ß-adrenergic modulation of discrimination learning and memory in the auditory cortex.

Despite vast literature on catecholaminergic neuromodulation of auditory cortex functioning in general, knowledge about its role for long-term memory formation is scarce. Our previous pharmacological studies on cortex-dependent frequency-modulated tone-sweep discrimination learning of Mongolian gerbils showed that auditory-cortical D1/5 -dopamine receptor activity facilitates memory consolidation and anterograde memory formation. Considering overlapping functions of D1/5 -dopamine receptors and ß-adrenoceptors, we hypothesised a role of ß-adrenergic signalling in the auditory cortex for sweep discrimination learning and memory. Supporting this hypothesis, the ß1/2 -adrenoceptor antagonist propranolol bilaterally applied to the gerbil auditory cortex after task acquisition prevented the discrimination increment that was normally monitored 1 day later. The increment in the total number of hurdle crossings performed in response to the sweeps per se was normal. Propranolol infusion after the seventh training session suppressed the previously established sweep discrimination. The suppressive effect required antagonist injection in a narrow post-session time window. When applied to the auditory cortex 1 day before initial conditioning, ß1 -adrenoceptor-antagonising and ß1 -adrenoceptor-stimulating agents retarded and facilitated, respectively, sweep discrimination learning, whereas ß2 -selective drugs were ineffective. In contrast, single-sweep detection learning was normal after propranolol infusion. By immunohistochemistry, ß1 - and ß2 -adrenoceptors were identified on the neuropil and somata of pyramidal and non-pyramidal neurons of the gerbil auditory cortex. The present findings suggest that ß-adrenergic signalling in the auditory cortex has task-related importance for discrimination learning of complex sounds: as previously shown for D1/5 -dopamine receptor signalling, ß-adrenoceptor activity supports long-term memory consolidation and reconsolidation; additionally, tonic input through ß1 -adrenoceptors may control mechanisms permissive for memory acquisition.

Late effect of dopamine D1/5 receptor activation on stimulus-induced BOLD responses in the hippocampus and its target regions depends on the history of previous stimulations.

fMRI was used to study late effects of dopamine D1/5 receptor activation on hippocampal signal processing and signal propagation to several target regions. The dopamine D1/5 receptor agonists SKF83959 and SKF38393 were intraperitoneally applied without, immediately before or 7 days after electrical stimulation of the right perforant pathway with bursts of high-frequency pulses. Control animals received a 0.9% NaCl solution. One day after D1/5 receptor activation, the perforant pathway was stimulated and the induced BOLD responses in the right hippocampus and its target regions, left hippocampus (l-HC) and medial prefrontal cortex (mPFC), were measured. Depending on the temporal relation between dopamine receptor activation and the first perforant pathway stimulation the induced BOLD response pattern differed. When applied without concurrent perforant pathway stimulation, the agonists caused region-selective increases in the induced BOLD responses: the effect of SKF83959 was evident in the mPFC whereas that of SKF38393 was confined to the l-HC. When applied in conjunction with perforant pathway stimulation, either agonist caused increased BOLD responses in both regions. In contrast, when applied 7 days after perforant pathway stimulation, neither SKF83959 nor SKF38393 modified the BOLD responses in the mPFC or l-HC 1day later. These findings suggest that (i) activation of dopamine D1/5 receptors alone is sufficient to modify stimulus-induced BOLD responses in target regions of the right hippocampus 24h later, and (ii), the history of previous stimulations crucially affects the impact of dopamine receptor activation on stimulus-induced BOLD responses.

Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.

Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins, and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area-specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing.

Differential effects of dopamine signalling on long-term memory formation and consolidation in rodent brain.

BACKGROUND: Using auditory discrimination learning in gerbils, we have previously shown that activation of auditory-cortical D1/D5 dopamine receptors facilitates mTOR-mediated, protein synthesis-dependent mechanisms of memory consolidation and anterograde memory formation. To understand molecular mechanisms of this facilitatory effect, we tested the impact of local pharmacological activation of different D1/D5 dopamine receptor signalling modes in the auditory cortex. To this end, protein patterns in soluble and synaptic protein-enriched fractions from cortical, hippocampal and striatal brain regions of ligand- and vehicle-treated gerbils were analysed by 2D gel electrophoresis and mass spectrometry 24 h after intervention. RESULTS: After auditory-cortical injection of SKF38393 - a D1/D5 dopamine receptor-selective agonist reported to activate the downstream effectors adenylyl cyclase and phospholipase C - prominent proteomic alterations compared to vehicle-treated controls appeared in the auditory cortex, striatum, and hippocampus, whereas only minor changes were detectable in the frontal cortex. In contrast, auditory-cortical injection of SKF83959 - a D1/D5 agonist reported to preferentially stimulate phospholipase C - induced pronounced changes in the frontal cortex. At the molecular level, we detected altered regulation of cytoskeletal and scaffolding proteins, changes in proteins with functions in energy metabolism, local protein synthesis, and synaptic signalling. Interestingly, abundance and/or subcellular localisation of the predominantly presynaptic protein α-synuclein displayed dopaminergic regulation. To assess the role of α-synuclein for dopaminergic mechanisms of memory modulation, we tested the impact of post-conditioning systemic pharmacological activation of different D1/D5 dopamine receptor signalling modes on auditory discrimination learning in α-synuclein-mutant mice. In C57BL/6JOlaHsd mice, bearing a spontaneous deletion of the α-synuclein-encoding gene, but not in the related substrains C57BL/6JCrl and C57BL/6JRccHsd, adenylyl cyclase-mediated signalling affected acquisition rates over future learning episodes, whereas phospholipase C-mediated signalling affected final memory performance. CONCLUSIONS: Dopamine signalling modes via D1/D5 receptors in the auditory cortex differentially impact protein profiles related to rearrangement of cytomatrices, energy metabolism, and synaptic neurotransmission in cortical, hippocampal, and basal brain structures. Altered dopamine neurotransmission in α-synuclein-deficient mice revealed that distinct D1/D5 receptor signalling modes may control different aspects of memory consolidation.

Synaptic proteome changes in mouse brain regions upon auditory discrimination learning.

Changes in synaptic efficacy underlying learning and memory processes are assumed to be associated with alterations of the protein composition of synapses. Here, we performed a quantitative proteomic screen to monitor changes in the synaptic proteome of four brain areas (auditory cortex, frontal cortex, hippocampus striatum) during auditory learning. Mice were trained in a shuttle box GO/NO-GO paradigm to discriminate between rising and falling frequency modulated tones to avoid mild electric foot shock. Control-treated mice received corresponding numbers of either the tones or the foot shocks. Six hours and 24 h later, the composition of a fraction enriched in synaptic cytomatrix-associated proteins was compared to that obtained from naïve mice by quantitative mass spectrometry. In the synaptic protein fraction obtained from trained mice, the average percentage (±SEM) of downregulated proteins (59.9 ± 0.5%) exceeded that of upregulated proteins (23.5 ± 0.8%) in the brain regions studied. This effect was significantly smaller in foot shock (42.7 ± 0.6% down, 40.7 ± 1.0% up) and tone controls (43.9 ± 1.0% down, 39.7 ± 0.9% up). These data suggest that learning processes initially induce removal and/or degradation of proteins from presynaptic and postsynaptic cytoskeletal matrices before these structures can acquire a new, postlearning organisation. In silico analysis points to a general role of insulin-like signalling in this process.

Dopamine modulates memory consolidation of discrimination learning in the auditory cortex.

In Mongolian gerbils, the auditory cortex is critical for discriminating rising vs. falling frequency-modulated tones. Based on our previous studies, we hypothesized that dopaminergic inputs to the auditory cortex during and shortly after acquisition of the discrimination strategy control long-term memory formation. To test this hypothesis, we studied frequency-modulated tone discrimination learning of gerbils in a shuttle box GO/NO-GO procedure following differential treatments. (i) Pre-exposure of gerbils to the frequency-modulated tones at 1 day before the first discrimination training session severely impaired the accuracy of the discrimination acquired in that session during the initial trials of a second training session, performed 1 day later. (ii) Local injection of the D1/D5 dopamine receptor antagonist SCH-23390 into the auditory cortex after task acquisition caused a discrimination deficit of similar extent and time course as with pre-exposure. This effect was dependent on the dose and time point of injection. (iii) Injection of the D1/D5 dopamine receptor agonist SKF-38393 into the auditory cortex after retraining caused a further discrimination improvement at the beginning of subsequent sessions. All three treatments, which supposedly interfered with dopamine signalling during conditioning and/or retraining, had a substantial impact on the dynamics of the discrimination performance particularly at the beginning of subsequent training sessions. These findings suggest that auditory-cortical dopamine activity after acquisition of a discrimination of complex sounds and after retrieval of weak frequency-modulated tone discrimination memory further improves memory consolidation, i.e. the correct association of two sounds with their respective GO/NO-GO meaning, in support of future memory recall.

Congenital lack of nNOS impairs long-term social recognition memory and alters the olfactory bulb proteome.

The gaseous neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal nitric oxide synthase (nNOS), is thought to play a major role in the modulation of memory. We tested adult nNOS-deficient and wild-type mice for their recognition memory abilities in the social discrimination paradigm, which is based on olfactory cues. Subsequently, proteomic investigation of the olfactory bulbs of both genotypes were performed under basal conditions and 6 h after learning, i.e., during the consolidation of long-term memory. Short-term and intermediate-term recognition memory was normal in nNOS-deficient mice. However, unlike wild-type mice, nNOS-deficient mice failed to consolidate an olfactory cued long-term recognition memory. Proteomic analysis revealed changes in glycolytic enzymes (e.g., fructose-bisphosphate aldolase C, glyceraldehyde-3-phosphate dehydrogenase), voltage-dependent anion-selective channels 1 and 2, alpha-synuclein, F-actin-interacting proteins (e.g., neuronal protein 25/transgelin 3), proteins of the ubiquitin proteasome system, and heterogeneous nuclear ribonucleoproteins implicated in the regulation of messenger RNA trafficking, stability and translation. Our data suggest that, in the mouse, NO of nNOS origin is critically involved in the regulation of protein synthesis-dependent olfactory long-term memory consolidation within relevant brain structures including the olfactory bulb.

Altered postsynaptic-density-levels of caldendrin in the para-chloroamphetamine-induced serotonin syndrome but not in the rat ketamine model of psychosis.

Caldendrin is a synaptic calcium sensor protein that is tightly associated with the postsynaptic density (PSD). Previous work has shown that the association of the protein with the synapse is highly dynamic and is increased in an activity-dependent manner. In the present study the caldendrin-association with the postsynaptic cytomatrix was analyzed in animal models of psychosis and drug abuse induced neurotoxicity. Subchronic administration of the N-methyl-D-aspartate (NMDA)-receptor antagonist ketamine, serving as a model of NMDA-receptor hypofunction and schizophrenia showed no significant effect on the PSD-levels of caldendrin, indicating that NMDA-receptor activity is not required to keep caldendrin at the synapse. However, administration of high doses of the serotonergic neurotoxin p-chloroamphetamine (PCA) lead to significant changes in the association of caldendrin with the PSD. These results underscore the dynamic association of caldendrin with the PSD and suggest a role of this synaptic calcium sensor in the PCA-induced serotonin syndrome.

Dopaminergic modulation of auditory cortex-dependent memory consolidation through mTOR.

Previous studies in the auditory cortex of Mongolian gerbils on discrimination learning of the direction of frequency-modulated tones (FMs) revealed that long-term memory formation involves activation of the dopaminergic system, activity of the protein kinase mammalian target of rapamycin (mTOR), and protein synthesis. This led to the hypothesis that the dopaminergic system might modulate memory formation via regulation of mTOR, which is implicated in translational control. Here, we report that the D1/D5 dopamine receptor agonist SKF-38393 substantially improved gerbils' FM discrimination learning when administered systemically or locally into the auditory cortex shortly before, shortly after, or 1 day before conditioning. Although acquisition performance during initial training was normal, the discrimination of FMs was enhanced during retraining performed hours or days after agonist injection compared with vehicle-injected controls. The D1/D5 receptor antagonist SCH-23390, the mTOR inhibitor rapamycin, and the protein synthesis blocker anisomycin suppressed this effect. By immunohistochemistry, D1 dopamine receptors were identified in the gerbil auditory cortex predominantly in the infragranular layers. Together, these findings suggest that in the gerbil auditory cortex dopaminergic inputs regulate mTOR-mediated, protein synthesis-dependent mechanisms, thus controlling for hours or days the consolidation of memory required for the discrimination of complex auditory stimuli.

Ablation of the presynaptic organizer Bassoon in excitatory neurons retards dentate gyrus maturation and enhances learning performance.

Bassoon is a large scaffolding protein of the presynaptic active zone involved in the development of presynaptic terminals and in the regulation of neurotransmitter release at both excitatory and inhibitory brain synapses. Mice with constitutive ablation of the Bassoon (Bsn) gene display impaired presynaptic function, show sensory deficits and develop severe seizures. To specifically study the role of Bassoon at excitatory forebrain synapses and its relevance for control of behavior, we generated conditional knockout (Bsn cKO) mice by gene ablation through an Emx1 promoter-driven Cre recombinase. In these animals, we confirm selective loss of Bassoon from glutamatergic neurons of the forebrain. Behavioral assessment revealed that, in comparison to wild-type littermates, Bsn cKO mice display selectively enhanced contextual fear memory and increased novelty preference in a spatial discrimination/pattern separation task. These changes are accompanied by an augmentation of baseline synaptic transmission at medial perforant path to dentate gyrus (DG) synapses, as indicated by increased ratios of field excitatory postsynaptic potential slope to fiber volley amplitude. At the structural level, an increased complexity of apical dendrites of DG granule cells can be detected in Bsn cKO mice. In addition, alterations in the expression of cellular maturation markers and a lack of age-dependent decrease in excitability between juvenile and adult Bsn cKO mice are observed. Our data suggest that expression of Bassoon in excitatory forebrain neurons is required for the normal maturation of the DG and important for spatial and contextual memory.

The dopaminergic midbrain participates in human episodic memory formation: evidence from genetic imaging.

Recent data from animal studies raise the possibility that dopaminergic neuromodulation promotes the encoding of novel stimuli. We investigated a possible role for the dopaminergic midbrain in human episodic memory by measuring how polymorphisms in dopamine clearance pathways affect encoding-related brain activity (functional magnetic resonance imaging) in an episodic memory task. In 51 young, healthy adults, successful episodic encoding was associated with activation of the substantia nigra. This midbrain activation was modulated by a functional variable number of tandem repeat (VNTR) polymorphism in the dopamine transporter (DAT1) gene. Despite no differences in memory performance between genotype groups, carriers of the (low expressing) 9-repeat allele of the DAT1 VNTR showed relatively higher midbrain activation when compared with subjects homozygous for the 10-repeat allele, who express DAT1 at higher levels. The catechol-O-methyl transferase (COMT) Val108/158Met polymorphism, which is known to modulate enzyme activity, affected encoding-related activity in the right prefrontal cortex (PFC) and in occipital brain regions but not in the midbrain. Moreover, subjects homozygous for the (low activity) Met allele showed stronger functional coupling between the PFC and the hippocampus during encoding. Our finding that genetic variations in the dopamine clearance pathways affect encoding-related activation patterns in midbrain and PFC provides strong support for a role of dopaminergic neuromodulation in human episodic memory formation. It also supports the hypothesis of anatomically and functionally distinct roles for DAT1 and COMT in dopamine metabolism, with DAT1 modulating rapid, phasic midbrain activity and COMT being particularly involved in prefrontal dopamine clearance.

Consolidation of auditory cortex-dependent memory requires N-methyl-D-aspartate receptor activation.

The pharmacological basis of sensory cortex-dependent learning and associated cortical reorganizations is only partially understood. In the Mongolian gerbil, the auditory cortex is critical for discriminating the directions of modulation of linearly frequency-modulated tones (FMs). To examine the role of N-methyl-D-aspartate (NMDA)-type glutamate receptors in FM discrimination learning, selective antagonists were used. Compared to vehicle-treated controls, both systemic administration of MK-801 before but not after training, and infusion of D-AP-5 into the auditory cortex after training caused retention deficits detectable 24h later. The amnesic actions were reversible and in a close temporal relation to memory formation. Acquisition performance and performance of an established FM discrimination reaction were not affected. These findings suggest that NMDA receptor activation is required for long-term memory consolidation in auditory cortex-dependent learning.

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

Kainate-induced epileptic seizures induce a recruitment of caldendrin to the postsynaptic density in rat brain.

Caldendrin defines a novel family of neuronal calcium-sensor proteins, the C-terminal moiety of which displays high similarity to calmodulin. We now report that the protein is recruited to the postsynaptic density (PSD) of cortical and hippocampal neurons in response to kainate-induced epileptic seizures, an animal model of human temporal lobe epilepsy. The translocation of caldendrin to the PSD did not occur in kainate-treated rats that did not develop seizures. The enhanced PSD levels of caldendrin are not due to increased protein synthesis and most likely reflect a recruitment from the soluble caldendrin protein pool. These findings suggest that the transduction of dendritic Ca2+-signals via caldendrin is altered by epileptic seizures and that caldendrin might be involved in the pathophysiology of temporal lobe epilepsy.

Learning-facilitated long-term depression requires activation of the immediate early gene, c-fos, and is transcription dependent.

De novo gene transcription is a prerequisite for long-term information storage in the brain. Learning-facilitated synaptic plasticity describes the ability of hippocampal synapses to respond with long-lasting synaptic plasticity to the coupling of afferent stimulation with a spatial learning experience. Strikingly, long-term depression (LTD) is facilitated by context-dependent spatial learning experiences suggesting it may play a role in information storage to enable spatial memory. Here, we investigated if learning-facilitated LTD requires the transcription factor, c-Fos and is transcription-dependent. Novel spatial learning about object-place configurations coupled with weak low frequency afferent stimulation induced robust LTD in control animals that persisted for >24h and was associated with elevations in hippocampal expression of c-Fos. Intracerebral application of a c-fos antisense oligonucleotide prevented the facilitation of LTD by novel spatial learning, inhibited elevations of c-Fos triggered by LTD and impaired spatial learning. The expression of the transcription factor zif268 was unaffected by the c-fos antisense oligonucleotide. Learning-facilitated LTD was prevented by a transcription inhibitor. These data support that learning-facilitated LTD requires elevations in c-Fos and is transcription dependent. The observation that LTD shares key regulatory mechanisms with learning and memory processes argues strongly for a role for this form of synaptic plasticity in long-term information storage in the hippocampus.

Behavioral semantics of learning and crossmodal processing in auditory cortex: the semantic processor concept.

Two phenomena of auditory cortex activity have recently attracted attention, namely that the primary field can show different types of learning-related changes of sound representation and that during learning even this early auditory cortex is under strong multimodal influence. Based on neuronal recordings in animal auditory cortex during instrumental tasks, in this review we put forward the hypothesis that these two phenomena serve to derive the task-specific meaning of sounds by associative learning. To understand the implications of this tenet, it is helpful to realize how a behavioral meaning is usually derived for novel environmental sounds. For this purpose, associations with other sensory, e.g. visual, information are mandatory to develop a connection between a sound and its behaviorally relevant cause and/or the context of sound occurrence. This makes it plausible that in instrumental tasks various non-auditory sensory and procedural contingencies of sound generation become co-represented by neuronal firing in auditory cortex. Information related to reward or to avoidance of discomfort during task learning, that is essentially non-auditory, is also co-represented. The reinforcement influence points to the dopaminergic internal reward system, the local role of which for memory consolidation in auditory cortex is well-established. Thus, during a trial of task performance, the neuronal responses to the sounds are embedded in a sequence of representations of such non-auditory information. The embedded auditory responses show task-related modulations of auditory responses falling into types that correspond to three basic logical classifications that may be performed with a perceptual item, i.e. from simple detection to discrimination, and categorization. This hierarchy of classifications determine the semantic "same-different" relationships among sounds. Different cognitive classifications appear to be a consequence of learning task and lead to a recruitment of different excitatory and inhibitory mechanisms and to distinct spatiotemporal metrics of map activation to represent a sound. The described non-auditory firing and modulations of auditory responses suggest that auditory cortex, by collecting all necessary information, functions as a "semantic processor" deducing the task-specific meaning of sounds by learning.

Memory consolidation for the discrimination of frequency-modulated tones in mongolian gerbils is sensitive to protein-synthesis inhibitors applied to the auditory cortex.

Differential conditioning of Mongolian gerbils to linearly frequency-modulated tones (FM) has recently received experimental attention. In the study of the role of cerebral protein synthesis for FM discrimination memory, gerbils received post-training bilateral injections of anisomycin into the auditory cortex under light halothane anesthesia. Compared with saline-treated controls, anisomycin-treated gerbils showed a discrimination decrement during the subsequent three days of training. They markedly improved their performance within training sessions, but started each session at low levels. When repeatedly trained gerbils received post-session injections of anisomycin, discrimination performance during subsequent sessions was similar to the pre-injection performance, indicating that retention, retrieval, reconsolidation, and expression of the established reaction were not affected. However, the improvement of a partially established discrimination reaction was impaired after this treatment. Intracortical injections of emetine confirmed this finding. Neither drug affected FM discrimination learning when given several days before the initial training. Our results suggest that protein-synthesis inhibitors applied to the auditory cortex of gerbils during the post-acquisition phase interfered with learning and memory-related aspects of FM processing. The resulting deficit was evident for a number of post-injection training days. This effect was probably due to impaired consolidation, i.e., processes required for long-term stabilization or retrieval of the memory trace while leaving short-term memory intact.

Rapamycin-sensitive signalling in long-term consolidation of auditory cortex-dependent memory.

New memories initially persist in a labile state and require protein synthesis-dependent processes of consolidation for long-term manifestation. Using differential conditioning to linearly frequency-modulated tones (FMs) we have recently shown that post-training injections of protein synthesis inhibitors into the auditory cortex of Mongolian gerbils interfere with long-term memory for a number of days. Here, we have used rapamycin as a pharmacological tool to elucidate signalling pathways that control the synthesis of proteins required for persistent memory storage. In mammalian cells, inhibition of target of rapamycin (TOR)-mediated pathways was shown to block the translation of distinct classes of mRNAs. Bilateral infusions of rapamycin into the gerbil auditory cortex shortly after FM discrimination training did not impair the maintenance of the newly acquired memory trace for 24 h, but caused profound retention deficits at 48 h after injection. Control experiments showed that the amnesic action is rapamycin-dependent, confined to the context of memory formation, and suppressed by the antagonist FK506. These data indicate that, in the mammalian brain, activation of rapamycin-sensitive signalling pathways contributes to long-term consolidation of a cerebral cortex-dependent form of memory. Moreover, the finding that rapamycin-induced amnesia parallels only late effects of conventional protein synthesis inhibitors on FM discrimination memory implies that at least two different protein synthesis-dependent processes control memory formation. Both are activated during or shortly after learning. Whereas one process is required for the initial maintenance of memory for about one day the second one is involved in the regulation of its long-lasting persistence in conditioning to FMs.