We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in >106-fold amplification of the original starting material, which is adequate for analysis-e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease.

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.Changes in synaptic connectivity are likely to be a key mechanism by which nervous system organization is permanently changed by experience. Local translation of some proteins in dendrites is increasingly considered to be important for changes in synaptic structure and receptor composition. Certain mRNAs are known to be targeted to dendrites (1, 2), and polyribosomal aggregates are observed in or near dendritic spines, more frequently at newly forming synapses (3-5). Dendrites have been shown to be equipped with components necessary for protein synthesis (6), and synthesis of proteins has been demonstrated directly in synaptoneurosomes (7,8) and in preparations of dendrites isolated from hippocampal neurons in culture (9). Local translation of transfected reporter-tagged mRNA has been demonstrated in transected dendrites (10).Protein translation induced by metabotropic receptor stimulation has previously been proposed to play a role in longterm potentiation (LTP), a model for synaptic plasticity (1,11,12). LTP induction also alters levels of specific mRNAs in tissue slices (13), and isotope-tagged leucine is taken up in dendritic regions of hippocampal slices in response to stimulation (14). We have demonstrated that phosphoinositidelinked metabotropic glutamate receptors (mGluR1), known to trigger a phosphorylation cascade, cause certain mRNAs to associate rapidly with protein translation complexes in synaptoneurosomes (15); this process is modulated by ionotropic receptors (16). Furthermore, we have shown that depolarization by 40 mM K ϩ or stimulation by phosphoinositide receptorspecific mGluR agonists increases [ 35 S]methionine incorporation into trichloroacetic acid-precipitable polypeptides (15, 17), indicating that de novo protein synthesis at the synapse occurs as a result of t...

The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.

Neurons are highly polarized cells with a mosaic of cytoplasmic and membrane proteins differentially distributed in axons, dendrites, and somata. In Drosophila and Xenopus, mRNA localization coupled with local translation is a powerful mechanism by which regionalized domains of surface or cytoplasmic proteins are generated. In neurons, there is substantial ultrastructural evidence positing the presence of protein synthetic machinery in neuronal processes, especially at or near postsynaptic sites. There are, however, remarkably few reports of mRNAs localized to these regions. We now present direct evidence that an unexpectedly large number of mRNAs, including members of the glutamate receptor family, second messenger system, and components of the translational control apparatus, are present in individual processes of hippocampal cells in culture.In central nervous system neurons, several lines of evidence suggest that proteins may be synthesized locally at or near postsynaptic sites independent of the cell body. First, ultrastructural studies have revealed the preferential localization of polyribosomes beneath postsynaptic sites and occasionally associated with membrane specializations on dendrites. It has been suggested that these anatomical structures represent the protein synthetic machinery necessary to translate and posttranslationally modify different classes of proteins (1,2 MATERIALS AND METHODS Hippocampal Cultures. Hippocampi were dissected from embryonic day 20-21 rat fetuses and cultured as described (12,13). Experiments were performed after 21-28 days in culture. In this study, cell bodies and their neurites were taken from cells cultured from different animals on three different days under similar conditions. During each of these sessions, a sample of culture medium was also aspirated and processed through antisense RNA (aRNA) processing to assess the possible presence of mRNAs in the culture medium from dying cells.Reverse Northern Blot Analyses. Samples were processed as described (14, 15)