The requirement of protein and messenger RNA synthesis for long-term memory suggests that neural activity induced by learning initiates a cascade of gene expression. Here we use differential screening to identify five immediate-early genes induced by neuronal activity. One of these is tissue-plasminogen activator (tPA), an extracellular serine protease, which is induced with different spatial patterns in the brain by three activity-dependent events: (1) convulsive seizure increases expression of tPA in the whole brain; (2) stimulation of the perforant path produces an epileptiform after-discharge that ultimately leads to kindling increases the levels of tPA throughout the hippocampus bilaterally; and (3) brief high-frequency stimulation of the perforant path that produces long-term potentiation (LTP) causes an NMDA (N-methyl-D-aspartate) receptor-mediated increase in the levels of tPA mRNA which is restricted to the granule cells of the ipsilateral dentate gyrus. As release of tPA is correlated with morphological differentiation, the increased expression of tPA may play a role in the structural changes that accompany activity-dependent plasticity.

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

Depolymerization of actin by latrunculin A transiently promotes neurotransmitter release. The mean rate of mEPSCs increases by a Ca2+-independent process, without a concomitant change in the mean amplitude. The readily releasable vesicle pool size and the rate of refilling of the readily releasable pool remain unaltered by latrunculin treatment. Evoked neurotransmitter release also increases in a manner consistent with an increase in vesicle release probability. The observed enhancement of neurotransmitter release is specific to actin depolymerization mediated by latrunculin A and is not caused by cytochalasin D. Our findings indicate that actin participates in a regulatory mechanism that restrains fusion of synaptic vesicles at the active zone.

a convergence point for synaptic activity acting through kinase A and impulse activity acting through Cam kinase. Together the two kinases could activate transcription in a synergistic manner, which could allow CRE-binding protein to couple short-term to long-term associative forms of synaptic plasticity.Studies of neuronal plasticity in vertebrates and invertebrates indicate that long-term changes in the functional properties of nerve cells are often accompanied by and perhaps mediated through changes in gene expression (1-7). The changes in gene expression commonly result from signal transduction at the membrane produced either by impulse activity, by synaptic activity, or by both (5, 8). These two types of signals are then conveyed from the membrane to the nucleus by independent second-messenger pathways: (l) by Ca2" influx through voltage-gated ion channels activated by the action potential and (ii) by cAMP or other second-messenger systems activated by synaptic transmitters binding to receptors coupled to guanine nucleotide-binding proteins (G proteins) (9-12). We here ask: Can these pathways converge and interact on common transcription factors? Can transcription factors detect the independent but coincident occurrence at the membrane of synaptic activity on the one hand and impulse activity on the other?In neurons, it initially appeared as if these two pathways might be independent. For example, transmitter receptors coupled to adenylyl cyclase were found to induce the expression of c-fos by means of CREB transcriptional activators that bind as dimers to the cAMP response element (CRE) (12)(13)(14). Conversely, c-fos was found to be activated independently by membrane depolarization and the consequent Ca2+ influx (9, 10). However, recent studies in PC12 (rat pheochromocytoma) cells by Sheng et al. (15) Tryptic peptide analysis of the phosphorylated protein shows that the two kinases phosphorylate a common peptide. Moreover, in vitro transcription from a CRE-containing fos promoter is enhanced when the CRE-binding protein is phosphorylated either by Cam kinase or by kinase A. Phosphorylation by both the kinases increases transcription to a greater extent than phosphorylation by either one alone. METHODSProtein Purification and Blotting. Bovine brain nuclear extracts were prepared essentially by the method of Yamamoto et al. (12). Briefly, fresh brains were homogenized and the cell nuclei were centrifuged, washed, and frozen. The nuclei were lysed and centrifuged, DNA was removed with DEAE-cellulose resin, and the protein was precipitated with ammonium sulfate. The nuclear proteins were fractionated by heparin affinity chromatography and the fractions were assayed for CRE binding by gel retardation assay. Active fractions were further purified by affinity chromatography on oligonucleotide-agarose resins-sequentially CRE, API, and CRE sequence. These resins were synthesized according to Kadonaga and Tjian (16). For analysis the purified active fractions were concentrated and subjected to standard SDS/ PAGE....