Memory consolidation in honey bees is enhanced by down-regulation of Down syndrome cell adhesion molecule and changes its alternative splicing

Abstract

Down syndrome cell adhesion molecule (Dscam) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention.

Keywords: Dscam; RNA interference (gene silencing); alternative splicing; honey bee; learning and memory.

FIGURE 1

Down syndrome cell adhesion molecule (Dscam) is required for learning and memory consolidation. (A) Western blot detecting Dscam (top) or tubulin (bottom) in honey bee central brains of control GFP and Dscam RNAi 48 h after injection of dsRNA into worker honey bees. (B) Schematic of the treatment to test for Dscam’s role in learning and memory consolidation. (C) Learning (left), short term (middle) and long-term memory (right) performances of control GFP RNAi (white) and Dscam RNAi (black) after injection of worker honey bees with dsRNA. CS: conditioned odor, N: novel odorant. *p < 0.05. (D) Schematic of the treatment control for Dscam’s role in learning and memory consolidation. (E) Learning (left) and memory (right) performances of control GFP RNAi (white, n = 39/n = 22 at 24 h) and Dscam RNAi (black, n = 46/n = 15 at 24 h) after injection of worker honey bees with dsRNA. CS, conditioned stimulus, N, novel odorant **p < 0.01. The source date underlying this figure are available in Supplementary Data 1.

FIGURE 2

Schematic of the honey bee Dscam variable exon clusters 4, 6, and 10. (A–C) Gene models of Apis mellifera Dscam variable exon clusters 4 (A, top), 6 (B, middle), and 10 (C, bottom) depicting constant flanking exons in orange and variable exons in light blue boxes. New variable exons are indicated by dark blue boxes. Alternative 5’ and 3’ splice sites are indicated by right and left directed flags, respectively. Intra-cluster splicing is indicated by solid lines and dashed lines indicate representative alternative splicing. Cryptic splice sites (<1%) are indicated by vertical green lines. Exon clusters are drawn to scale with scale bars are shown at the bottom. (D) Alternative 5’ splice sites in exons 3 and 5 are shown as small arrows.

FIGURE 3

Down syndrome cell adhesion molecule (Dscam) variable exon 4 alternative splicing analysis during memory consolidation. (A) Representative 5% denaturing polyacrylamide gel separating ³²P-labeled alternative splice products from worker brains conditioned with unpaired (left) and paired odors and reward. Identity of splice variants are indicated at the right. M, Marker. (B–D) Analysis of alternative splicing unpaired control (green), fast (light blue) and slow learners (dark blue) indicating exon inclusion frequency (in%) for the different variables immediately after training (0 h), 2 or 4 h after training. Significant changes are indicated by vertical arrows (*p < 0.05; **p < 0.01). The source date underlying this figure are available in Supplementary Data 1.

FIGURE 4

Down syndrome cell adhesion molecule (Dscam) variable exon 10 alternative splicing analysis during memory consolidation. (A) Representative 5% denaturing polyacrylamide gel separating ³²P-labeled alternative splice products from worker brains conditioned with unpaired (left) and paired odors and reward. Identity of splice variants are indicated at the right. M, Marker. (B–D) Analysis of alternative splicing unpaired control (green), fast (light blue) and slow learners (dark blue) indicating exon inclusion frequency (in%) for the different variables immediately after training (0 h), 2 or 4 h after training. Significant changes are indicated by vertical arrows (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The source date underlying this figure are available in Supplementary Data 1.

FIGURE 5

Down syndrome cell adhesion molecule (Dscam) variable exon 6 alternative splicing analysis during memory consolidation. (A,B) Analysis of alternative splicing unpaired control (green), fast (light blue) and slow learners (dark blue) indicating exon inclusion frequency (in%) from amplicon sequencing for the different variables 2 h after training for main variable (A) and alternative splice sites (B). Significant changes are indicated by vertical arrows (*p < 0.05; **p < 0.01). The source date underlying this figure are available in Supplementary Data 1.