Expression of the core exon-junction complex factor eukaryotic initiation factor 4A3 is increased during spatial exploration and striatally-mediated learning

Abstract

Regulation of dendritically localized mRNAs offers an important means by which neurons can sculpt precise signals at synapses. Arc is one such dendritically localized mRNA, and it has been shown to contain two exon-junction complexes (EJCs) within its 3'UTR. The EJC has been postulated to regulate cytoplasmic Arc mRNA availability through translation-dependent decay and thus contribute to synaptic plasticity. Core proteins of the EJC include eIF4A3, an RNA helicase, and Magoh, which stabilizes the interaction of eIF4A3 with target mRNAs. Arc mRNA expression is activity-regulated in numerous brain regions, including the dorsal striatum and hippocampus. Therefore in this study, the in vivo expression of these core EJC components was investigated in adult Sprague-Dawley rats to determine whether there are also behaviorally regulated changes in their expression. In the present work, there was no change in the expression of Magoh mRNA following spatial exploration, a paradigm previously reported to robustly and reliably upregulate Arc mRNA expression. Interestingly, however, there were increases in eIF4A3 mRNA levels in the dorsal striatum and hippocampus following spatial exploration, similar to previous reports for Arc mRNA. Furthermore, there were activity-dependent changes in eIF4A3 protein distribution and expression within the striatum following spatial exploration. Importantly, eIF4A3 protein colocalized with Arc mRNA in vivo. Like Arc mRNA expression, eIF4A3 mRNA expression in the dorsomedial striatum, but not dorsolateral striatum or hippocampus, significantly correlated with behavioral performance on a striatally-mediated, response-reversal learning task. This study provides direct evidence that a core EJC component, eIF4A3, shows activity-dependent changes in both mRNA and protein expression in the adult mammalian brain. These findings thus further implicate eIF4A3 as a key mediator of Arc mRNA availability underlying learning and memory processes in vivo.

Figure 1. Expression of EJC components in dorsal striatum of rats engaged in spatial exploration for 5 min

(A) Mean expression (arbitrary gray value) of eIF4A3 mRNA (± SEM; n=4-10/group) in dorsal striatum analyzed via radioactive in situ hybridization histochemistry and expressed as a percent of basal values in caged control (CC) animals. Rats in the CC group were sacrificed immediately upon removal from their home cage. Rats in the remaining groups explored a novel spatial environment (see Methods) for 5 min and were then either sacrificed immediately (“5 min” group) or returned to the home cage for 25 min before sacrifice (“30 min” group); or 55 min before sacrifice (“60 min” group). *Significantly different from CC (p=0.03). (B) Mean expression (arbitrary gray value) of Magoh mRNA (± SEM; n=4-7/group) in dorsal striatum analyzed via radioactive in situ hybridization histochemistry and expressed as a percent of basal values in CC animals. (C) A sense ribonucleotide probe for eIF4A3 gave no signal. (D) Striatal section from a CC rat labeled with the anti-sense ribonucleotide probe for eIF4A3.. (E) Striatal section from a “30 min” group rat labeled with the anti-sense ribonucleotide probe for eIF4A3.

Figure 2. Expression of EJC components in dorsal hippocampus of rats engaged in spatial exploration for 5 min

(A-D) Mean expression (arbitrary gray value) of eIF4A3 mRNA (± SEM; n=4-10/group) in the CA1 (A), CA3 (B), upper blade of the dentate gyrus (DGub; C), and lower blade of the dentate gyrus (DGlb; D) subregions of dorsal hippocampus analyzed via radioactive in situ hybridization histochemistry and expressed as a percent of basal values in caged control (CC) animals. *Significantly different from CC. (E-H) Mean expression (arbitrary gray value) of Magoh mRNA (± SEM; n=10/group) in CA1 (E), CA3 (F), DGub (G), and DGlb (H) analyzed via radioactive in situ hybridization histochemistry and expressed as a percent of basal values in CC animals.

Figure 3. Expression of eIF4A3 protein in dorsal striatum of rats engaged in spatial exploration for 5 min

(A) Mean (± SEM, n=4-10/group) eIF4A3 protein distribution in dorsal striatum measured as percent of total image area with signal (i.e. percentage of total pixel area in the field with eIF4A3-labeled pixels). *Significantly different from caged controls (CC; p<0.01). (B) Signal intensity (average gray area), expressed as mean percent of control (± SEM, n=4-10/group), of eIF4A3 protein-labeled pixels in dorsal striatum *Significantly different from CC (p=0.02). (C) Significant inverse correlation (p<0.05) between percent of total field with eIF4A3 protein signal above threshold and the average signal intensity (mean gray value) of the labeled.

Figure 4. Arc mRNA colocalizes with eIF4A3 protein in vivo following response-reversal learning on a T-maze

(A) Representative photomicrograph of Arc mRNA in situ hybridization histochemical staining (red) and eIF4A3 protein immunofluorescence (green) in dorsal striatum of a rat sacrificed 5 min after reaching criterion on a striatally-mediated, response-reversal learning task (see Methods). Scale bar = 20 μm. (B-E) Higher magnification images of the region delineated in the box in (A) showing colocalization of eIF4A3 protein expression and Arc mRNA colocalization (B) and the individual channels for the DAPI nuclear counter stain (C), Arc mRNA (D), and eIF4A3 protein (E). Arrows highlight points of colocalization of Arc mRNA and eIF4A3 signal.. Scale bar in B-E = 2 μm.

Figure 5. Expression of eIF4A3 mRNA in the brains of rats undergoing response-reversal learning

(A) Rats trained to perform on a response-reversal learning task on a T-maze showed significant increases in eIF4A3 mRNA expression in the dorsomedial (DM) and dorsolateral (DL) striatum compared to caged control (CC) rats (n = 11 per group). *Significantly different from CC (p=0.0001). (B-G) Degree of correlation between behavioral performance (trials to criterion) on the response-reversal task and eIF4A3 mRNA expression (average gray value from densitometric analysis) in DM striatum (A), DL striatum (B), CA1 of dorsal hippocampus (C), CA3 of dorsal hippocampus (D), upper blade of dentate gyrus (DGub; E), and lower blade of dentate gyrus (DGlb; F). *Significant correlation (p<0.05).