Localization and local translation of Arc/Arg3.1 mRNA at synapses: some observations and paradoxes

Abstract

Arc is a unique immediate early gene whose expression is induced as synapses are being modified during learning. The uniqueness comes from the fact that newly synthesized Arc mRNA is rapidly transported throughout dendrites where it localizes near synapses that were recently activated. Here, we summarize aspects of Arc mRNA translation in dendrites in vivo, focusing especially on features of its expression that are paradoxical or that donot fit in with current models of how Arc protein operates. Findings from in vivo studies that donot quite fit include: (1) Following induction of LTP in vivo, Arc mRNA and protein localize near active synapses, but are also distributed throughout dendrites. In contrast, Arc mRNA localizes selectively near active synapses when stimulation is continued as Arc mRNA is transported into dendrites; (2) Strong induction of Arc expression as a result of a seizure does not lead to a rundown of synaptic efficacy in vivo as would be predicted by the hypothesis that high levels of Arc cause glutamate receptor endocytosis and LTD. (3) Arc protein is synthesized in the perinuclear cytoplasm rapidly after transcriptional activation, indicating that at least a pool of Arc mRNA is not translationally repressed to allow for dendritic delivery; (4) Increases in Arc mRNA in dendrites are not paralleled by increases in levels of exon junction complex (EJC) proteins. These results of studies of mRNA trafficking in neurons in vivo provide a new perspective on the possible roles of Arc in activity-dependent synaptic modifications.

Keywords: Arc/Arg3.1; LTP; dendrite; dendritic mRNA; dendritic spines; immediate early genes; protein synthesis; synaptic plasticity.

FIGURE 1

Activity-dependent induction and Arc mRNA and localization near active synapses as revealed by fluorescent in situ hybridization (FISH). (A) Arc mRNA is expressed at low levels under basal conditions. The image illustrates the distribution of Arc mRNA (red) on the non-stimulated side of an anesthetized rat. (B) Induction of Arc expression after an electroconvulsive seizure. The rat was euthanized 2 h after the ECS. Note: that Arc is expressed by large numbers of dentate granule cells and Arc mRNA is transported throughout dendrites. (C) Selective targeting of Arc mRNA to active synapses. The panel illustrates the selective localization of Arc mRNA in the middle molecular layer of the dentate gyrus (DG) after high frequency stimulation of the MPP. (D–F) are high magnification views of the fields shown in (A–C) respectively. Selective targeting of Arc mRNA to the activated portions of dendrites was initially described by Steward et al. (1998). (G) Image of a dendrite from a neuron in culture in which immobile Arc/MS2 mRNA particles are identified by image averaging over time. (H) Higher power view of an Arc/MS2 particle parked at the base of a dendritic spine (spine is indicated by the arrow in G). The image was smoothed by Gaussian blurring function in Photoshop. (G,H) Are from Dynes and Steward (2012). (I) Non-isotopic in situ hybridization preparation illustrating the distribution of Arc mRNA 2 h after inducing LTP with 20 high frequency trains (a minimal stimulation paradigm). Arrows indicate band of increased mRNA levels in the middle molecular layer, but note also that Arc mRNA is distributed throughout the dendrites of dentate granule cells.

FIGURE 2

Changing patterns of Arc mRNA localization after discontinuation of synaptic stimulation. (A) Arc mRNA distribution as revealed by NRISH following 2 h of high frequency stimulation of the perforant path to cause localization and 10 min of no stimulation. (B) Arc mRNA distribution as revealed by NRISH following 2 h of high frequency stimulation of the perforant path to cause localization and 30 min of no stimulation. (C) Arc mRNA distribution as revealed by NRISH following 2 h of high frequency stimulation of the perforant path to cause localization and 2 h of no stimulation.

FIGURE 3

Absence of depression of synaptic responses as Arc protein levels increase in dendrites. (A) Perforant path responses before and at various times after ECS. (B) Plot of average population excitatory postsynaptic potential (EPSP) slope over time after ECS (n = 3 experiments). Note: post-ictal depression immediately after the ECS, rapid recovery of response amplitude and stability of responses for more than 2 h during which Arc protein levels are increasing in dendrites.

FIGURE 4

Appearance of newly synthesized Arc protein after induction by brief exposure to a novel enriched environment. The panels illustrate immunostaining for Arc protein in the CA1 region of the hippocampus (A–C), dentate gyrus (DG, panels D–F) and CA3 region of the hippocampus (G–I) in cage controls and 30 and 60 min after exposure to an enriched environment. Note prominent staining of cell bodies at 30 min. Scale bar in I = 100 μm.

FIGURE 5

Distribution of Arc and Bdnf mRNAs on polyribosome gradients. (A) Post-mitochondrial adult mouse brain lysate from two littermates was spun on 20–50% sucrose gradients to separate mRNP, monosome and polysome fractions. 0.72 ml fractions were collected with UV monitoring of RNA levels at A254. Fraction numbers are indicated below the panel. (B) Quantitative RT-PCR was used to measure Arc (solid lines) and BDNF (dotted lines) mRNA level in each fraction as described in Methods. Data are plotted as a fraction of the total recovered from the gradient, and are normalized for RNA recovery from each fraction. Two biologic replicates are individually shown on the graph. Error bars reflect standard deviation of three technical replicates.

FIGURE 6

Distribution of NMD proteins after synaptic stimulation. (A,C,E,G,I) Basal levels of Arc mRNA, eIF4AIII, Barentz, Staufen2, and Upf1expression on the contralateral side to stimulation, respectively. (B,D,F,H,J) Arc mRNA, eIF4AIII, Barentz, Staufen2, and Upf distribution after 1 h of synaptic stimulation, respectively. Scale bar is 100 μm.