Using pharmacological approaches, several recent studies suggest that local protein synthesis is required for synaptic plasticity. Convincing demonstrations of bona fide dendritic protein synthesis in mammalian neurons are rare, however. We developed a protein synthesis reporter in which the coding sequence of green fluorescent protein is flanked by the 5' and 3' untranslated regions from CAMKII-alpha, conferring both dendritic mRNA localization and translational regulation. In cultured hippocampal neurons, we show that BDNF, a growth factor involved in synaptic plasticity, stimulates protein synthesis of the reporter in intact, mechanically, or "optically" isolated dendrites. The stimulation of protein synthesis is blocked by anisomycin and not observed in untreated neurons. In addition, dendrites appear to possess translational hot spots, regions near synapses where protein synthesis consistently occurs over time.

The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.

Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of ␣-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 4D optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis.cytokinesis ͉ mannosidase ͉ photoconversion ͉ ReAsH ͉ tetracysteine

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction.

In vivo imaging has revolutionized our understanding of biological processes in brain physiology and pathology. However, breathing-induced movement artifacts have impeded the application of this powerful tool in studies of the living spinal cord. Here we describe in detail a method to image stably and repetitively, using two-photon microscopy, the living spinal tissue in mice with dense fluorescent cells or axons, without the need for animal intubation or image post-processing. This simplified technique can greatly expand the application of in vivo imaging to study spinal cord injury, regeneration, physiology and disease.

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X 2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X 2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X 2 receptor distribution on the seconds time scale.ion channel ͉ ATP ͉ filopodia C ationic P2X receptors mediate the ''fast'' milliseconds time scale actions of ATP in the nervous system (1, 2). The identity of most natively expressed P2X receptors is unclear, but many neurons express P2X 2 mRNA, P2X 2 proteins, and functional P2X 2 -like receptors (2). For example, ATP mediates synaptic transmission in a portion of CA1 neurons (3), and postnatal hippocampal neurons express P2X receptors, which include P2X 2 subunits (3-7). Moreover, cytosolic ATP concentration is 1-5 mM, and ATP released during tissue damage activates neuronal P2X receptors in the periphery (1). ATP released as a synaptic transmitter and during ischemia of brain neurons may contribute to pathophysiology, but there are no available data on the cellular consequences of P2X 2 receptor activation or on the dynamic aspects of P2X 2 receptor distribution in brain neurons.This study used P2X 2 receptors tagged with green fluorescent protein (GFP) in a quantitative method to study receptors expressed with recombinant Sindbis virus in embryonic hippocampal neurons. We report (i) the properties of functional GFP-tagged P2X 2 receptors, (ii) an optical and electrophysiological approach to measuring receptor numbers in living cells, and (iii) the cellular effects of P2X receptor activation. Materials and MethodsMolecular Biology. By PCR the P2X 2 stop codon was removed and the FLAG (f) epitope was inserted in frame with the P2X 2 cDNA cDNA (9). In the same PCR we inserted an XhoI site in the DNA. We generated GFP37 (10) with a XhoI site before the start codon and subcloned it into P2X 2 -f between the XhoI site 3Ј of the FLAG epitope and HindIII in the pcDNA3 polylinker to yield P2X 2 -GFP. The P2X 2 -f-GFP fragment was inserted into pSinRep5 between the StuI and ApaI sites, and infective Sindbis particles were generated with the use of the Sindbis Expression System (http:͞͞www.invitrogen.com͞). Site-directed mutagenesis was performed on the cDNAs with the use of synthetic oligonucleotides to generate K69A and T18A mutants (Quick Change; Stratagene).Electrophysiology and Imaging. All cell preparations, twoelectrode voltage-clamp record...

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Alu sequences from GC-rich DNA are likely to be harmful and prevented from spreading in the population by natural selection. This implies no functional importance for an Alu sequence itself, but merely that, as the deletions of Alus are very unlikely to be precise, a deletion event removing an Alu is also likely to remove valuable sequences around it, and the chromosome bearing the deletion will be lost by selection.The explanation favoured by the authors for Alu enrichment in generich regions is that of positive selection in favour of Alus in GC-rich DNA. This theory, however, cannot explain the observations. The data show that Alu sequences up to five million years old are not enriched in GC-rich regions. But in human population genetics, estimated times to common ancestry of typical genomic regions show that Alu sequences which are five million years old have already been fixed (found in all individuals) in the population. This observation is also what would be expected from neutrality and genetic drift, given the human effective population size. (Alu sequences which are truly advantageous will spread to fixation much more quickly.) Earlier human ancestors would also be expected to have had similar fixation times for Alu insertions. Yet it is only during the spread to fixation of Alu sequences that positive natural selection has any opportunity to act. Thus, an increasing abundance of Alu sequences in GC-rich DNA as they age beyond five million years cannot be the result of natural selection for positive functions of Alu insertions.