Neuroanatomical, molecular, and paleontological evidence is examined in light of human brain evolution. The brain of extant humans differs from the brains of other primates in its overall size and organization, and differences in size and organization of specific cortical areas and subcortical structures implicated into complex cognition and social and emotional processing. The human brain is also characterized by functional lateralizations, reflecting specializations of the cerebral hemispheres in humans for different types of processing, facilitating fast and reliable communication between neural cells in an enlarged brain. The features observed in the adult brain reflect human-specific patterns of brain development. Compared to the brains of other primates, the human brain takes longer to mature, promoting an extended period for establishing cortical microcircuitry and its modifications. Together, these features may underlie the prolonged period of learning and acquisition of technical and social skills necessary for survival, creating a unique cognitive and behavioral niche typical of our species. The neuroanatomical findings are in concordance with molecular analyses, which suggest a trend toward heterochrony in the expression of genes implicated in different functions. These include synaptogenesis, neuronal maturation, and plasticity in humans, mutations in genes implicated in neurite outgrowth and plasticity, and an increased role of regulatory mechanisms, potentially promoting fast modification of neuronal morphologies in response to new computational demands. At the same time, endocranial casts of fossil hominins provide an insight into the timing of the emergence of uniquely human features in the course of evolution. We conclude by proposing several ways of combining comparative neuroanatomy, molecular biology and insights gained from fossil endocasts in future research.

Background

Formalin fixation (FF) is the standard and most common method for preserving postmortem brain tissue. FF stabilizes cellular morphology and tissue architecture, and can be used to study the distinct morphologic and genetic signatures of different cell types. Although the procedure involved in FF degrades messenger RNA over time, an alternative approach is to use small RNAs (sRNAs) for genetic analysis associated with cell morphology. Although genetic analysis is carried out on fresh or frozen tissue, there is limited availability or impossibility on targeting specific cell populations, respectively.

New method

The goal of this study is to detect miRNA and other classes of sRNA stored in formalin or in paraffin embedded for over decades. Two brain samples, one formed by a mixed population of cortical and subcortical cells, and one formed by pyramidal shaped cells collected by laser-capture microdissection, were subjected to sRNA sequencing.

Results

Performing bioinformatics analysis over the sequenced sRNA from brain tissue, we detected several classes of sRNA, such as miRNAs that play key roles in brain neurodevelopmental and maintenance pathways, and hsa-mir-155 expression in neurons. Comparison with existing method: Our method is the first to combine the approaches for: laser-capture of pyramidal neurons from long-term formalin-fixed brain; extract sRNA from laser-captured pyramidal neurons; apply a suite of bioinformatics tools to detect miRNA and other classes of sRNAs on sequenced samples having high levels of RNA degradation.

Conclusion

This is the first study to show that sRNA can be rescued from laser-captured FF pyramidal neurons.

Objectives

The serotonergic system is involved in the regulation of socio-emotional behavior and heavily innervates the amygdala, a key structure of social brain circuitry. We quantified serotonergic axon density of the four major nuclei of the amygdala in humans, and examined our results in light of previously published data sets in chimpanzees and bonobos.

Materials and methods

Formalin-fixed postmortem tissue sections of the amygdala from six humans were stained for serotonin transporter (SERT) utilizing immunohistochemistry. SERT-immunoreactive (ir) axon fiber density in the lateral, basal, accessory basal, and central nuclei of the amygdala was quantified using unbiased stereology. Nonparametric statistical analyses were employed to examine differences in SERT-ir axon density between amygdaloid nuclei within humans, as well as differences between humans and previously published data in chimpanzees and bonobos.

Results

Humans displayed a unique pattern of serotonergic innervation of the amygdala, and SERT-ir axon density was significantly greater in the central nucleus compared to the lateral nucleus. SERT-ir axon density was significantly greater in humans compared to chimpanzees in the basal, accessory basal, and central nuclei. SERT-ir axon density was greater in humans compared to bonobos in the accessory basal and central nuclei.

Conclusions

The human pattern of SERT-ir axon distribution in the amygdala complements the redistribution of neurons in the amygdala in human evolution. The present findings suggest that differential serotonergic modulation of cognitive and autonomic pathways in the amygdala in humans, bonobos, and chimpanzees may contribute to species-level differences in social behavior.

Williams syndrome (WS) is a rare neurodevelopmental disorder caused by the hemideletion of approximately 25-28 genes at 7q11.23. Its unusual social and cognitive phenotype is most strikingly characterized by the disinhibition of social behavior, in addition to reduced global IQ, with a relative sparing of language ability. Hypersociality and increased social approach behavior in WS may represent a unique inability to inhibit responses to specific social stimuli, which is likely associated with abnormalities of frontostriatal circuitry. The striatum is characterized by a diversity of interneuron subtypes, including inhibitory parvalbumin-positive interneurons (PV+) and excitatory cholinergic interneurons (Ch+). Animal model research has identified an important role for these specialized cells in regulating social approach behavior. Previous research in humans identified a depletion of interneuron subtypes associated with neuropsychiatric disorders. Here, we examined the density of PV+ and Ch+ interneurons in the striatum of 13 WS and neurotypical (NT) subjects. We found a significant reduction in the density of Ch+ interneurons in the medial caudate nucleus and nucleus accumbens, important regions receiving cortical afferents from the orbitofrontal and ventromedial prefrontal cortex, and circuitry involved in language and reward systems. No significant difference in the distribution of PV+ interneurons was found. The pattern of decreased Ch+ interneuron densities in WS differs from patterns of interneuron depletion found in other disorders.

Comparative analyses of neuronal phenotypes in closely related species can shed light on neuronal changes occurring during evolution. The study of post-mortem brains of nonhuman primates (NHPs) has been limited and often does not recapitulate important species-specific developmental hallmarks. We utilize induced pluripotent stem cell (iPSC) technology to investigate the development of cortical pyramidal neurons following migration and maturation of cells grafted in the developing mouse cortex. Our results show differential migration patterns in human neural progenitor cells compared to those of chimpanzees and bonobos both in vitro and in vivo, suggesting heterochronic changes in human neurons. The strategy proposed here lays the groundwork for further comparative analyses between humans and NHPs and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species.

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes, with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioural pathologies in humans, remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome, we narrowed this cellular phenotype to a single gene candidate, frizzled 9 (FZD9). At the neuronal stage, layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.