Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway.

Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway.

Enhancement of death receptor 4-mediated apoptosis and cytotoxicity in renal cell carcinoma cells by anisomycin.

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies, and an effective therapy is lacking for metastatic RCC. Anisomycin is known to inhibit protein synthesis and induce ribotoxic stress. The aim of this study was to explore whether anisomycin enhances the cytotoxic effects of mapatumumab, a human agonistic monoclonal antibody specific for death receptor 4 (DR4), in human RCC cells. We examined the cytotoxicity of anisomycin alone and in combination with mapatumumab in human RCC cell lines and primary RCC cell cultures. RCC cells treated with anisomycin showed cytotoxicity in a dose-dependent manner. Anisomyin in combination with mapatumumab showed a synergistic effect not only in two human RCC cell lines but also in five primary RCC cell cultures. The synergy between anisomycin and mapatumumab for cytotoxicity was also observed for apoptosis. Interestingly, anisomycin significantly increased DR4 expression at both the mRNA and the protein level. Furthermore, the combination-induced cytotoxicity was significantly suppressed by a human recombinant DR4:Fc chimeric protein. The combination of anisomycin and mapatumumab also enhanced the activity of caspases 8 and 3, the downstream molecules of death receptors. These findings indicate that anisomycin sensitizes RCC cells to DR4-mediated apoptosis through the induction of DR4, suggesting that combinational treatment with anisomycin and mapatumumab might represent a novel therapeutic strategy for the treatment of RCC.

Anisomycin-induced GATA-6 degradation accompanying a decrease of proliferation of colorectal cancer cell.

Transcription factor GATA-6 plays a key role in normal cell differentiation of the mesoderm and endoderm. On the other hand, GATA-6 is abnormally overexpressed in many clinical gastrointestinal cancer tissue samples, and accelerates cell proliferation or an anti-apoptotic response in cancerous tissues. We previously showed that activation of the JNK signaling cascade causes proteolysis of GATA-6. In this study, we demonstrated that anisomycin, a JNK activator, stimulates nuclear export of GATA-6 in a colorectal cancer cell line, DLD-1. Concomitantly, anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest in a plate culture. However, it did not induce apoptosis under growth arrest conditions. Furthermore, the growth of DLD-1 cells in a spheroid culture was suppressed by anisomycin. Although 5-FU showed only a slight inhibitory effect on 3D spheroid cultures, the same concentration of 5-FU together with a low concentration of anisomycin exhibited strong growth inhibition. These results suggest that the induction of GATA-6 dysfunction may be more effective for chemotherapy for colorectal cancer, although the mechanism underlying the synergistic effect of 5-FU and anisomycin remains unknown.

Intrahippocampal infusion of spermidine improves memory persistence: Involvement of protein kinase A.

Spermidine (SPD) is an endogenous aliphatic amine that modulates GluN2B-containing NMDA receptors and improves memory. Recent evidence suggests that systemic SPD improves the persistence of the long term memory of fear. However, the role of hippocampal polyamines and its binding sites in the persistence of fear memory is to be determined, as well as its putative underlying mechanisms. This study investigated whether the intrahippocampal (i.h.) infusion of spermidine or arcaine, modulators of polyamine binding site at GluN2B-containing NMDA receptors, alters the persistence of the memory of contextual fear conditioning task in rats. We also investigated whether protein synthesis and cAMP dependent protein kinase (PKA) play a role in SPD-induced improvement of the fear memory persistence. While 12h post-training infusion of spermidine facilitated, arcaine and the inhibitor of protein synthesis (anisomycin) impaired the memory of fear assessed 7days after training. The infusion of arcaine, anisomycin or a selective PKA inhibitor (H-89), at doses that have no effect on memory per se, prevented the SPD-induced improvement of memory persistence. H-89 prevented the stimulatory effect of SPD on phospho-PKA/total-PKA ratio. These results suggests that the improvement of fear memory persistence induced by spermidine involves GluN2B-containing NMDA receptors, PKA pathway and protein synthesis in rats.

Long non-coding RNA BACE1-AS is a novel target for anisomycin-mediated suppression of ovarian cancer stem cell proliferation and invasion.

Human ovarian cancer stem cells (OCSCs) are one of the main factors affecting ovarian cancer cell metastasis, recurrence, prognosis and tolerance to chemotherapy drugs. However, the mechanisms of OCSC proliferation and invasion are not clear. Recent studies suggest that anisomycin can inhibit the proliferative and invasive ability of various tumor cells by increasing the production of the toxic amyloid ß (Aß1-42) peptides from the amyloid precursor protein (APP). We explored whether anisomycin could also suppress human OCSC proliferation and invasion. The CD44+/CD117+ OCSCs were enriched from human clinical ovarian tumor tissues. OCSCs treated with anisomycin showed reduced proliferation compared to controls. Moreover, anisomycin significantly suppressed the invasive capacity of OCSCs in vitro, as indicated by cell migration assays. The mRNA expression levels of long non-coding RNA (lncRNA) ß-site APP cleaving enzyme 1 antisense strand (BACE1-AS) were significantly increased in anisomycin-treated OCSCs compared to controls. In addition, mRNA and protein levels of BACE1 and Aß1-42 were increased in anisomycin-treated OCSCs compared to controls. We confirmed that anisomycin suppressed the growth of xenograft tumors formed by OCSCs in vivo. Finally, when expression of lncRNA BACE1-AS was silenced using siRNA, BACE1 expression was downregulated and the antiproliferative and anti-invasive effects of anisomycin were reduced. Overall, we identified lncRNA BACE1-AS as a novel target for anisomycin. Elevation of lncRNA BACE1-AS expression is a potential mechanism for suppressing human OCSC proliferation and invasion.

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty.

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2-related inhibitory peptide; or the blocker of L-voltage-dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by ß-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.

Low-dose anisomycin sensitizes glucocorticoid-resistant T-acute lymphoblastic leukemia CEM-C1 cells to dexamethasone-induced apoptosis through activation of glucocorticoid receptor and p38-MAPK/JNK.

Glucocorticoid (GC) resistance in children with acute lymphoblastic leukemia (ALL) usually resulted in the failure of treatment. Exploring new agents to overcome GC resistance is important. Here we reported for the first time that low-dose anisomycin has the potential to sensitize GC-resistant T-ALL CEM-C1 cells to dexamethasone (DEX). Compared with the use of DEX or low-dose anisomycin alone, co-treatment with them resulted in a significant increase of growth inhibition, apoptosis and cell cycle arrest in CEM-C1 cells through induction of activated caspase-3 and up-regulation of Bim, p21and p27, and down-regulation of Mcl-1, Bcl-2, c-myc, cyclin A and cyclin D1. Furthermore, co-treatment remarkably activated glucocorticoid receptor (GR), p38-MAPK and JNK, and all of them were canceled only by the GR inhibitor RU486, indicating GR might be an at the upstream of GR-p38-MAPK/JNK pathway. We conclude that low-dose anisomycin sensitizes GC-resistant CEM-C1 cells to DEX and this effect is mediated, at least in part, by activation of the GR-p38-MAPK/JNK signaling pathway.

Anisomycin suppresses Jurkat T cell growth by the cell cycle-regulating proteins.

BACKGROUND: Recent studies have shown that anisomycin significantly inhibits mammalian cell proliferation, but its mechanism remains unclear. In this study, Jurkat T cells were used to first explore a relationship between effect of anisomycin on them and alteration of cell cycle-regulating proteins. METHODS: Cell colony formation, CCK-8 assay, flow cytometry, RT-PCR and western blot were employed to evaluate correlation of ten cell cycle-regulating proteins with suppression of the cell proliferation and arrest of the cell cycle by anisomycin. RESULTS: Our data showed that anisomycin inhibited the colony-formation and proliferation of Jurkat T cells in a dose-dependent manner, and arrested the cells into S and G2/M phases with the production of sub-diploid cells. The levels of P21, P-P27 and P53/P-P53 reached their peaks 4 h after anisomycin treatment, presenting a positive correlation with anisomycin concentration, and P16, P-P21, P27, P57, P73/P-P73 and P-Rb changed little with the prolonged exposure time or increased concentrations of anisomycin. But the level of Rb protein was increased at 24 h after the treatment of anisomycin. The expression of an inverted CCAAT box binding protein (ICBP90) in Jurkat T cells came to decrease 12 h after the treatment of anisomycin, presenting a negative correlation with anisomycin concentration. Subsequently, the expression of P-CDK2 was also decreased at 24 h, presenting an obviously negative correlation, whereas P-CDK1 showed no differences among the differently treated Jurkat T cells. Furthermore, the level of P21 and P53 mRNA was increased with the enhanced concentrations of anisomycin. CONCLUSION: The results indicate that anisomycin may activate the P53/P21/P27 signaling to decrease the expression of ICBP90, inhibit expression of P-CDK2 to block the cells into S and G2/M phases, and finally result in proliferation inhibition of Jurkat T cells.

Synergistic induction of TRAIL-mediated apoptosis by anisomycin in human hepatoma cells via the BH3-only protein Bid and c-Jun/AP-1 signaling pathway.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super-family, and it has been shown that many human cancer cell lines are refractory to TRAIL-induced cell death. However, the molecular mechanisms underlying resistance are unclear. In the present study, we show that TRAIL-resistance is reversed in human hepatoma cells by anisomycin, which is known to inhibit protein synthesis and induce ribotoxic stress. Synergistic induction of apoptosis in cells treated with anisomycin plus TRAIL was associated with activation of caspases and cleavage of Bid, a pro-apoptotic BH3-only protein. Silencing of Bid expression by small interfering RNA (siRNA) significantly attenuated the loss of mitochondrial membrane potential (MMP, Δψm) and significantly increased induction of apoptosis in cells treated with anisomycin and TRAIL, confirming that Bid cleavage is required for the response. In addition, c-Jun/AP-1 was rapidly activated upon stimulation with anisomycin; however, the knockdown of c-Jun/AP-1 expression by c-Jun siRNA markedly reduced anisomycin plus TRAIL-induced loss of MMP and apoptosis. Taken together, the findings show that anisomycin sensitizes TRAIL-mediated hepatoma cell apoptosis via the mitochondria-associated pathway, involving the cleavage of Bid and activation of the c-Jun/AP-1 pathway, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.

[Protein phosphatase MKP-1 participates in c-fos gene derepression under the action of stress factors on fibroblasts transformed with E1A and cHA-ras oncogenes].

Immediate-early response gene c-fos expression is repressed and not activated after serum stimulation of serum-starved fibroblasts transformed with E1A and cHa-ras oncogenes. We have previously shown that such stress factors as an anisomycin are able to activate c-fos gene transcription in E1A + cHa-ras transformants, wherein MEK/ERK signal pathway plays a major role in the activation. In the present paper, we investigated the role of MKP-1-dependent regulation of c-fos gene by dephosphorylation of ERK kinases. It has been shown that MKP-1 gene transcription in E1A + ras transformants is activated by anisomycin for a maximum of 1 h, and then a reduction in the level of transcription occurs. Use of inhibitors of MAP-kinase has revealed that MKP-1 gene transcription depends on MEK/ERK and JNK kinase cascades, but not om p38 cascade. The anisomycin-induced c-fos gene transcription intensified after transfection of siRNA MKP-1 into the cells. Thus, protein phosphatase MKP-1 carries a negative regulation of c-fos gene transcription by dephosphorylation of ERK kinases that are a key signal component under the action of such stress reagent as anisomycin on the E1A + ras-transformed cells.

Disruption of Nox2 and TNFRp55/p75 eliminates cardioprotection induced by anisomycin.

Transient activation of p38 through anisomycin is demonstrated to precondition the heart against myocardial injury. However, it remains unknown whether specific TNF-α receptor (TNFR) p55/p75 and Nox2, a subunit of NADPH oxidase, are involved in this event. We sought to investigate whether the genetic disruption of TNFRp55/p75 and Nox2 eliminated cardioprotection elicited by anisomycin and whether p38-dependent activation of Nox2 stimulated TNFR to ultimately achieve protective effects. Adult wild-type and TNFR p55/p75(-/-) and Nox2(-/-) mice received intraperitoneal injections of anisomycin (0.1 mg/kg), a potent activator of p38. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after 24 h. Left ventricular function was measured, and infarct size was determined. Myocardial TNF-α protein, Nox2, and superoxides releases were detected. Gel kinase assay was employed to detect the effect of p38 on Nox2 phosphorylation. Activation of p38 through anisomycin produces marked improvements in left ventricular functional recovery, and the reduction of myocardial infarction, which were abrogated by disruption of Nox2 and TNFR p55/p75. Disruption of Nox2 and TNFR p55/p75 abolished the effect of anisomycin-induced reduction of infarct size. Anisomycin induced the production of TNF-α, which was abrogated in Nox2(-/-) mice and by treatment with SB203580, but not by disruption of p55/p75. Anisomycin treatment resulted in an increase in Nox2 protein and the phosphorylation of Nox2, which was blocked by inhibition of p38. Taken together, these results indicate that stimulation of the Nox2 and TNFR p55/p75 pathway is a novel approach to anisomycin-induced cardioprotection.

Surprising similarity between mechanisms mediating low (1 Hz)-and high (100 Hz)-induced long-lasting synaptic potentiation in CA1 of the intact hippocampus.

It is generally assumed that long lasting synaptic potentiation (long-term potentiation, LTP) and depression (long-term depression, LTD) result from distinct patterns of afferent activity, with high and low frequency activity favouring LTP and LTD, respectively. However, a novel form of N-methyl-d-aspartate (NMDA) receptor-dependent synaptic potentiation in the hippocampal CA1 area in vivo induced by low frequency afferent stimulation has recently been demonstrated. Here, we further characterize the mechanisms mediating this low frequency stimulation (LFS)-induced LTP in area CA1 of intact, urethane-anesthetized preparations. Consistent with previous reports, alternating, low frequency (1 Hz) stimulation of CA1 afferents originating in the contralateral CA3 area and the medial septum resulted in gradually developing, long lasting (>2 h) LTP of field excitatory postsynaptic potentials (fEPSPs) recorded in CA1. Local application of the protein synthesis inhibitor anisomycin in CA1 blocked LFS-induced LTP, as did application of H89, an inhibitor of protein kinase A. Given the apparent overlap in molecular mechanisms mediating LFS-LTP and "classic" high-frequency stimulation (HFS)-induced LTP in CA1, we examined the relation between these forms of LTP by means of occlusion experiments. LFS, delivered to synapses saturated by initial HFS, resulted in a gradually developing LTD, rather than the normally seen LTP. Conversely, initial induction of LFS-LTP reduced the amount of subsequent HFS-LTP. Together, these experiments reveal a surprising similarity in the molecular mechanisms (dependence on NMDA receptors, protein kinase A, protein synthesis) mediating LTP induced by highly distinct (1 vs. 100 Hz) induction protocols. Importantly, these findings further challenge the "high-frequency-LTP, low-frequency LTD" dogma by demonstrating that this dichotomy does not account for all types of plasticity phenomena at central synapses.

Proteasome inhibition enhances the induction and impairs the maintenance of late-phase long-term potentiation.

Protein degradation by the ubiquitin-proteasome pathway plays important roles in synaptic plasticity, but the molecular mechanisms by which proteolysis regulates synaptic strength are not well understood. We investigated the role of the proteasome in hippocampal late-phase long-term potentiation (L-LTP), a model for enduring synaptic plasticity. We show here that inhibition of the proteasome enhances the induction of L-LTP, but inhibits its maintenance. Proteasome inhibitor-mediated enhancement of the early part of L-LTP requires activation of NMDA receptors and the cAMP-dependent protein kinase. Augmentation of L-LTP induction by proteasome inhibition is blocked by a protein synthesis inhibitor anisomycin and is sensitive to the drug rapamycin. Our findings indicate that proteasome inhibition increases the induction of L-LTP by stabilizing locally translated proteins in dendrites. In addition, our data show that inhibition of the proteasome blocks transcription of brain-derived neurotrophic factor (BDNF), which is a cAMP-responsive element-binding protein (CREB)-inducible gene. Furthermore, our results demonstrate that the proteasome inhibitors block degradation of ATF4, a CREB repressor. Thus, proteasome inhibition appears to hinder CREB-mediated transcription. Our results indicate that blockade of proteasome activity obstructs the maintenance of L-LTP by interfering with transcription as well as translation required to sustain L-LTP. Thus, proteasome-mediated proteolysis has different roles during the induction and the maintenance of L-LTP.

Time course and efficiency of protein synthesis inhibition following intracerebral and systemic anisomycin treatment.

Since its discovery in the 1960s, anisomycin has been used for studying the impact of protein synthesis for manifold cerebral processes such as long-term plastic changes after learning. The common limitation of nearly all pharmacological experiments, including anisomycin treatment, is to precisely verify the affected brain regions. Here we illustrate anisomycin effects on protein synthesis in distinct brain regions of mice (C57BL/6JOlaHsd), revealing differences between three modes of anisomycin application (subcutaneous, s.c.; intraperitoneal, i.p.; local microinfusions into the hippocampus). Our method is based on inhibition of the incorporation of the radioactively-labelled amino acids [(35)S]-Methionine/Cysteine into newly synthesised proteins. Washing the brain slices before autoradiography removes pools of amino acids, which have not been incorporated into newly synthesised proteins, thus, illustrating pure protein synthesis. By comparing different routes of systemic anisomycin application (i.p. versus s.c.; 150 mg/kg) it became evident that the effect of i.p. injection of anisomycin is fully reversed after 6 h, whereas s.c. injection is inhibiting protein synthesis in the hippocampus even 9 h after application. Local microinfusions of anisomycin into the hippocampus were shown to have long-lasting effects as well, which reversed as late as 9 h after injection. The diffusion of anisomycin was maximal at 3 h after injection and more precisely confined to the intended area using a lower dose (20 microg/site) instead of the commonly used dose of 62.5 microg/site. The broad time window of anisomycin action, as revealed in our study, has to be considered, if it comes to the interpretation of time course studies within the context of protein synthesis-dependent processes.

Protein synthesis inhibitors, gene superinduction and memory: too little or too much protein?

To date, the effects of protein synthesis inhibitors (PSI) in learning and memory processes have been attributed to translational arrest and consequent inhibition of de novo protein synthesis. Here we argue that amnesia produced by PSI can be the direct result of their abnormal induction of mRNA-a process termed gene superinduction. This action exerted by PSI involves an abundant and prolonged accumulation of mRNA transcripts of genes that are normally transiently induced. We summarize experimental evidence for the multiple mechanisms and signaling pathways mediating gene superinduction and consider its relevance for PSI-induced amnesia. This mechanistic alternative to protein synthesis inhibition is compared to models of electroconvulsive seizures and fragilexsyndrome associated with enhanced mRNA/protein levels and cognitive deficits.

Amnesia produced by altered release of neurotransmitters after intraamygdala injections of a protein synthesis inhibitor.

Amnesia produced by protein synthesis inhibitors such as anisomycin provides major support for the prevalent view that the formation of long-lasting memories requires de novo protein synthesis. However, inhibition of protein synthesis might disrupt other neural functions to interfere with memory formation. Intraamygdala injections of anisomycin before inhibitory avoidance training impaired memory in rats tested 48 h later. Release of norepinephrine (NE), dopamine (DA), and serotonin, measured at the site of anisomycin infusions, increased quickly by approximately 1,000-17,000%, far above the levels seen under normal conditions. NE and DA release later decreased far below baseline for several hours before recovering at 48 h. Intraamygdala injections of a beta-adrenergic receptor antagonist or agonist, each timed to blunt effects of increases and decreases in NE release after anisomycin, attenuated anisomycin-induced amnesia. In addition, similar to the effects on memory seen with anisomycin, intraamygdala injections of a high dose of NE before training impaired memory tested at 48 h after training. These findings suggest that altered release of neurotransmitters may mediate amnesia produced by anisomycin and, further, raise important questions about the empirical bases for many molecular theories of memory formation.

[Role of c-Jun NH (2)-terminal kinase in insulin resistance after burn].

OBJECTIVE: To investigate the role of c-Jun NH (2)-terminal kinase (JNk) in insulin resistance after burn and its mechanism. METHODS: Twenty-four Sprague-Dawley rats were randomized to control, burn and burn + anisomycin groups. The rats in control group received sham burn trauma, and burn and burn + anisomycin groups received 30% total body surface area (TBSA) full thickness burn injury. Anisomycin (5 mg/kg) together with 250 microl dimethyl sulfoxide (DMSO) was injected to the rats in anisomycin group intravenously, and only 250 microl DMSO in the other two groups. Euglycemic-hyperinsulinemic glucose clamps was performed 2 hours after the injection. The changes of phospho-serine 307, phospho-tyrosine of insulin receptor substrate (IRS)-1 and phospho-JNK in muscle tissues were determined and compared using immunoprecipitation and Western blot analysis or immunohistochemistry in the three groups. RESULTS: The infusing rates of total 10% glucose (mg x kg(-1) x min(-1)) in control, burn and burn + anisomycin group were 12.3 +/- 0.4, 6.6 +/- 0.3, 6.5 +/- 0.4, respectively. The level of IRS-1 Serine 307 phosphorylation and phospho-JNK in muscle increased significantly, while insulin-induced tyrosine phosphorylation of IRS-1 decreased markedly after burn. CONCLUSIONS: The activation of JNK elevates the level of IRS-1 phospho-serine 307 and might play a role in insulin resistance after burn in rats.

Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis.

Memory, as measured by changes in an animal's behavior some time after learning, is a reflection of many processes. Here, using a trace paradigm, in mice we show that de novo protein synthesis is required for acquisition, consolidation, reconsolidation, and extinction of classically conditioned eyelid responses. Two critical periods of protein synthesis have been found: the first, during training, the blocking of which impaired acquisition; and the second, lasting the first 4 h after training, the blocking of which impaired consolidation. The process of reconsolidation was sensitive to protein synthesis inhibition if anisomycin was injected before or just after the reactivation session. Furthermore, extinction was also dependent on protein synthesis, following the same temporal course as that followed during acquisition and consolidation. This last fact reinforces the idea that extinction is an active learning process rather than a passive event of forgetting. Together, these findings demonstrate that all of the different stages of memory formation involved in the classical conditioning of eyelid responses are dependent on protein synthesis.

Consolidation of fear extinction requires protein synthesis in the medial prefrontal cortex.

Extinction of conditioned fear is thought to form a long-term memory of safety, but the neural mechanisms are poorly understood. Consolidation of extinction learning in other paradigms requires protein synthesis, but the involvement of protein synthesis in extinction of conditioned fear remains unclear. Here, we show that rats infused intraventricularly with the protein synthesis inhibitor anisomycin extinguished normally within a session but were unable to recall extinction the following day. Anisomycin-treated rats showed no savings in the rate of re-learning of extinction, consistent with amnesia for extinction training. The identical effect was observed when anisomycin was microinfused into the medial prefrontal cortex (mPFC) but not the insular cortex. Furthermore, we observed that extinction training increased c-Fos levels in the mPFC but not in the insular cortex, consistent with extinction-induced gene expression in the mPFC. These findings extend previous lesion and unit-recording data by demonstrating that the mPFC is a critical storage site for extinction memory, rather than simply a pathway for expression of extinction. Understanding consolidation of fear extinction could lead to new treatments for anxiety disorders in which fear extinction is thought to be compromised.

Antagonism of estrogenic effects on feeding behavior by central implants of anisomycin.

The following experiment determined whether the estrogenic suppression of food intake is dependent upon changes in protein synthesis within neurons of the paraventricular nucleus of the hypothalamus (PVN). Ovariectomized rats were treated centrally with anisomycin-filled or empty (control) cannulae in the PVN. Females were injected with either 2.0 micrograms of estradiol benzoate (EB) or the oil vehicle and the inner cannulae were removed 2 h later. EB injections significantly lowered food and water intake in the central control group but not in animals given PVN implants of anisomycin. Body weight gain decreased for all females. EB induced comparable levels of female sexual behavior in both groups, demonstrating that anisomycin implants did not affect the ability of estradiol to stimulate lordosis. These findings indicate that the effects of estradiol on food intake require the activation of protein synthesis in estrogen-sensitive PVN neurons.