Fast, convenient, and effective method to transiently transfect primary hippocampal neurons.

Transfection of primary neurons in culture has proven to be experimentally challenging in the past. To overcome these limitations, we present a detailed transfection protocol for hippocampal neurons based on DNA/Ca(2+)-phosphate coprecipitation. The main advantages being (1) the speed and convenience, (2) the remarkable efficiency of transfection for mature neurons, and (3) consistent health of the neurons upon transfection allowing subsequent manipulations. The strength of this protocol is convincingly demonstrated by the fact that the expressed protein can be detected biochemically on Western blots.

Microtubule-dependent recruitment of Staufen-green fluorescent protein into large RNA-containing granules and subsequent dendritic transport in living hippocampal neurons.

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP-labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 microm/min with a maximum velocity of 24. 3 microm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Purification and characterization of rat hippocampal CA3-dendritic spines associated with mossy fiber terminals.

We report a revised and improved isolation procedure for CA3-dendritic spines, most of them still in association with mossy fiber terminals resulting in a 7.5-fold enrichment over nuclei and a 29-fold enrichment over myelin. Additionally, red blood cells, medullated fibers, mitochondria and small synaptosomes were significantly depleted. We show by high resolution electron microscopy that this subcellular fraction contains numerous dendritic spines with a rich ultrastructure, e.g. an intact spine apparatus, membranous organelles, free and membrane-bound polyribosomes, endocytic structures and mitochondria. This improved experimental system will allow us to study aspects of post-synaptic functions at the biochemical and molecular level.

The mammalian staufen protein localizes to the somatodendritic domain of cultured hippocampal neurons: implications for its involvement in mRNA transport.

In hippocampal neurons, certain mRNAs have been found in dendrites (), and their localization and translation have been implicated in synaptic plasticity (). One attractive candidate to achieve transport of mRNAs into dendrites is Staufen (Stau), a double-stranded RNA-binding protein, which plays a pivotal role in mRNA transport, localization, and translation in Drosophila (). Using antibodies raised against a peptide located in the RNA-binding domain IIa and a polyclonal antibody raised against a recently cloned human Staufen homolog, we identify a 65 kDa rat homolog in cultured rat hippocampal neurons. In agreement with the exclusive somatodendritic localization of mRNAs in these cells, we find that Staufen is restricted to the same domain. By immunoelectron microscopy, we show enrichment of the mammalian homolog of Stau (mStau) in the vicinity of smooth endoplasmic reticulum and microtubules near synaptic contacts. Finally, the association of the mStau with neuronal mRNAs is suggested by the colocalization with ribonucleoprotein particles specifically in distal dendrites known to contain mRNA, ribosomes, and translation factors (). These results suggest a role for mStau in the polarized transport and localization of mRNAs in mammalian neurons.

Focal adhesion kinase in the brain: novel subcellular localization and specific regulation by Fyn tyrosine kinase in mutant mice.

Signaling by tyrosine kinases is required for the induction of synaptic plasticity in the central nervous system. Comparison of fyn, src, yes, and abl nonreceptor tyrosine kinase mutant mice shows a specific requirement for Fyn in the induction of long-term potentiation at CA1 synapses in the hippocampus. To identify components of a Fyn-dependent pathway that may be involved with hippocampus function we examined tyrosine-phosphorylated proteins in kinase mutant mice. We found that nine proteins were hypophosphorylated specifically in fyn mutants. One of the hypophosphorylated proteins was focal adhesion tyrosine kinase (FAK). FAK also showed reduced activity in immunocomplex kinase assays only in fyn mutants. FAK is expressed at very high levels in the brain but in contrast to non-neural cells, FAK was not restricted to focal adhesion contacts. FAK was found in axons, dendrites, and the intermediate filament cytoskeleton of astrocytes. Brain extracts from the mutants also show specific patterns of compensatory changes in the activity of the remaining Src family kinases. Tyrosine phosphorylation is a critical regulator of FAK, and impairments in FAK signal transduction in fyn mutants may contribute to the mutant neural phenotype.