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PubMed2004

Activation of p38 MAP kinase enhances sensitivity of muscle glucose transport to insulin.

Muscle contractile activity is followed by an increase in the sensitivity of glucose transport to insulin. There is evidence suggesting that activation of p38 MAP kinase (p38) is involved in the stimulation of glucose transport by insulin and contractions. Exercise results in an increase in p38 phosphorylation that lasts for hours. In this context, we tested the hypothesis that activation of p38 results in an increase in insulin sensitivity. Muscles were exposed to anisomycin for 30 min to activate p38. Anisomycin increased p38 phosphorylation approximately 2.5-fold and glucose transport activity 2- to 3-fold. Three hours after anisomycin treatment, by which time the acute effect on glucose transport had partially worn off, sensitivity of muscle glucose transport to 60 microU/ml insulin was markedly increased. Both the activation of p38 and the increase in insulin sensitivity induced by anisomycin were completely prevented by pretreatment of muscles with the p38 inhibitor SB-202190. However, in contrast to the finding with anisomycin, inhibition of p38 activation did not prevent the contraction-induced increase in insulin sensitivity. Thus our results show that activation of p38 is followed by an increase in insulin sensitivity of muscle glucose transport. However, activation of p38 is not necessary for induction of an increase in muscle insulin sensitivity by contractions. This finding provides evidence that contractions have an additional effect that makes p38 activation unnecessary for enhancement of insulin sensitivity by contractile activity.

Animals↗
PubMed1975

Growth hormone effects on sleep and wakefulness in the rat.

The sleep-wakefulness pattern and brain protein levels were determined in rats for 3 h following these conditions: administration of an inhibitor of protein synthesis (anisomycin), administration of several doses of rat growth hormone (GH) or thyrotropin, and administration of a combination of anisomycin + GH or thyrotropin. Anisomycin inhibited sleep and increased wakefulness, GH increased REM sleep, and thyrotropin produced no change. The combined administration of GH and anisomycin returned sleep to control levels. Anisomycin produced a time-dependent decrease in brain protein levels, GH produced changes in brain protein levels as compared to controls in the second hour only, and thyrotropin had no effect upon brain protein levels. The results are discussed in terms of the possible relationship between sleep and proteins.

Animals↗
PubMed2002

Comparison of the apoptosis-inducing abilities of various protein synthesis inhibitors in U937 cells.

We compared the abilities of ricin, diphtheria toxin, cycloheximide, and anisomycin to induce apoptosis, using human myeloid leukemia U937 cells at the concentration of each toxin at which almost complete protein synthesis inhibition was attained within 3 h. Among these toxins, anisomycin was found to be the most potent apoptosis inducer. After a 6-h exposure to anisomycin (1 microg/ml), nearly 95% of the cells had apoptotic nuclear morphological changes, while 53%, 30%, and 10% of the cells showed apoptotic changes after exposure to ricin (0.1 microg/ml), diphtheria toxin (10 microg/ml), and cycloheximide (10 microg/ml), respectively. Furthermore, a rapid increase in caspase-3-like activity was observed in anisomycin-treated cells. A similar increase in caspase-3-like activity was also observed in ricin-treated cells on a slower time schedule. However, only a slight increase in the protease activity was induced by diphtheria toxin or cycloheximide even after 6 h of incubation. Since both ricin and anisomycin are known to act on 28S ribosomal RNA, our results suggest that this action mechanism may be responsible for their potent apoptosis induction, and protein synthesis inhibition alone is not sufficient to induce apoptosis.

Anisomycin↗
PubMed1999

Sensitization of tumor cells to ribotoxic stress-induced apoptotic cell death: a new therapeutic strategy.

We describe a procedure that sensitizes chemotherapy-and tumor necrosis factor-resistant human tumor cell populations in vitro and in nude mouse transplants to the immediate triggering of high rates of cell death by anisomycin, an agent causing activation of stress-activated protein kinases [SAPKs, as defined by P. Cohen (Trends Cell Biol., 7: 353-361, 1997)] including p38/RK and c-jun NH2-terminal kinase homologues, following its binding to ribosomal 28S RNA (M. S. Iordanov et al, Mol. Cell. Biol., 17: 3373-3381, 1997). Sensitization is effected by successive application of an inhibitor of histone deacetylation (trichostatin A, butyrate) and of flavopiridol, known as an inhibitor of cyclin dependent kinases and evaluated presently in clinical trials. Effective concentrations of anisomycin, flavopiridol, and trichostatin A are in the submicromolar range. Tumor cell death can be prevented by epidermal growth factor (EGF), if added before flavopiridol or after anisomycin but not if applied between the additions of these agents, suggesting that flavopiridol interrupts an EGF-activated survival pathway and that anisomycin, besides triggering cell death, guards this pathway against the interference by flavopiridol. In contrast to EGF, dibutyryl-cAMP exerts protection that is flavopiridol-insensitive. For triggering cell death, anisomycin cannot be replaced by DNA- or mitotic spindle-targeted drugs in this system. The present findings, that a combination of transcriptional and signal transduction-targeted modulators sensitizes tumor cells synergetically to stress-mediated triggering of cell death and that ribotoxic stress is more efficient in this respect than genotoxic or spindle-targeted stress, bear important implications for the therapeutic exploitation of cellular stress responses. The stepwise sensitization and triggering of cell death in the present system allow separate analysis and manipulation of processes contributing to cellular death susceptibility and of the mechanism responsible for triggering cell death, thus providing the operational basis for further development of this therapeutic approach.

Animals↗
PubMed2005

Novel signaling stimulated by arsenite increases cholesterol metabolism through increases in unphosphorylated steroidogenic acute regulatory (StAR) protein.

Cholesterol metabolism to pregnenolone is dependent on the steroidogenic acute regulatory protein (StAR), which activates mitochondrial transfer of cholesterol to cytochrome CYP450scc. In mouse Y-1 adrenal cells and testis MA10 cells stimulation by 8-Bromo-cAMP (Br-cAMP) is augmented by a novel signaling initiated by low concentrations of arsenite (3-20 microM) and anisomycin (0.2 microM), a more selective stress agent. Each elevated StAR mRNA (three-fold after 6 h treatment) even with simultaneous stimulation by Br-cAMP. Arsenite produced parallel increases in StAR protein expression and cholesterol metabolism, but not for P450scc-mediated metabolism of 20alpha-hydroxycholesterol. Although arsenite and anisomycin each stimulated the phosphorylation of p38, the p38 inhibitor SB203580 (SB) produced additive increases in StAR expression. Cholesterol metabolism increased in parallel but without the increased StAR protein phosphorylation produced by Br-cAMP. Arsenite and anisomycin each elevated StAR mRNA but preferentially increased the 3.5 kb form relative to the 1.6 kb form. Arsenite and anisomycin each enhanced the stability of the more labile 3.5 kb mRNA which contains AU-rich elements that control mRNA stability. Although there were increases in both forms of StAR mRNA, arsenite did not stimulate a StAR promoter-reporter that exhibited a typical three-fold response to Br-cAMP. Arsenite and anisomycin may therefore activate a novel SB-independent MAP kinase which in part increases StAR expression through stabilizing the 3.5 kb mRNA but which may also activate a mechanism that by-passes transcription factors detected by the reporter. SB stimulation, which was completely blocked by a MEK inhibitor, was also selective towards the 3.5 kb StAR mRNA suggesting a second pathway for mRNA stabilization. These activations contrast with inhibition of StAR expression by arsenite at higher concentrations or longer incubation times.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Regulation of GLUT5, GLUT2 and intestinal brush-border fructose absorption by the extracellular signal-regulated kinase, p38 mitogen-activated kinase and phosphatidylinositol 3-kinase intracellular signalling pathways: implications for adaptation to diabetes.

We have investigated the role of the extracellular signal-regulated kinase (ERK), p38 and phosphatidylinositol 3-kinase (PI 3-kinase) pathways in the regulation of intestinal fructose transport. Different combinations of anisomycin, PD98059 and wortmannin had very different effects on fructose transport in perfused isolated loops of rat jejunum. Transport was stimulated maximally by anisomycin (2 microM) and blocked by SB203580 (20 microM), confirming involvement of the p38 pathway. PD98059 (50 microM) alone had little effect on fructose transport. However, it had a dramatic effect on stimulation by anisomycin, diminishing the K(a) 50-fold from 1 microM to 20 nM to show that the ERK pathway restrains the p38 pathway. The K(a) for diabetic jejunum was 30 nM and PD98059 had no effect. Transport in the presence of anisomycin was 3.4-fold that for anisomycin plus PD98059 plus wortmannin. Transport was mediated by both GLUT5 and GLUT2. In general, GLUT2 levels increased up to 4-fold within minutes and with only minimal changes in GLUT5 or SGLT1 levels, demonstrating that GLUT2 trafficks by a rapid trafficking pathway distinct from that of GLUT5 and SGLT1. GLUT2 intrinsic activity was regulated over a 9-fold range. It is concluded that there is extensive cross-talk between the ERK, p38 and PI 3-kinase pathways in their control of brush-border fructose transport by modulation of both the levels and intrinsic activities of GLUT5 and GLUT2. The potential of the intracellular signalling pathways to regulate short-term nutrient transport during the assimilation of a meal and longer-term adaptation to diabetes and hyperglycaemia is discussed.

Animals↗
PubMed1992

Failure of a protein synthesis inhibitor to modify glutamate receptor-mediated neurotoxicity in vivo.

The delayed neuronal death (DND) resulting from brief forebrain ischemia has recently been reported to be markedly attenuated by parenteral administration of the reversible protein synthesis inhibitor, anisomycin. Previous work suggests that ischemia-induced DND is mediated by glutamate acting at one or more glutamate receptors, since glutamate receptor antagonists have been reported to reduce ischemia-induced DND. Consequently, we tested whether anisomycin could modify DND induced by direct intracerebral administration of the excitotoxins, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxasole (AMPA) or kainic acid. Anisomycin, administered parenterally, in multiple doses did not alter DND induced by any of these excitotoxins, nor did combined parenteral and direct intracerebral injection of anisomycin protect against DND induced by AMPA. Thus, neurotoxicity induced by direct intracerebral administration of NMDA, AMPA or kainic acid does not appear to require de novo protein synthesis, and, therefore, is not likely to be mediated by the expression of a programmed cell death cascade.

Animals↗
PubMed2006

Protein synthesis inhibition in the basolateral amygdala following retrieval does not impair expression of morphine-associated conditioned place preference.

Conditioned place preference is an animal model used to evaluate the affective properties of natural rewards and drugs of abuse. This animal model is a kind of classical conditioning that depends on learning and memory. The basolateral amygdala (BLA) plays an important role in the consolidation and extinction of memory for this task. However, there is a lack of evidence demonstrating protein synthesis dependent reconsolidation following retrieval in conditioned animals. In other words, is it possible to observe morphine-associated place preference if recall of this preference is disrupted? Accordingly, we investigated this hypothesis by BLA infusion of protein synthesis inhibitor, anisomycin, immediately after retrieval (test) in conditioned place preference paradigm. In the first experiment, the conditioned animals were exposed to the two sides of the apparatus for 15 min in a drug-free state during retrieval. In the second experiment, the animals received an injection of morphine (7.5 mg/kg, i.p.) and immediately after, they were exposed to the two sides of the apparatus for 15 min. Finally in the third experiment, after habituation and training in the conditioned place preference task, the animals received an injection of the unconditioned stimulus (morphine, i.p.; 7.5 mg/kg) followed by confinement for 10 min in the morphine-paired compartment (conditioned stimulus) during memory retrieval. For the three experiments the animals were subsequently exposed in a free-drug state to the two sides of the apparatus for the retest. Our results show that the protein synthesis inhibition in all of these experimental designs had no effect on conditioned place preference memory under conditions that would initiate reconsolidation, suggesting that if reconsolidation of a conditioned place preference task exists it is not mediated by protein synthesis in basolateral amygdala. The effect of anisomycin on consolidation of contextual fear conditioning was also investigated as a positive control to assure that the negative results were not due to methodological problems. Using the same dose of anisomycin (62.5 microg/1 microl) in morphine-associated place preference procedures, we have found that this anisomycin dose blocks the consolidation of contextual fear memory, ruling out the possibility that these negative results can be attributed to methodological problem of some sort.

Amygdala↗
PubMed2005

Roles of mTOR and JNK in serine phosphorylation, translocation, and degradation of IRS-1.

In 3T3-L1 adipocytes, insulin or anisomycin stimulated phosphorylation of IRS-1 at Ser(307) and Ser(636/639), both of which were partially reduced by the mTOR inhibitor, rapamycin, or the JNK inhibitor, SP600125, and were further inhibited by a combination of them. Interestingly, anisomycin-induced p70(S6K) phosphorylation was reduced by SP600125, while insulin-induced p70(S6K) phosphorylation was not. Furthermore, unlike insulin, anisomycin failed to elicit translocation or degradation of IRS-1. These results indicate that mTOR and JNK play roles in phosphorylating IRS-1 serine residues, and that insulin and anisomycin are different in terms of the relationship of activation between mTOR and JNK, and the effects on IRS-1 localization and stability.

3T3-L1 Cells↗
PubMed2002

Activation of the mitochondrial caspase cascade in the absence of protein synthesis does not require c-Jun N-terminal kinase.

Prolonged activation of the c-Jun N-terminal kinase (JNK) has been suggested as a signal for apoptosis in response to a wide variety of stimuli. Using three cytocidal RNA or protein synthesis inhibitors (actinomycin D, anisomycin, and emetine), the potential role of JNK in activation of the mitochondrial apoptotic cascade was investigated in A549-S cells. Protein synthesis inhibition per se was not the cause of cell death as cycloheximide induced only growth arrest. All the cytocidal inhibitors induced cytochrome c release and caspases 9 activation within hours, but only anisomycin caused persistent JNK activation. Although, the JNK inhibitor, SP600125, inhibited JNK-dependent anisomycin-induced c-Jun phosphorylation, it was ineffective in preventing anisomycin-induced caspase activation and cell death. Thus, all three lethal macromolecule synthesis inhibitors can activate the mitochondrial apoptotic machinery independent of JNK activation, demonstrating that the mitochondrial apoptotic pathway can be activated independently of the JNK pathway in the absence of protein synthesis.

Anisomycin↗
PubMed2003

Ribotoxic stress activates p38 and JNK kinases and modulates the antigen-presenting activity of dendritic cells.

Initiation of adaptive immunity requires activation of dendritic cells (DC) by "danger" signals. This study examines the functional consequences of activating a cellular stress response in human DC. Anisomycin, a potent inducer of this "stress" response, selectively activates p38 kinase in DC at low concentrations, and both p38 kinases and JNKs at higher concentrations. Activation of p38, was accompanied by an increase in the potency of dendritic cells to activate T cells. In contrast to LPS, anisomycin had no effect on the expression of several DC activation markers. Anisomycin synergised with LPS in driving release of IL-12 and TNF-alpha. Anisomycin also enhanced the formation of clusters between DC and T cells. Enhanced cytokine release and clustering were both inhibited by the selective p38 alpha and p38 beta inhibitor SB203580. This study demonstrates that the cellular stress response, mediated via p38 kinases, plays an important role in the regulation of several aspects of DC function.

Anisomycin↗
PubMed1997

Protein synthesis inhibitors delay transneuronal death in the piriform cortex of young adult rats.

It has been demonstrated that apoptotic cell death is an active process that is dependent on RNA and protein synthesis. The question remains as to whether neuronal death in adult, mammalian brains can also be demonstrated in vivo to be dependent on protein synthesis. To address this question we have analysed transneuronal death in the piriform (olfactory) cortex. Following unilateral olfactory bulb ablation in young adult rats, layer IIa of the piriform cortex undergoes rapid degeneration, that commences 12 h after ablation and that is almost complete at 48 h. In order to block protein synthesis, three to six subcutaneous injections of the short acting protein synthesis inhibitor anisomycin, were given at 2 h intervals beginning just before the ablation of the olfactory bulb. In other cases a single injection of the long acting protein synthesis inhibitor emetine were made intracerebrally just before or after olfactory bulb ablation. The number of dying cells was then counted in sections through the rostrocaudal extent of the piriform cortex. Both anisomycin and emetine injections markedly reduced the number of pyknotic cells in layer IIa of the piriform cortex after olfactory bulb ablation. The effect of anisomycin was dose-dependent, near lethal doses leading to an almost complete absence of cell death (six injections of 100 mg/kg). As the doses of anisomycin were reduced, more dying cells were observed. Emetine was only effective at near lethal doses (10 mg/kg) and showed a greater capacity to reduce the levels of cell death when injected into structures near the piriform cortex (e.g., accumbens nucleus) than when injected into more distant structures. To further confirm that the cell death observed was due to apoptosis, we analysed sections by tunel staining to demonstrate DNA fragmentation. We found that tunel-positive cells were also always pyknotic, one of the landmarks of apoptosis. The appearance of pyknotic cells labelled by the tunel method demonstrated that the dying cells in the piriform cortex did indeed undergo apoptosis.

Animals↗
PubMed2003

Time windows for effects of protein synthesis inhibitors on Pavlovian conditioning in Hermissenda: behavioral aspects.

Inhibitors of protein and mRNA syntheses inhibit long-term memory (LTM), but we lack information about the time windows during which those inhibitors are effective. Anisomycin (a protein synthesis translation inhibitor) was given to Hermissenda crassicornis which had received sufficient Pavlovian conditioning to produce LTM. When tested after 90 min, LTM recall was blocked when anisomycin was administered until 13 min after conditioning and from 65 to 75 min. There was good recall from 16 to 60 min. When tested at 240 min, two periods of sensitivity to anisomycin were revealed: one finished before 20 min, while the other lasted from 60 to 220 min.A brief study of the transcription inhibitor actinomycin-D found its action similar to that of anisomycin, except not inhibiting recall from about 160 to 220 min.

Animals↗
PubMed2001

Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells.

Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP-70 kinase and the TCR/CD3 gamma-chain mutants, but did not protect the src p56(lck)-deficient cells. Similarly, MHC-I ligation did not protect anisomycin-treated src p56(lck)-deficient cells against apoptosis. These data suggest that MHC-I-induced inhibition of apoptosis depends on intact src p56(lck) activity, but not on major secondary messenger molecules associated with TCR signaling. Overall the results support the idea that signal transduction by MHC-I molecules is involved in homeostatic processes of importance for T-cell survival and death.

Anisomycin↗
PubMed1995

The activation of distinct mitogen-activated protein kinase cascades is required for the stimulation of 2-deoxyglucose uptake by interleukin-1 and insulin-like growth factor-1 in KB cells.

The uptake of 2-deoxyglucose into KB cells was stimulated about 2-fold by interleukin-1 (IL1), anisomycin or insulin-like growth factor-1 (IGF1). Stimulation by IL1 and anisomycin was prevented by SB 203580, a specific inhibitor of the mitogen-activated protein (MAP) kinase homologue termed 're-activating kinase' [RK; also known as p38, p40 and CSBP (cytokine synthesis anti-inflammatory-drug-binding protein)], but was unaffected by PD 98059, a specific inhibitor of the activation of the classical MAP kinase pathway. In contrast, the stimulation of 2-deoxyglucose uptake by IGF1 was blocked by PD 98059 and unaffected by SB 203580. Consistent with these observations, IL1 and anisomycin were potent activators of MAP kinase-activated protein (MAPKAP) kinase-2, a physiological substrate of RK, whereas IGF1 was only a very weak activator of MAPKAP kinase-2. Conversely, IGF1 was a stronger activator of p42 MAP kinase than IL1 or anisomycin. These results imply that the activation of distinct MAP kinase pathways is required for the stimulation of glucose transport by IL1/anisomycin and IGF1 in KB cells, and suggest that the combined use of SB 203580 and PD 98059 is a powerful new approach to explore the roles of different MAP kinase cascades in cell regulation.

Anisomycin↗
PubMed1999

Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis.

The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g. anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis.

Anisomycin↗
PubMed2003

CD40 signaling is impaired in L. major-infected macrophages and is rescued by a p38MAPK activator establishing a host-protective memory T cell response.

Leishmania, a protozoan parasite, lives and multiplies as amastigote within macrophages. It is proposed that the macrophage expressed CD40 interacts with CD40 ligand on T cells to induce IFN-gamma, a Th1-type cytokine that restricts the amastigote growth. Here, we demonstrate that CD40 cross-linking early after infection resulted in inducible nitric oxide synthetase type-2 (iNOS2) induction and iNOS2-dependent amastigote elimination. Although CD40 expression remained unaltered on L. major-infected macrophages, delay in the treatment of macrophages or of mice with anti-CD40 antibody resulted in significant reduction in iNOS2 expression and leishmanicidal function suggesting impaired CD40 signaling in Leishmania infection. The inhibition of CD40-induced iNOS2 expression by SB203580, a p38-mitogen activated protein kinase (p38MAPK)-specific inhibitor, and the reversal of the inhibition by anisomycin, a p38MAPK activator, suggested a crucial role of p38MAPK in CD40 signaling. Indeed, the CD40-induced p38MAPK phosphorylation, iNOS2 expression and anti-leishmanial function were impaired in Leishmania-infected macrophages but were restored by anisomycin. Anisomycin's effects were reversed by SB203580 emphasizing the role of p38MAPK in CD40-induced iNOS2-dependent leishmanicidal function. Anisomycin administration in L. major-infected BALB/c mice resulted in significant reduction in the parasite load and established a host-protective Th1-type memory response. Also implicated in these findings is a scientific rationale to define novel anti-parasite drug targets and to bypass the problem of drug resistance.

Animals↗
PubMed2003

Protein synthesis in the amygdala, but not the auditory thalamus, is required for consolidation of Pavlovian fear conditioning in rats.

The amygdala is an essential neural substrate for Pavlovian fear conditioning. Nevertheless, long-term synaptic plasticity in amygdaloid afferents, such as the auditory thalamus, may contribute to the formation of fear memories. We therefore compared the influence of protein synthesis inhibition in the amygdala and the auditory thalamus on the consolidation of Pavlovian fear conditioning in Long-Evans rats. Rats received three tone-footshock trials in a novel conditioning chamber. Immediately after fear conditioning, rats were infused intra-cranially with the protein synthesis inhibitor, anisomycin. Conditional fear to the tone and conditioning context was assessed by measuring freezing behaviour in separate retention tests conducted at least 24 h following conditioning. Post-training infusion of anisomycin into the amygdala impaired conditional freezing to both the auditory and contextual stimuli associated with footshock. In contrast, intra-thalamic infusions of anisomycin or a broad-spectrum protein kinase inhibitor [1-(5'-isoquinolinesulphonyl)-2-methylpiperazine, H7] did not affect conditional freezing during the retention tests. Pre-training intra-thalamic infusion of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV), which blocks synaptic transmission in the auditory thalamus, produced a selective deficit in the acquisition of auditory fear conditioning. Autoradiographic assays of cerebral [14C]-leucine incorporation revealed similar levels of protein synthesis inhibition in the amygdala and thalamus following intra-cranial anisomycin infusions. These results reveal that the establishment of long-term fear memories requires protein synthesis in the amygdala, but not the thalamus, after auditory fear conditioning. Forms of synaptic plasticity that depend on protein synthesis, such as long-term potentiation, are likely candidates for the encoding and long-term storage of fear memories in the amygdala.

Amygdala↗