Calcium-evoked dendritic exocytosis (CEDE), demonstrated in cultured hippocampal neurons, is a novel mechanism that could play a role in synaptic plasticity. A number of forms of neuronal plasticity are thought to be mediated by calcium/calmodulin-dependent protein kinase II (CaMKII). Here, we investigate the role of CaMKII in CEDE. We find that the developmental time course of CEDE parallels the expression of alphaCaMKII, a dominant subunit of CaMKII. An inhibitor of this enzyme, KN-62, blocks CEDE. Furthermore, 7 d in vitro neurons (which normally do not express alphaCaMKII nor show CEDE) can undergo CEDE when infected with a recombinant virus producing alphaCaMKII. Expression of a constitutively active CaMKII produces dendritic exocytosis in the absence of calcium stimulus, and this exocytosis is blocked by nocodazole, an inhibitor of microtubule polymerization that also blocks CEDE. These results indicate that CEDE is mediated by the activation of CaMKII, consistent with the view that CEDE plays a role in synaptic plasticity.

The Alzheimer's amyloid protein (A␤) is released from the larger amyloid ␤-protein precursor (APP) by unidentified enzymes referred to as ␤-and ␥-secretase. ␤-Secretase cleaves APP on the amino side of A␤ producing a large secreted derivative (sAPP␤) and an A␤-bearing C-terminal derivative that is subsequently cleaved by ␥-secretase to release A␤. Alternative cleavage of the APP by ␣-secretase at A␤16/17 releases the secreted derivative sAPP␣. In yeast, ␣-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPIanchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of A␤40 and A␤42 in the medium but had no effect on sAPP␣. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of A␤40, A␤42, and sAPP␤ than its parental line (GPI؉). When this parental line was treated with PI-PLC, A␤40, A␤42, and sAPP␤ decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in ␤-secretase activity and A␤ secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in A␤ biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.The amyloid that is invariably deposited in Alzheimer's disease (AD) 1 is composed of an approximately 4-kDa peptide (amyloid ␤-peptide, A␤) that is derived from a larger protein referred to as the amyloid ␤-protein precursor (APP) (1, 2). APP is a type I integral membrane glycoprotein with a large Nterminal extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. The A␤ peptide begins 99 amino acids from the C terminus of APP, and it extends from the extracellular region to a point half-way through the APP membrane-spanning domain (1). A␤ is released from APP by cleavage on its N-and C-terminal ends by ␤-and ␥-secretase, respectively. ␤-Secretase cleavage before residue 1 of A␤ (672 of APP770) also releases the secreted derivative sAPP␤, whereas an alternative cleavage before residue 17 by ␣-secretase releases sAPP␣. In most culture systems tested, the predominant cleavage product is sAPP␣, and this may serve to prevent the production of A␤ (1). The proteolytic processing of APP to sAPP and A␤ is regulated by protein kinase C (3), protein tyrosine kinase (4), muscarinic receptors (5), and estrogens (6). The regulatory pathways involved are cell type-dependent, have little or no effect in some cell types, and normally stimulate the secretion of sAPP␣ while simultaneously reducing the secretion of A␤ (2). A metalloproteinase related to the tumor necrosis factor-␣ converting enzyme (7,8) can cleave APP to sAPP␣ upon activation of PKC by phorbol esters 2 (9, 10). Strong evidence that A␤ plays...

Vaccinia virus can be used to infect cells in the CNS of frogs, Xenopus laevis, and Rana pipiens, both in vivo and in vitro. In vivo infections were accomplished by injection of viral solution into the tectal ventricle of stage 40-48 tadpoles or by local injections into distinct neural regions. Infections with high titer of virus injected into the ventricle resulted in the majority of cells in the brain expressing foreign protein, while cells in the retina and optic nerve showed no expression. Infection with lower viral titers resulted in fewer infected cells that were distributed throughout the otherwise normal tissue. Intense expression of foreign protein in the brain was observed 36 hr after injection and remained high for at least 4 days. Infected animals developed normally and had the same number of cells in the optic tectum as control animals. Infection with a recombinant virus carrying the gene for Green Fluorescent Protein labels neurons, so that infected cells can be observed in vivo. Vaccinia virus provides a versatile means to alter proteins in distinct populations of neurons in amphibia.

Here we describe a technique that uses a recombinant vaccinia virus to transfect neurons in rat hippocampal slices. This technique allows the use of molecular biological manipulations on neuronal tissue while maintaining intact synaptic function. This method should be useful in testing specific hypotheses regarding the role of synaptic proteins.