompanied by structural changes in the intermediate medial hyperstriatum ventrale, a brain structure known to be essential for different types of visual learning in chicks. However, the question whether protein synthesis inhibition also blocks these changes has not yet been investigated.55

Passive Avoidance Learning

Newly hatched chicks will also peck at water droplets that form at beads of different color and shape. When one of the beads is associated with a bitter tasting fluid, the chicks quickly learn to avoid pecking at beads of that color and shape.103,105 Injection of ANI immediately before or up to 30min after initial training blocks the formation of LTM in this task.37,133 Pretraining ANI injection can also prevent structural changes in the lobus parolfactorius that accompany the retention of passive avoidance.120 A second time window of critical protein synthesis has been identified with ANI injections 4-5h post-training.132 Inhibitors of protein fucosylation similarly prevent LTM formation when applied during this second time window, indicating the generation of glycoproteins that are essential for the learning process.104,106

Song Learning

When zebra finches listen to the song of a conspecific, neuronal activity and expression of an immediate early gene (zenk, synonym to zif268) is increased in the caudomedial striatum.24,78,79,86 With repeated listening to the same song, neuronal activity declines and zenk expression disappears. However, novel calls or complex sounds reinduce neuronal activity and zenk expression. This 'stimulus-specific habituation' can last for up to two days.23,24 Injection of CXM immediately after song presentation (0.5-3h) or at multiple subsequent periods (5.5-7h, 14-15h, and 17-18.5h) blocks the reduction in neuronal activity and prevents the decline of zenk expression.23,24,140

Mammals

Since the early 1960s a wealth of data has accumulated that supports a critical role of protein synthesis for LTM formation in mice and rats (for a review see ref. 29). The data have been collected in paradigms that range from classical conditioning to complex spatial learning tasks.

Fear Conditioning

In recent years, the involvement of protein synthesis in auditory cued and contextual fear conditioning has been extensively studied; this paradigm appears to be particularly well suited for molecular, cellular and biochemical analyses since a robust association of the auditory CS or the training context with the aversive US can be achieved within one brief training session. In different experiments with ANI, CXM and actinomycin-D it has been shown that the formation of such fear memory can be prevented with systemic, intra-ventricular or intra-amygdalar blockade of protein synthesis.6'11 >n5,n6>127 Contextual fear conditioning was further found to involve either one or two PSI-sensitive consolidation periods, depending on the intensity of training.13 In the latter and in other studies, a striking similarity was observed to the effects of PKA and MAPK inhibition.114,115 It has further been shown that fear memories require amygdalar protein synthesis also for reconsolidation after their retrieval.84 Extinction of contextual fear memory, on the other hand, can take place in the presence of systemically injected PSIs.65

Conditioned Taste Aversion

Similar to fear conditioning, conditioned taste aversion learning is characterized by a brief training episode, in this case with a novel taste that is followed by the induction of sickness (e.g., through administration of lithium chloride). Consolidation of taste aversion memory can be prevented by intra-ventricular injections of CXM,56,137 and by bilateral ANI injections into the gustatory cortex before or during trainingal.108 Recent studies furthermore have shown that ANI injection into the gustatory cortex can also block the extinction of conditioned taste aversion.10

Avoidance Learning

It has long been established that administration of PSIs before or immediately after training prevents passive avoidance learning in rodents.35,36,66,95,96,124 The same treatment does not affect STM, nor the performance during acquisition of the task. However, the amnesic action of protein synthesis inhibition was shown to decrease with the strength of training and to increase with longer duration of protein synthesis inhibition. The involvement of hippocampus and amygdala in this task is illustrated by the amnesic effects of local actinomycin-D and CXM injections, respecitively.11 On the other hand, protein synthesis inhibition over a period of 6-8h is necessary to block active avoidance learning or the extinction of this task in mice.33,34

Discrimination Learning

Injections of CXM or ANI during and following training interfere with the establishment of LTM for an object discrimination task.122 Moreover, rat olfactory discrimination learning in two different social situations was found to be blocked by protein synthesis inhibition.70 ANI, however, does not prevent memory formation after olfactory discrimination learning,124 possibly due to residual protein synthesis or the occurrence of protein-synthesis independent synap-tic changes outlasting the training-test interval. Two critical waves of protein synthesis for brightness discrimination learning , one around training (between 10min pre and 80min post-) and one 4-6h post training,50,51 could be identified with hippocampal ANI injections. Injections between these two time windows are without effect.

Spatial Learning

Few studies have investigated the effects of protein synthesis inhibition in spatial learning tasks. However, the formation of spatial LTM in the water maze can be disturbed by subcutaneous or intra-ventricular injection of ANI before, but not after each training session.65,77 Extinction of spatial memory, in contrast, has proven insensitive to ANI.65

Electrophysiological Models

Long-term potentiation (LTP) and long-term depression (LTD) are electrophysiological models of synaptic plasticity that can be tested in vivo as well as in vitro. Physiological and molecular events that characterize LTP and LTD resemble those observed in learning paradigms, i.e., strong parallels exist between hippocampal LTP and spatial learning,12,10 and amygdala LTP and fear conditioning.73,102 Accordingly, a late phase of LTP (L-LTP), which is sensitive to PSIs in vitro and in vivo,38,39,57,63,85, 9,92,123 can be distinguished from a PSI-insensitive early phase (E-LTP). A similar protein synthesis-dependent late phase of cerebellar LTD has also been described.69,71 The maintenance of LTP for up to 3h has been shown to require protein synthesis, but not mRNA synthesis,92 suggesting that - as in ITM - proteins which can support a limited maintenance of LTP are synthesized from preexisting mRNA.

Principle Findings and Future Perspectives in Protein Synthesis Inhibitor Research

Investigations in both vertebrates and invertebrates have provided ample evidence for the dependence of LTM formation on de novo protein synthesis. Findings from protein synthesis inhibition are supported by recent molecular and genetic studies which interfere with the function of specific transcription factors during memory formation.8,14,60,61,93,131,145,146 PSIs have been particularly valuable tools in determining spatial and temporal characteristics, i.e., the "Where? And When?" of learning-induced gene-expression.

The "When"

In essence, it seems that at least two critical waves of protein synthesis exist during memory consolidation; only when applied within these time windows are protein synthesis inhibitors able to block the formation of LTM.13,50,51,104,132,134 Moreover, the finding that a limited LTM can still be induced in the presence of transcription inhibitors during an initial phase following training92,130 has led to the concept of an intermediate-term memory (ITM), which is supported primarily through translation from preexisting mRNAs. Other studies have identified unique biochemical processes during this transitional phase of information storage, such as activation of PKA and calcineurin. 2 The biochemical processes that underly ITM and LTM appear to be activated in parallel; the induction of early gene transcription factors for example is essential for LTM, but not for ITM, and occurs within minutes after training.

Protein synthesis is further involved in the reconsolidation and continuous refinement of existing memories. Interestingly, opposite effects of PSIs were observed in different aversive learning tasks: ANI blocked the extinction of an active avoidance memory after systemic injection34 and the extinction of conditioned taste aversion when infused to the insular cortex,10 but strongly interfered with the reconsolidation of auditory cued fear memory after injection into the amygdala.84 These observations may illustrate the complexity of memory reconsolidation and the contribution of different brain areas therein.

The "Where"

By disrupting memory consolidation with local injections of PSIs, it was in fact possible to identify brain areas that undergo neural plasticity following training, such as the amygdala during Pavlovian fear conditioning114 or the gustatory cortex during aversive taste conditioning.10 Moreover, PSIs may be used to address the intracellular localization of protein synthesis. It has also become evident in recent years that some critical protein constituents are synthesised synaptically during memory transport of mRNA into dendrites ap pears to be common for plasticity-related molecules including microtubule-associated pro-tein2 (MAP2),44'138 CamKII,76 arg3.1,68 brain spectrin,45 trkB and BDNF.135 Such synaptic protein synthesis may speed up local reorganization processes, allow localized and transcription-independent regulation of protein synthesis through retrograde messengers82 and possibly provide new "synaptic tags".16,40,41 As one of the best examples, CamKII mRNA is actively transported into dendrites and associates with synaptic polysomes upon depolarization.3,76 Specific cis-acting elements in the 3' untranslated region of CaMKII mRNA are responsible for its localization and for its synaptic translation, which involves the cytoplasmic polyadenylation element binding protein, CpEB.76,143 Initiation of translation, elongation of the peptide chain and the abundance of translation machinery components including CPEB are regulated by the TOR protein, which has become an exciting new target in memory re-

search.16

PSIs have also been helpful for the identification of signal transduction pathways that control gene-expression during formation of LTM and to identify the factors that are induced. For instance, a striking parallel has been observed between protein synthesis inhibition and the effect of glucocorticoid inhibition during chick passive avoidance learning.111,112 In Pavlovian fear memory, a temporal coincidence of PSI- with PKA- and MAPK inhibitor-sensitive phases of memory consolidation has been observed. e.g.,113 As the PKA and MAPK pathways converge in CREB activation, these findings support the idea that CREB-mediated gene expression may be critical for fear memory activation in both vertebrates and invertebrates has been implicated in numerous other learning paradigms.7,31,52,58,64,141

Downstream of CREB, several genes are induced during a first PSI-sensitive phase immediately after training. In this phase, so-called immediate early gene transcription factors (e.g., c-fos, c-jun, zif268) and neurotrophic factors, the latter of which can initiate activity-dependent functional and structural reorganization in presynaptic and neighboring cells, are expressed.2,18,30,53,78,79,107 Target genes of these transcriptional activators are expressed in later waves of protein synthesis and likely participate in the modification of synaptic structure and signal transduction.9,19,20,87,119,128

In summary, PSIs have been valuable tools for investigations on the brain structures and time windows that are critical for memory formation. They are used to dissect signalling pathways that control gene expression during learning and to identify the factors that are critical in these processes. The application of PSIs to questions of intracellular control mechanisms of synaptic plasticity, such as dendritic protein synthesis and synaptic tagging, and to the question of memory reconsolidation are promising new fields of research.

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