Decreased density of cholinergic interneurons in striatal territories in Williams syndrome.

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.

Neurodevelopmental disorders of the prefrontal cortex in an evolutionary context.

The prefrontal cortex consists of several cytoarchitectonically defined areas that are involved in higher-order cognitive and emotional processing. The areas are highly variable in terms of organization of cortical layers and distribution of specific neuronal classes, and are affected in neurodevelopmental and psychiatric disorders. Here the focus is on microstructural anatomical characteristics of human prefrontal cortex in an evolutionary context with special emphasis on Williams syndrome. We include a pilot analysis of distribution of neurons labeled with an antibody to non-phosphorylated neurofilament protein (SMI-32) in the frontal pole of Williams syndrome to further examine microstructural characteristics of the prefrontal cortex in Williams syndrome and implications of the distribution of SMI-32 immunoreactive neurons for connectivity between the frontal pole and other cortical areas in the disorder.

Serotonergic innervation of the human amygdala and evolutionary implications.

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.

Species-specific maturation profiles of human, chimpanzee and bonobo neural cells.

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.

Biphasic dendritic growth of dorsolateral prefrontal cortex associative neurons and early cognitive development.

AIM: To analyze postnatal development and life-span changes of apical dendrite side branches (oblique dendrites) from associative layer IIIC magnopyramidal neurons in the human dorsolateral prefrontal cortex and to compare the findings with the previously established pattern of basal dendrite development. METHODS: We analyzed dendritic morphology from 352 rapid-Golgi impregnated neurons (10-18 neurons per subject) in Brodmann area 9 from the post-mortem tissue of 25 subjects ranging in age from 1 week to 91 years. Data were collected in the period between 1994 and 1996, and the analysis was performed between September 2017 and February 2018. Quantitative dendritic parameters were statistically analyzed using one-way analysis of variance and two-tailed t tests. RESULTS: Oblique dendrites grew rapidly during the first postnatal months, and the increase in the dendrite length was accompanied by the outgrowth of new dendritic segments. After a more than one-year-long "dormant" period of only fine dendritic rearrangements (2.5-16 months), oblique dendrites displayed a second period of marked growth, continuing through the third postnatal year. Basal and oblique dendrites displayed roughly the same growth pattern, but had considerably different topological organization in adulthood. CONCLUSION: Our analysis confirmed that a biphasic pattern of postnatal dendritic development, together with a second growth spurt at the age of 2-3 years, represents a unique feature of the associative layer IIIC magnopyramidal neurons in the human dorsolateral prefrontal cortex. We propose that these structural changes relate to rapid cognitive development during early childhood.

Increased glia density in the caudate nucleus in williams syndrome: Implications for frontostriatal dysfunction in autism.

Williams syndrome (WS) is a rare neurodevelopmental disorder with a well-described, known genetic etiology. In contrast to Autism Spectrum Disorders (ASD), WS has a unique phenotype characterized by global reductions in IQ and visuospatial ability, with relatively preserved language function, enhanced reactivity to social stimuli and music, and an unusual eagerness to interact socially with strangers. A duplication of the deleted region in WS has been implicated in a subset of ASD cases, defining a spectrum of genetic and behavioral variation at this locus defined by these opposite extremes in social behavior. The hypersociability characteristic of WS may be linked to abnormalities of frontostriatal circuitry that manifest as deficits in inhibitory control of behavior. Here, we examined the density of neurons and glia in associative and limbic territories of the striatum including the caudate, putamen, and nucleus accumbens regions in Nissl stained sections in five pairs of age, sex, and hemisphere-matched WS and typically-developing control (TD) subjects. In contrast to what is reported in ASD, no significant increase in overall neuron density was observed in this study. However, we found a significant increase in the density of glia in the dorsal caudate nucleus, and in the ratio of glia to neurons in the dorsal and medial caudate nucleus in WS, accompanied by a significant increase in density of oligodendrocytes in the medial caudate nucleus. These cellular abnormalities may underlie reduced frontostriatal activity observed in WS, with implications for understanding altered connectivity and function in ASD. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 531-545, 2018.

Basal Dendritic Morphology of Cortical Pyramidal Neurons in Williams Syndrome: Prefrontal Cortex and Beyond.

Williams syndrome (WS) is a unique neurodevelopmental disorder with a specific behavioral and cognitive profile, which includes hyperaffiliative behavior, poor social judgment, and lack of social inhibition. Here we examined the morphology of basal dendrites on pyramidal neurons in the cortex of two rare adult subjects with WS. Specifically, we examined two areas in the prefrontal cortex (PFC)-the frontal pole (Brodmann area 10) and the orbitofrontal cortex (Brodmann area 11)-and three areas in the motor, sensory, and visual cortex (BA 4, BA 3-1-2, BA 18). The findings suggest that the morphology of basal dendrites on the pyramidal neurons is altered in the cortex of WS, with differences that were layer-specific, more prominent in PFC areas, and displayed an overall pattern of dendritic organization that differentiates WS from other disorders. In particular, and unlike what was expected based on typically developing brains, basal dendrites in the two PFC areas did not display longer and more branched dendrites compared to motor, sensory and visual areas. Moreover, dendritic branching, dendritic length, and the number of dendritic spines differed little within PFC and between the central executive region (BA 10) and BA 11 that is part of the orbitofrontal region involved into emotional processing. In contrast, the relationship between the degree of neuronal branching in supra- versus infra-granular layers was spared in WS. Although this study utilized tissue held in formalin for a prolonged period of time and the number of neurons available for analysis was limited, our findings indicate that WS cortex, similar to that in other neurodevelopmental disorders such as Down syndrome, Rett syndrome, Fragile X, and idiopathic autism, has altered morphology of basal dendrites on pyramidal neurons, which appears more prominent in selected areas of the PFC. Results were examined from developmental perspectives and discussed in the context of other neurodevelopmental disorders. We have proposed hypotheses for further investigations of morphological changes on basal dendrites in WS, a syndrome of particular interest given its unique social and cognitive phenotype.

A human neurodevelopmental model for Williams syndrome.

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.

A dual comparative approach: integrating lines of evidence from human evolutionary neuroanatomy and neurodevelopmental disorders.

The evolution of the human brain has been marked by a nearly 3-fold increase in size since our divergence from the last common ancestor shared with chimpanzees and bonobos. Despite increased interest in comparative neuroanatomy and phylogenetic methods, relatively little is known regarding the effects that this enlargement has had on its internal organization, and how certain areas of the brain have differentially expanded over evolutionary time. Analyses of the microstructure of several regions of the human cortex and subcortical structures have demonstrated subtle changes at the cellular and molecular level, suggesting that the human brain is more than simply a 'scaled-up' primate brain. Ongoing research in comparative neuroanatomy has much to offer regarding our understanding of human brain evolution. Through analysis of the neuroanatomical phenotype at the level of reorganization in cytoarchitecture and cellular morphology, new data continue to highlight changes in cell density and organization associated with volumetric changes in discrete regions. An understanding of the functional significance of variation in neural circuitry can further be approached through studies of atypical human development. Many neurodevelopmental disorders cause disruption in systems associated with uniquely human features of cognition, including language and social cognition. Understanding the genetic and developmental mechanisms that underlie variation in the human cognitive phenotype can help to clarify the functional significance of interspecific variation. By uniting approaches from comparative neuroanatomy and neuropathology, insights can be gained that clarify trends in human evolution. Here, we explore these lines of evidence and their significance for understanding functional variation between species as well as within neuropathological variation in the human brain.

Micro RNA detection in long-term fixed tissue of cortical glutamatergic pyramidal neurons after targeted laser-capture neuroanatomical microdissection.

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.

Novel tools, classic techniques: evolutionary studies using primate pluripotent stem cells.

Recent applications of genomic tools on the analysis of alterations unique to our species coupled with a growing number of neuroanatomical studies across primates provide an unprecedented opportunity to compile different levels of human brain evolution into a complex whole. Applications of induced pluripotent stem cell (iPSC) technology, capable of reprogramming somatic tissue of different species and generating species-specific neuronal phenotypes, for the first time offer an opportunity to test specific evolutionary hypotheses in a field of inquiry that has been long plagued by the limited availability of research specimens. In this review, we will focus specifically on the experimental role of iPSC technology as applied to the analysis of neocortical pyramidal neurons. Pyramidal neurons emerge as particularly suitable for testing evolutionary scenarios, since they form the most common morphological class of neurons in the cortex, display morphological variations across different cortical areas and cortical layers that appear species-specific, and express unique molecular signatures. Human and nonhuman primate iPSC-derived neurons may represent a unique biological resource to elucidate the phenotypic differences between humans and other hominids. As the typical morphology of pyramidal neurons tends to be compromised in neurological disorders, application of iPSC technology to the analysis of pyramidal neurons could not only bring new insights into human adaptation but also offer opportunities to link biomedical research with studies of the origins of the human species.

Evolution, development, and plasticity of the human brain: from molecules to bones.

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.