A cellular mechanism for targeting newly synthesized mRNAs to synaptic sites on dendrites

Abstract

Long-lasting forms of activity-dependent synaptic plasticity involve molecular modifications that require gene expression. Here, we describe a cellular mechanism that mediates the targeting newly synthesized gene transcripts to individual synapses where they are locally translated. The features of this mechanism have been revealed through studies of the intracellular transport and synaptic targeting of the mRNA for a recently identified immediate early gene called activity-regulated cytoskeleton-associated protein Arc. Arc is strongly induced by patterns of synaptic activity that also induce long-term potentiation, and Arc mRNA is then rapidly delivered into dendrites after episodes of neuronal activation. The newly synthesized Arc mRNA localizes selectively at synapses that recently have been activated, and the encoded protein is assembled into the synaptic junctional complex. The dynamics of trafficking of Arc mRNA reveal key features of the mechanism through which synaptic activity can both induce gene expression and target particular mRNA transcripts to the active synapses.

Figure 1

Newly synthesized Arc mRNA is selectively targeted to dendritic domains that have been synaptically activated. The photomicrographs illustrate the distribution of Arc mRNA as revealed by nonisotopic in situ hybridization in nonactivated dentate gyrus (A), 2 h after a single electroconvulsive seizure (B), and after delivering high-frequency trains to the medial perforant path over a 2-h period (C). Note the uniform distribution of Arc mRNA across the dendritic laminae after an ECS and the prominent band of labeling in the middle molecular layer after high-frequency stimulation of the perforant path. (D) Schematic illustration of the dendrites of a typical dentate granule cell and the pattern of termination of medial perforant path projections. HF, hippocampal fissure; GCL, granule cell layer. (A and B) [Reproduced with permission from ref. (Copyright 2001, Elsevier Science)]. (D) [Reproduced with permission from ref. (Copyright 1998, Elsevier Science)].

Figure 2

Analysis of the distribution of Arc mRNA after various periods of synaptic stimulation. The graph illustrates the distribution of Arc mRNA in the molecular layer of the dentate gyrus after various periods of stimulation of the medial perforant path. After 30 min of stimulation, Arc mRNA is still confined to the cell body layer. With continued stimulation, levels of labeling increase progressively in the activated dendritic lamina, whereas there is minimal if any increase in labeling in the nonactivated distal dendritic segments (the outer molecular layer). Thus, newly synthesized Arc mRNA appears to be captured by active synapses, preventing the further migration of the mRNA into more distal segments. [Reproduced with permission from ref. (Copyright 1998, Elsevier Science)].

Figure 3

Redistribution of Arc mRNA after localized synaptic activation. In these experiments, Arc expression was induced by delivering an ECS. Then, the rat was anesthetized, and stimulation and recording electrodes were positioned so as to activate the medial perforant path on one side. High frequency trains (400-hz trains, eight pulses per train) were delivered for 30 or 15 min, respectively just before the animals were killed and perfused for in situ hybridization (in both cases, perfusion occurred 2 h after the ECS). As little as 15 min of synaptic stimulation was sufficient to produce a prominent band of labeling for Arc mRNA in the middle molecular layer of the dentate gyrus (B). After 30 min of stimulation, the band became more distinct as levels of labeling decreased in the nonactivated laminae, especially in the outer molecular layer (E and F). [Reproduced with permission from ref. (Copyright 2001, Elsevier Science)].

Figure 4

Arc protein accumulates in activated dendritic laminae in the same pattern as Arc mRNA. (A) Immunostaining of tissue sections from stimulated animals using an Arc-specific antibody revealed a band of newly synthesized protein in the same dendritic laminae in which Arc mRNA was concentrated. Note the sharp boundary (arrows) between the middle molecular layer (mml, the site of termination of the synapses that were activated) and the inner molecular layer. The fact that synaptic activation leads to the selective targeting of both recently synthesized mRNA and protein suggests that the targeting of the mRNA underlies a local synthesis of the protein. HF, hippocampal fissure; gcl, granule cell layer. (B) Newly synthesized Arc protein also is concentrated in the nucleus. Short arrows indicate examples of labeled nuclei.

Figure 5

Evidence from subcellular fractionation experiments that Arc protein is concentrated at the synaptic junction. Shown is a slot blot of protein samples from subcellular fractions prepared according to the procedure of ref. that have been stained with various antibodies. Band 1 contains myelin; band 2 contains nonsynaptic plasma membrane; band 3 contains synaptic plasma membranes (SPM). SJC is the fraction enriched in postsynaptic densities obtained by detergent extraction of band 3. Note that Arc protein is present at the highest relative levels in the synaptic plasma membrane and SJC fractions as are α and β isoforms of CAMKII and fodrin, which are highly enriched in psd.

Figure 6

Ultrastructural evidence for spine motility in synapses that have experienced intense synaptic activation. Illustrated are synapses in the middle molecular layer of the dentate gyrus on the control nonstimulated side (A) and after 2 h of high-frequency stimulation of the medial perforant path (B). Note that on the stimulated side, spines exhibit a chalise-like form that is remarkably similar to the form of highly motile spines. Animals received medial perforant path stimulation as described for 2 h and then were perfused with 2% paraformaldehyde/2% glutaraldehyde and prepared for electron microscopy. Photomicrographs then were taken in the middle molecular layer on the stimulated and control nonstimulated sides. den, dendrite; s, spine; t, terminal.