Identification of tool use acquisition-associated genes in the primate neocortex

Abstract

Japanese macaques are able to learn how to use rakes to take food after only a few weeks of training. Since tool-use training induced rapid morphological changes in some restricted brain areas, this system will be a good model for studying the neural basis of plasticity in human brains. To examine the mechanisms of tool-use associated brain expansion on the molecular and cellular level, here, we performed comprehensive analysis of gene expressions with microarray. We identified various transcripts showing differential expression between trained and untrained monkeys in the region around the lateral and intraparietal sulci. Among candidates, we focused on genes related to synapse formation and function. Using quantitative reverse transcription-polymerase chain reaction and histochemical analysis, we confirmed at least three genes (ADAM19, SPON2, and WIF1) with statistically different expression levels in neurons and glial cells. Comparative analysis revealed that tool use-associated genes were more obviously expressed in macaque monkeys than marmosets or mice. Thus, our findings suggest that cognitive tasks induce structural changes in the neocortex via gene expression, and that learning-associated genes innately differ with relation to learning ability.

Keywords: gene expression; macaque; microarray; parietal cortex; tool use.

Figure 1

Tool use training of Rhesus macaque monkeys (A, B) and collected brain areas for gene expression analysis (C–E). Schematic representation of tool use training (A). Experimental procedure and learning curve of tool use training (B). Horizontal axis indicates learning periods. Vertical axis indicates the score of the tool use test. The score of 50 timed trial tests was calculated as follows: monkeys retrieved pieces of food on the first attempt (score 3), second attempt (score 2), multiple attempts (score 1) or more than five attempts or failed (score 0). Dissection and sample preparation of the macaque brain (C–E). Examples of dissected area from the lateral sulcus (LS) and the intraparietal sulcus (IPS) region (marked area) (D, E). RNAs were purified from the anterior region of the surrounding area of LS including SII and insula (Is), and from the posterior region of the surrounding area of the IPS. We collected both superior and inferior LS and IPS regions, because it was difficult to dissect half of these areas.

Figure 2

Quantitative analysis of tool use associate candidates by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) (A–D). qRT–PCR was performed with five control and five experimental animals for the lateral sulcus (LS) and M1, and four experimental and four control animals for the intraparietal sulcus (IPS) region. Note that SPON2, ADAM19, and WIF1 in the LS and IPS showed higher expression in experimental animals than control animals, although no significant differences were seen in M1. *P < 0.05.

Figure 3

In situ hybridization of adult macaque superior lateral sulcus (LS) (A–D) and inferior intraparietal sulcus (IPS) regions (E, F) for ADAM19 (A, B), SPON2 (C, D), and WIF1 (E, F) of experimental (A, C, E) or control neocortex (B, D, F). Note that many SPON2‐ or WIF1‐expressing cells were located in the upper layers, which are particularly evolved in the primate brain. Expressions of all these genes were seen in the control monkeys, although their expression levels looked different between control and experimental monkeys. Scale bar is 100 μm.

Figure 4

In situ hybridization for ADAM19, SPON2 and WIF1 (A, D, G, J), subsequent co‐immunostaining with cell type specific markers (B, E, H, K), and overlay images of in situ hybridization (green, pseudo‐colour) and IHC (red) (C, F, I, L) of sections derived from experimental monkeys. In situ hybridization of adult macaque superior lateral sulcus (LS) (A–F) and inferior intraparietal sulcus (IPS) regions (G–L) for ADAM19 (A–C), SPON2 (D–F), and WIF1 (G–L) with immunostaining for the neuronal marker NeuN (A–I) or oligodendrocyte marker OSP (J–L). ADAM19 and SPON2‐expressing cells were NeuN‐positive (A–F), and WIF1‐expressing cells were NeuN‐negative (G–I) but OSP‐positive (J–L). IHC, immunohistochemistry; OSP, oligodendrocyte specific protein. Scale bar is 100 μm.

Figure 5

ADAM19, SPON2 and WIF1 expressions in the mouse and marmoset neocortex. Similar ADAM19 expression levels among the macaque, marmoset and mouse (A–E). In situ hybridization for ADAM19 in marmoset (A, B) and mouse (C) somatosensory cortex. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis of ADAM19 expression in developing marmoset (D) and mouse (E). Differential SPON2 expression between the macaque and the other species (F–P). In situ hybridization for SPON2 in the marmoset (F, K, L, M) and mouse (N). In situ hybridization for SPON2 (F) and subsequent immunostaining with a Tbr2 antibody (G) in GW12 marmoset neocortex. Overlay image of SPON2 (green, pseudo‐colour) and Tbr2 (red) staining (H, I, J). H, I, and J are magnified images of the areas indicated by arrows (F). In situ hybridization for SPON2 of the lateral sulcus (LS) area (K) and cerebellum (L) in P0 marmoset, and the LS area in adult marmoset (M) and adult mouse somatosensory cortex (N). qRT–PCR analysis of SPON2 expression in developing marmoset (O) and mouse (P). Note that SPON2 expression was reduced during development and disappeared by the adult stage for both species, in contrast to macaques. Differential WIF1 expression between marmoset and mouse neocortex (Q–T). In situ hybridization for WIF1 in marmoset (Q) and mouse caudal parietal cortex (R, left) and habenula (R, right). Note that in situ hybridization staining for WIF1 was seen in the habenula but no staining was seen in the neocortex. qRT–PCR analysis of WIF1 expression in developing marmoset (S) and mouse (T). qRT–PCR was performed with three animals at each stage. For qRT–PCR experiments, tissues were derived from the whole brain for E14 mouse embryos, the parietal cortex for P14 and adult mice, the whole neocortex for GW12 marmoset embryos, and the parietal cortex for P0 and adult marmosets. Scale bars are 500 μm (K, L, Q), 200 μm (A–C), 100 μm (N, R), 50 μm (F, G), and 10 μm (J).

Figure 6

Summary of temporal change of gene expressions among mouse, marmoset and macaque neocortex (A) and hypothetical model of tool use associated changes (B) and brain expansion of primates (C). Tool use induces gene expressions (e.g. SPON2 and ADAM19) in efferent neurons of the SII area. Their gene products function in the intraparietal sulcus (IPS) and induce cortical changes. In turn, this makes subsequent gene expression changes (e.g. WIF1) in the neurons or oligodendrocytes of the IPS area. Reciprocal interaction of these gene products may cause plastic changes such as axogenesis, myelination or synaptic changes (B). Acquisition of activity‐dependent gene expressions might have accelerated brain expansion by both innate and behavior‐dependent changes simultaneously acquired (C).