Analysis of synaptic gene expression in the neocortex of primates reveals evolutionary changes in glutamatergic neurotransmission

Abstract

Increased relative brain size characterizes the evolution of primates, suggesting that enhanced cognition plays an important part in the behavioral adaptations of this mammalian order. In addition to changes in brain anatomy, cognition can also be regulated by molecular changes that alter synaptic function, but little is known about modifications of synapses in primate brain evolution. The aim of the current study was to investigate the expression patterns and evolution of 20 synaptic genes from the prefrontal cortex of 12 primate species. The genes investigated included glutamate receptors, scaffolding proteins, synaptic vesicle components, as well as factors involved in synaptic vesicle release and structural components of the nervous system. Our analyses revealed that there have been significant changes during primate brain evolution in the components of the glutamatergic signaling pathway in terms of gene expression, protein expression, and promoter sequence changes. These results could entail functional modifications in the regulation of specific genes related to processes underlying learning and memory.

Keywords: brain evolution; glutamatergic neurotransmission; prefrontal cortex; synapse.

Figure 1.

(A) PCA plot with samples plotted in 2 dimensions using their projections onto the first 2 principal components. Each dot represents an individual brain sample and the colors identify the different phylogenetic groups. PC1 and PC2 refer to the first and second principal component. (B) Fraction of variance (histogram) of the principal components when PCA is applied to all 20 genes. PC1 and PC2 represent 17 and 13%, respectively. (C) The strongest loadings in the first 2 PC are depicted.

Figure 2.

Boxplots of the gene expression in PFC among the phylogenetic groups. In all cases data were log-transformed after using a human sample as a relative expression of 1, and then all the values were normalized relative to it. The genes most strongly represented in the loadings of the first 2 PCs are shown. In (A) and (B) boxplots of gene expression for the 5 genes in first PC and in the second PC are represented, respectively. For all the genes, the P-value obtained in the nonparametric Kruskal–Wallis test (df = 6) is: NEFL (P = 0.0001), DLG1 (P = 0.077), DLG4 (P = 0.043), PPP1R9B (P = 0.026), SLC17A7 (P = 0.0081), CNTNAP2 (P = 0.0055), CAMK2A (P = 0.0363), GRIA4 (P = 0.0006), NRXN1 (P = 0.051), and GRIN2B (P = 0.004). The significant paired contrasts were (P < 0.05): NEFL (OWM vs. all the other groups), DLG1 (OWM–lorises and OWM–NWM), DLG4 (human–lorises), PPP1R9B (human–chimpanzee), SLC17A7 (chimpanzee–lorises and OWM–loris), CAMK2A (chimpanzee–lorises and chimpanzee–OWM), GRIA4 (chimpanzee–NWM, chimpanzee–OWM, and human–NWM, human–OWM), NRXN1 (chimpanzee–OWM, OWM–lorises, and human–lorises), and GRIN2B (chimpanzee–lorises, human–lorises, human–NWM, human–OWM, and human–chimpanzee). Only CNTNAP2 was observed to be significantly different at Kruskal–Wallis test but then not significant after the Mann–Whitney U test for any population. Open circles represent values that fall outside the upper and lower quartiles. The asterisks (*) represent a P < 0.05 in the paired comparisons using Mann–Whitney contrasts. Note that the groups that concentrate almost the totality of the significance are chimpanzees and OWMs due to greater number of samples on them (n = 11). OWM: Old World monkeys; NWM: New World monkeys.

Figure 3.

Ancestral character state reconstruction. A phylogenetic tree of the primates used in the study and the inferred ancestral gene expression values for the genes most weighted in PC1 and PC2. The top row of trees corresponds to PC1 elements and the bottom row shows PC2 elements. Numbers on the nodes are the estimated levels of gene expression in the genes along the different evolutionary lineages. Numbers at the tips are average values from each phylogenetic group. The final expression values represent relative values; there is no attempt at quantifying the absolute level of expression. NWM: New World monkeys; OWM: Old World monkeys.

Figure 4.

Western blot analysis of proteins involved in the Ionotropic Glutamate Receptor pathway. (A) For each sample, 15 μg of PFC homogenate were loaded into a 4–12% gel and blotted against CAMK2A, SLC17A7, GRIN2B, and GRIA4 proteins. For all the cases, β-actin was used as a loading control. (B) Densitometric analysis for the genes studied. The samples were grouped and the results are presented as means ± SD. The samples used for the western blot analysis are summarized in Table 2. NWM, New World monkeys; OWM, Old World monkeys. AU, arbitrary units.