Early Cellular Events Research Articles

Preparing the “Soil”: The Premetastatic Niche

Current focus on cancer metastasis has centered on the intrinsic factors regulating the cell autonomous homing of the tumor cells to the metastatic site. Specific up-regulation of fibronectin and clustering of bone marrow-derived cellular infiltrates coexpressing matrix metalloproteinases in distant tissue sites before tumor cell arrival are proving to be indispensable for the initial stages of metastasis. These bone marrow-derived hematopoietic progenitors that express vascular endothelial growth factor receptor 1 mobilize in response to the unique array of growth factors produced by the primary tumor. Their arrival in distant sites represents early changes in the local microenvironment, termed the "premetastatic niche," which dictate the pattern of metastatic spread. Focus on the early cellular and molecular events in cancer dissemination and selectivity will likely lead to new approaches to detect and prevent metastasis at its earliest inception.

Mechanisms of action of the carcinogenic heterocyclic amine PhIP

Formed during the cooking of meat, the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4-5- b]pyridine (PhIP) is mutagenic and carcinogenic. Although the metabolism and mutational effects of PhIP are well defined, the early cellular and genomic events by which it can induce neoplastic transformation are not yet fully characterised. These early cellular responses to genotoxic doses of PhIP were examined in a human mammary epithelial cell, MCF10A. Using Western blotting, PhIP was shown to induce expression of the DNA damage response proteins p53 and p21 WAF1/CIP1, and to inhibit cell growth while activating G1 cell cycle checkpoint, a consequence of PhIP-induced DNA damage. Using low doses of PhIP (previously shown to activate oestrogenic signalling), PhIP increased proliferation in the oestrogen receptor (ER)-negative MCF10A cell line and to activate the mitogen-activated protein kinase (MAPK) pathway. Inhibition of this pathway significantly reduced the PhIP-induced cell growth of MCF10A cells. The work presented here suggests that, further to its genotoxic properties, at levels close to human exposure PhIP stimulates cellular signalling pathways that are linked to the promotion and progression of neoplastic disease. It is possible that a combination of these DNA damaging and growth promoting properties provide a mechanism for the tumourigenicity of PhIP, and may be key determinants for the tissue specificity of PhIP-induced carcinogenesis.

Plasticity of Neurons and Glia Following Neonatal Hypoxic-Ischemic Brain Injury in Rats

Periventricular white matter injury in premature infants is linked to chronic neurological dysfunction. Periventricular white matter injury is caused by many mechanisms including hypoxia-ischemia (HI). Animal models of HI in the neonatal rodent brain can replicate some important features of periventricular white matter injury. Most rodent studies have focused upon early cellular and tissue events following unilateral neonatal HI that is elicited by unilateral carotid artery ligation and followed by timed exposure to moderate hypoxia. Milder hypoxic-ischemic insults elicit preferential white matter injury. Little information is available about long-term cellular effects of unilateral HI. One month after unilateral neonatal hypoxia ischemia, we show that all the components for structural reorganization of the brain are present in moderately injured rats. These components in the injured side include extensive influx of neurites, axonal and dendritic growth cones, abundant immature synapses, and myelination of many small axons. Surprisingly, this neural recovery is often found in and adjacent to cysts that have the ultrastructural features of bone extracellular matrix. In contrast, brains with severe hypoxia ischemia one month after injury still undergo massive neuronal degeneration. While massive destruction of neurons and glia are striking events shortly after brain HI, neural cells re-express their intrinsic properties and attempt an anatomical recovery long after injury.

Full Length Article

Attenuation by Metallothionein of Early Cardiac Cell Death via Suppression of Mitochondrial Oxidative Stress Results in a Prevention of Diabetic Cardiomyopathy Lu Cai, Yuehui Wang, Guihua Zhou, Teresa Chen, Ye Song, Xiaokun Li, Y. James Kang Diabetes was induced by streptozotocin (STZ) in both cardiac-specific, metallothionein (MT)-overexpressing transgenic mice and wild-type (WT) controls. However, cardiac apoptosis along with a release of mitochondrial cytochrome c , as well as a depletion of mitochondrial reduced glutathione, was observed in the WT, but not in the cardiac-specific, MT-overexpressing transgenic (MT-TG) diabetic mice 7 to 21 days after STZ treatment. Morphological abnormalities, revealed by electron microscope examination, and fibrosis, shown by Sirius-red staining, were also observed in the WT, but not in the MT-TG diabetic mice 6 months after STZ treatment. These results suggest that cardiac apoptosis is a major early cellular event in diabetic hearts and that its attenuation results in a prevention of diabetic cardiomyopathy. We aimed to test whether attenuation of early-phase cardiac cell death can prevent diabetic cardiomyopathy. Our previous study showed that cardiac apoptosis as a major early cellular response to diabetes is induced by hyperglycemia-derived oxidative stress that activates a mitochondrial cytochrome c -mediated caspase-3 activation pathway. Metallothionein (MT) as a potent antioxidant prevents the development of diabetic cardiomyopathy. Diabetes was induced by a single dose of streptozotocin (STZ) (150 mg/kg) in cardiac-specific, metallothionein-overexpressing transgenic (MT-TG) mice and wild-type (WT) controls. On days 7, 14, and 21 after STZ treatment, cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activation. Cardiomyopathy was evaluated by cardiac ultrastructure and fibrosis in the diabetic mice 6 months after STZ treatment. A significant reduction in diabetes-induced increases in TUNEL-positive cells, caspase-3 activation, and cytochrome c release from mitochondria was observed in the MT-TG mice as compared to WT mice. Cardiac protein nitration (3-nitrotyrosine [3-NT]) and lipid peroxidation were significantly increased, and there was an increase in mitochondrial oxidized glutathione and a decrease in mitochondrial reduced glutathione in the WT, but not in the MT-TG, diabetic mice. Double staining for cardiomyocytes with alpha sarcomeric actin and caspase-3 or 3-NT confirmed the cardiomyocyte-specific effects. A significant prevention of diabetic cardiomyopathy and enhanced animal survival were observed in the MT-TG diabetic mice as compared to WT diabetic mice. These results suggest that attenuation of early-phase cardiac cell death by MT results in a significant prevention of the development of diabetic cardiomyopathy. This process is mediated by MT suppression of mitochondrial oxidative stress.

Early Vascular and Neuronal Changes in a VEGF Transgenic Mouse Model of Retinal Neovascularization

To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR). Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy. Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest. Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease.

A short primer on early molecular and cellular events in thymus organogenesis and replacement.

Haematopoietic precursors have to undergo a complex series of maturational steps in the thymus before they exit into the periphery as functional T lymphocytes. Thymic stroma cells, the majority being of epithelial origin, provide the functional partners for the maturational progression along this differentiation pathway. Here we review some of the molecular and cellular mechanisms that account for thymus organogenesis and discuss a strategy to use thymic epithelial precursor cells for the regeneration of the thymic microenvironment.

Early Cellular Responses in Cortical Bone Healing Around Unloaded Titanium Implants: An Animal Study

A clear understanding of the early cellular events leading to osseointegration of implants is currently lacking. To gain better insight, titanium implants were inserted in a rabbit model and histologic and histomorphometric analyses were performed at early time points after insertion. Thirty-six cylindrical implants were inserted in the tibial diaphysis of six rabbits and left to heal for 1 to 42 days. Samples were processed into paraffin or methylmethacrylate sections, on which the surface of new bone, region of altered nuclear morphology, relative surface of basic multicellular units (BMUs) and blood vessels, and bone-to-implant contact were measured. After coagulum formation, osteoclasts and osteoblasts were observed at the bone surface 1 week after healing. In the preexisting bone, osteocytic lacunae appeared to be devoid of cells. This region of altered nuclear morphology continued to extend for 28 days (P <0.05) after implant insertion. This expansion was accompanied by an invasion of the damaged bone by BMUs that initiated intensive bone remodeling, which reached its maximum after 4 weeks (P <0.05) but was ongoing after 6 weeks of implant insertion. This study evaluated the early cellular events in cortical bone surrounding titanium implants. The insertion of an implant into bone initiates a series of biologic processes, including the formation of a hematoma, altered nuclear morphology of the osteocytes surrounding the implantation site, intensive bone remodeling, and the formation of new bone, eventually leading to the osseointegration of the implant.

Early cellular events in multiple sclerosis: Intimations of an extrinsic myelinolytic antigen

In a previous immunohistological study of tissues from unusually early cases of MS cluster analysis revealed a progression of demyelination through five distinct stages [Gay F, Drye T, Dick G, et al. The application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis. Identification and characterization of the primary demyelinating lesion. Brain 1997;120:1461–83]. Tissues from six of the earliest cases in this series contained regions of normal appearing white and grey matter in which well developed inflammatory events, concentrated in perivascular spaces, were found to extend locally into the perivascular parenchyma to envelop ostensibly intact myelin sheaths. The beginnings of myelin sheath lysis and phagocytosis were subsequently detected within these lesions and similar foci were found in subpial and in subependymal tissues. They were characterised by a spreading HLA Class II antigen expression on microglia, and by the presence of co-locating C3 complement–IgG complexes on capillary basement membranes, on microglial cell membranes and within the cytoplasm of large bodied activated astrocytes. Parenchymal lesions contained significantly few CD4+ T cells and showed no evidence of capillary leakage of plasma proteins. Despite the presence of complexed immunoglobulin and complement, opsonization of the myelin sheath could not be demonstrated. These observations point to the presence in early MS of a diffusing, complement-fixing, myelinolytic antigen, processed mainly within the Virchow-Robin spaces and distributed in the cerebrospinal and extracellular fluid compartments of the central nervous system.

Critical Role of the Ubiquitin Ligase Activity of UHRF1, a Nuclear RING Finger Protein, in Tumor Cell Growth

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

Anionic Peptide Factor/Phosphatidylcholine Particles Promote the Inhibition of Vascular Cell Adhesion Molecule-1 in Human Umbilical Vein Endothelial Cells

Objective: High-density lipoproteins (HDLs) have significant cardiovascular benefits by retarding the progression of atherosclerosis. One of the mechanisms is the inhibition by HDLs of the vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells. Our objective was to test the effect on VCAM-1 expression by the human umbilical vein endothelial cells (HUVEC) of a minor HDL₂ and HDL₃ apolipoprotein, the anionic peptide factor (APF). The peptide has previously been found to develop some beneficial effects against atherosclerosis, i.e. by promoting the cholesterol efflux from endothelial cells. Methods: We examined the effects of two HDL apolipoproteins A-I and APF, either in presence or absence of phosphatidylcholines (PCs), or free PCs, on the expression of VCAM-1 by HUVEC. The cells were stimulated with either the tumor necrosis factor-α (TNFα, 500 pg/ml) or the calcium bound to heparin (10 µg Ca²⁺/ml, 50 µg heparin/ml). Results: In the presence of TNFα, only the free PCs (0.25 and 1 mM) developed an inhibitory effect (up to 50%). In the absence of TNFα and in the presence of calcium bound to heparin, either the lipid-free APF (3.5 µM) or the APF/PC complexes (1:57 molar ratio) or the free PCs (0.25 mM) exhibited a substantial inhibitory effect (72, 71 and 42%, respectively). Conclusion: Our present findings suggest for the first time that one of the mechanisms of the antiatherogenic action of APF involves the inhibition of VCAM-1 expression by HUVEC. The peptide, through its phospholipid-binding and its calcium antagonist abilities, appears to confer on the HDLs a protective effect against the early cellular event of the inflammatory process.

Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation

Using primary explant cultures of mouse bladder, the early response of the urothelium after superficial and full-thickness injuries was investigated. In such an in vitro wound healing model, explant surfaces with a mostly desquamated urothelial superficial layer represented superficial wounds, and the exposed lamina propria at the cut edges of the explants represented full-thickness wounds. The urothelial cell ultrastructure, the expression and subcellular distribution of the tight junctional protein occludin, and differentiation-related proteins CK 20, uroplakins, and actin were followed. Since singular terminally differentiated superficial cells remained on the urothelium after superficial injury (i.e., original superficial cells), we sought to determine their role during the urothelial wound-healing process. Ultrastructural and immunocytochemical studies have revealed that restored tight junctions are the earliest cellular event during the urothelial superficial and full-thickness wound-healing process. Occludin-containing tight junctions are developed before the new superficial cells are terminally differentiated. New insights into the urothelium wound-healing process were provided by demonstrating that the original superficial cells contribute to the urothelium wound healing by developing tight junctions with de novo differentiated superficial cells and by stretching, thus providing a large urothelial surface with asymmetric unit membrane plaques.

Reorganization of cytoskeleton induced by 5‐aminolevulinic acid‐mediated photodynamic therapy and its correlation with mitochondrial dysfunction

This study investigated the early cellular events which occurred after mitochondrial photodamage induced by 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT). Subcellular localization of protoporphyrin IX (PpIX) in NIH3T3 cells was studied by confocal microscopy. Mitochondrial damage was assessed by measuring mitochondrial transmembrane potential and ATP contents, and confirmed by characteristic appearance on transmission electron microscopy. Cellular adhesion was measured by the level of resistance to trypsinization. Cytoskeletal studies were performed by fluorescent staining of cytoskeletal components. Following ALA-PDT, mitochondrial damage was found in NIH3T3 cells as judged by the decrease of membrane potential and ATP contents. Mitochondrial photodamage was further confirmed by electron microscopy. Resistance to trypsinization after ALA-PDT was shown to be light dose-dependent. The increase of cellular adhesion after ALA-PDT was correlated with mitochondrial photodamage and reorganization of cytoskeletal components in NIH3T3 cells. This study has demonstrated that mitochondrial dysfunctions induced by ALA-PDT results in alterations of cellular morphology and cellular adhesion.

Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-γ, and Eradicate Tumor Cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-gamma. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45(+) leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-alpha. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4(+) effector memory T cell. We conclude that quiescent CD4(+) effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-gamma, leading to tumor cell eradication.

Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens

Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in lower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT 50 value from –2 °C to over –10 °C. We examined physiological and morphological alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to –4 °C, freezing injury-associated ultrastructural changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells.

Early expression of adhesion molecules after lung transplantation: Evidence for a role of aggregated P-selectin-positive platelets in human primary graft failure

Background Primary graft failure (PGF) secondary to ischemia-reperfusion injury is the main cause of death in the first month after lung transplantation. The aim of this study was to identify early cellular and immunologic events associated with PGF in human lung transplants. Methods Induction of P-selectin, E-selectin and intercellular adhesion molecule-1 (ICAM-1) and evaluation of leukocytes and platelets accumulation were investigated in 18 post-reperfusion surgical specimens of lung allografts by an immunohistochemical technique. Results Selectins were restricted to the venular plexus after reperfusion as in the normal lung, whereas ICAM-1 was induced in all cases on alveolar capillaries. Numerous polymorphonuclear cells (18 of 18 cases) and aggregated platelets (7 of 18 cases) were identified along the venular plexus after reperfusion. Compared with the other patients, those with aggregated P-selectin-positive platelets were characterized by a longer duration of mechanical ventilation ( p < 0.01), a lower Pa o 2/Fi o 2 ratio ( p < 0.01) and the presence of radiologic edema ( p < 0.05) within the first 3 post-operative days. Conclusions We showed in the reperfused lung a distinct expression of adhesion molecules on venous and capillary pulmonary endothelia that may influence the role of leukocytes and platelets during the early course of transplantation. Furthermore, the knowledge of an association between the presence of P-selectin-positive platelet aggregates and PGF criteria might have implications for graft management and therapeutic strategies.

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFkappaB in human macrophages.

Although much has been learned about signal transduction mechanisms and binding proteins involved in lipopolysaccharides (LPS)-induced activation of monocytes/macrophages, little is known about the ability of internalized LPS to activate cells. To approach this question we either exposed macrophages to LPS or microinjected the cells with LPS before studying early cellular events associated with LPS-mediated macrophage activation. We measured membrane currents and translocation of NFkappaB to the nucleus. Using the whole-cell patch clamp technique ion channels were analyzed and characterized as K+ sensitive inward and outward currents. Exogenous LPS was shown to increase the voltage-dependent outward current whereas the voltage-dependent inward current was unaffected. However when cells were microinjected with LPS both inward and outward current were completely abolished. The presence of LPS within the cells did not prevent them to perform phagocytosis or to respond to fMLP with an appropriate increase in [Ca2+]i. The immunocytological detection of NFkappaB p65 translocation revealed that exogenous LPS led to the nuclear localization of the p65 subunit of NFkappaB, whereas only the cytoplasmic localization of p65 was observed following microinjection of LPS. These data show that one major process in macrophage activation, the NFkappaB dependent transcription of a number of genes encoding for many inflammatory mediators cannot be induced by intracellular LPS but requires the interaction of LPS with external membrane components. However intracellular LPS causes a drastic decrease in potassium currents which by keeping the cell membrane at a depolarized potential may result in changed biological answers of these cells.

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Neosis: A Novel Type of Cell Division in Cancer

Using computerized video time-lapse microscope, we studied early cellular events during carcinogen-induced transformation of C3H10T1/2 cells. Multinucleate / polyploid giant cells (MN/PGs) formed due to DNA damage is thought to die via mitotic catastrophe. Before they die, some MN/PGs undergo a novel type of cell division, termed neosis, characterized by karyokinesis via nuclear budding followed by asymmetric, intracellular cytokinesis, producing several small mononuclear cells, termed the Raju cells, with extended mitotic life span (MLS). Mitotic derivatives of Raju cells give rise to transformed cell lines, inherit genomic instability, display a phenotype and transcriptome different from the neosis mother cell, and display anchorage-independent growth. Neosis of MN/PGs also precedes spontaneous transformation of p53-/- mouse cells. Rodent neotic clones, and primary and metastatic human tumor cells undergo spontaneous or induced secondary/tertiary neosis. Neosis seems to extend the MLS of cells under conditions of genetic duress not favoring mitosis. It precedes tumorigenesis, occurs several times during tumor progression, yielding tumor-initiating Raju cells and introducing tumor cell heterogeneity subject to natural selection during tumor progression. Events during neosis, and its relevance to origin of established cell lines, multistep carcinogenesis, cancer stem cells, and therapeutic advantages of anti-neotic agents (neosicides) are discussed.

Pathophysiology of early ischaemia reperfusion injury of the colon in aortic surgery

Ischaemia-reperfusion (I-R) injury of the colon has been implicated in the systemic inflammatory response syndrome, which may lead to multiple organ dysfunction syndrome and death. The aim of this study was to investigate the early cellular events in I-R injury using elective aortic surgery as a model.

Acute Induction of Conserved Synaptic Signaling Pathways inDrosophilaMelanogaster

Analyses of early molecular and cellular events associated with long-term plasticity remain hampered in Drosophila by the lack of an acute procedure to activate signal transduction pathways, gene expression patterns, and other early cellular events associated with long-term synaptic change. Here we describe the development and first use of such a technique. Bursts of neural activity induced in Drosophila comatosets and CaP60A Kumts mutants, with conditional defects in N-ethylmaleimide-sensitive fusion factor 1 and sarco-endoplasmic reticulum Ca2+ ATPase, respectively, result in persistent (>4 hr) activation of neuronal extracellular signal-regulated kinase (ERK). ERK activation at the larval neuromuscular junction coincides with rapid reduction of synaptic Fasciclin II; in soma, nuclear translocation of activated ERK occurs together with increased transcription of the immediate-early genes Fos and c/EBP (CCAAT element binding protein). The effect of "seizure-stimulation" on ERK activation requires neural activity and is mediated through activation of MEK (MAPK/erk kinase), the MAPKK (mitogen-activated protein kinase kinase) that functions upstream of ERK. Our results (1) provide direct proof for the conservation of synaptic signaling pathways in arthropods, (2) demonstrate the utility of a new genetic tool for analysis of synaptic plasticity in Drosophila, and (3) potentially enable new proteomic and genomic analyses of activity-regulated molecules in an important model organism.