SUMMARY Normal brain function requires that the overall synaptic activity in neural circuits be kept constant. Long-term alterations of neural activity leads to homeostatic regulation of synaptic strength by a process known as synaptic scaling. The molecular mechanisms underlying synaptic scaling are largely unknown. Here we report that all-trans retinoic acid (RA), a well-known developmental morphogen, unexpectedly mediates synaptic scaling in response to activity blockade. We show that activity blockade increases RA synthesis in neurons, and that acute RA treatment enhances synaptic transmission. The RA-induced increase in synaptic strength is occluded by activity blockade-induced synaptic scaling. Suppression of RA synthesis prevents synaptic scaling. This novel form of RA signaling operates via a translation-dependent but transcription-independent mechanism, causes an up-regulation of postsynaptic glutamate receptor levels, and requires RARα receptors. Together, our data suggest that RA functions in homeostatic plasticity as a signaling molecule that increases synaptic strength by a protein synthesis-dependent mechanism.

The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapsespecific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 m pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.

Retinoic acid (RA) plays important roles in development by modulating gene transcription through nuclear receptor activation. Increasing evidence supports a role for RA and RA receptors (RARs) in synaptic plasticity in the brain. We have recently reported that RA mediates a type of homeostatic synaptic plasticity through activation of dendritic protein synthesis, a process that requires dendritically localized RARα and is independent of transcriptional regulation. The molecular basis of this translational regulation by RA/RARα signaling, however, is unknown. Here we show that RARα is actively exported from the nucleus. Cytoplasmic RARα acts as an RNA-binding protein that associates with a subset of mRNAs, including dendritically localized glutamate receptor 1 (GluR1) mRNA. This binding is mediated by the RARα carboxyl terminal F-domain and specific sequence motifs in the 5′UTR of the GluR1 mRNA. Moreover, RARα association with the GluR1 mRNA directly underlies the translational control of GluR1 by RA: RARα represses GluR1 translation, while RA binding to RARα reduces its association with the GluR1 mRNA and relieves translational repression. Taken together, our results demonstrate a ligand-gated translational regulation mechanism mediated by a non-genomic function of RA/RARα signaling.

Homeostatic plasticity is thought to play an important role in maintaining the stability of neuronal circuits. During one form of homeostatic plasticity, referred to as synaptic scaling, activity blockade leads to a compensatory increase in synaptic transmission by stimulating in dendrites the local translation and synaptic insertion of the AMPA receptor subunit GluR1. We have previously shown that all-trans retinoic acid (RA) mediates activity blockadeinduced synaptic scaling by activating dendritic GluR1 synthesis and that this process requires RAR␣, a member of the nuclear RA receptor family. This result raised the question of where RAR␣ is localized in dendrites and whether its localization is regulated by RA and/or activity blockade. Here, we show that activity blockade or RA treatment in neurons enhances the concentration of RAR␣ in the dendritic RNA granules and activates local GluR1 synthesis in these RNA granules. Importantly, the same RNA granules that contain RAR␣ also exhibit an accumulation of GluR1 protein but with a much slower time course than that of RAR␣, suggesting that the former regulates the latter. Taken together, our results provide a direct link between dendritically localized RAR␣ and local GluR1 synthesis in RNA granules during RA-mediated synaptic signaling in homeostatic synaptic plasticity.FMRP ͉ GluR1 ͉ local protein translation ͉ synaptic scaling H omeostatic synaptic plasticity plays important roles in maintaining the stability of neural networks through the adjustment of global synaptic strength (1, 2). A particular form of homeostatic plasticity, referred to as synaptic scaling, modifies all synapses onto a neuron concurrently in a multiplicative fashion, with greater synaptic adjustment at stronger synapses than at weaker ones, allowing global changes of synaptic activity while preserving relative synaptic strength between individual synapses (3, 4). One well characterized form of synaptic scaling is the increase in synaptic strength induced by chronic blockade of neuronal activity with tetradotoxin (TTX) and the NMDA receptor antagonist APV. This form of synaptic scaling, manifested by an increase in synaptic glutamate receptor response, is mediated by the local synthesis and synaptic insertion of homomeric GluR1 receptors (5-7).We recently found that all-trans retinoic acid (RA), the active form of retinoids in the brain, mediates activity blockade-induced synaptic scaling (8). We demonstrated that activity blockadewhich induces homeostatic plasticity-strongly stimulates neuronal RA synthesis, which in turn activates dendritic GluR1 synthesis. We thus described a function of RA as a synaptic signal that operates during homeostatic plasticity to up-regulate synaptic strength by increasing the size of the postsynaptic glutamate receptor response. Additionally, we suggested that RAR␣ may be critically involved in this form of RA signaling because RAR␣ is present in dendrites, and knocking down RAR␣ blocks synaptic scaling. These results were surprising, given our previous under...

Local translation of dendritic mRNAs plays an important role in neuronal development and synaptic plasticity. Although several hundred putative dendritic transcripts have been identified in the hippocampus, relatively few have been verified by in situ hybridization and thus remain uncharacterized. One such transcript encodes the protein neuronatin. Neuronatin has been shown to regulate calcium levels in non-neuronal cells such as pancreatic or embryonic stem cells, but its function in mature neurons remains unclear. Here we report that neuronatin is translated in hippocampal dendrites in response to blockade of action potentials and NMDA-receptor dependent synaptic transmission by TTX and APV. Our study also reveals that neuronatin can adjust dendritic calcium levels by regulating intracellular calcium storage. We propose that neuronatin may impact synaptic plasticity by modulating dendritic calcium levels during homeostatic plasticity, thereby potentially regulating neuronal excitability, receptor trafficking, and calcium dependent signaling.