Inhibitory Systems in Brain Evolution: Pathways of Vulnerability in Neurodevelopmental Disorders.

BACKGROUND: The evolution of the primate brain has been characterized by the reorganization of key structures and circuits underlying derived specializations in sensory systems, as well as social behavior and cognition. Among these, expansion and elaboration of the prefrontal cortex has been accompanied by alterations to the connectivity and organization of subcortical structures, including the striatum and amygdala, underlying advanced aspects of executive function, inhibitory behavioral control, and socioemotional cognition seen in our lineages. At the cellular level, the primate brain has further seen an increase in the diversity and number of inhibitory GABAergic interneurons. A prevailing hypothesis holds that disruptions in the balance of excitatory to inhibitory activity in the brain underlies the pathophysiology of many neurodevelopmental and psychiatric disorders. SUMMARY: This review highlights the evolution of inhibitory brain systems and circuits and suggests that recent evolutionary modifications to GABAergic circuitry may provide the substrate for vulnerability to aberrant neurodevelopment. We further discuss how modifications to primate and human social organization and life history may shape brain development in ways that contribute to neurodivergence and the origins of neurodevelopmental disorders. KEY MESSAGES: Many brain systems have seen functional reorganization in the mammalian, primate, and human brain. Alterations to inhibitory circuitry in frontostriatal and frontoamygdalar systems support changes in social behavior and cognition. Increased complexity of inhibitory systems may underlie vulnerabilities to neurodevelopmental and psychiatric disorders, including autism and schizophrenia. Changes observed in Williams syndrome may further elucidate the mechanisms by which alterations in inhibitory systems lead to changes in behavior and cognition. Developmental processes, including altered neuroimmune function and age-related vulnerability of inhibitory cells and synapses, may lead to worsening symptomatology in neurodevelopmental and psychiatric disorders.

Altered dendritic morphology in dorsolateral prefrontal cortex of nonhuman primates prenatally exposed to maternal immune activation.

Women who contract a viral or bacterial infection during pregnancy have an increased risk of giving birth to a child with a neurodevelopmental or psychiatric disorder. The effects of maternal infection are likely mediated by the maternal immune response, as preclinical animal models have confirmed that maternal immune activation (MIA) leads to long lasting changes in offspring brain and behavior development. The present study sought to determine the impact of MIA-exposure during the first or second trimester on neuronal morphology in dorsolateral prefrontal cortex (DLPFC) and hippocampus from brain tissue obtained from MIA-exposed and control male rhesus monkey (Macaca mulatta) during late adolescence. MIA-exposed offspring display increased neuronal dendritic branching in pyramidal cells in DLPFC infra- and supragranular layers relative to controls, with no significant differences observed between offspring exposed to maternal infection in the first and second trimester. In addition, the diameter of apical dendrites in DLPFC infragranular layer is significantly decreased in MIA-exposed offspring relative to controls, irrespective of trimester exposure. In contrast, alterations in hippocampal neuronal morphology of MIA-exposed offspring were not evident. These findings demonstrate that a maternal immune challenge during pregnancy has long-term consequences for primate offspring dendritic structure, selectively in a brain region vital for socioemotional and cognitive development.

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.

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.

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.

Biodegradable poly(caprolactone fumarate) 3D printed scaffolds for segmental bone defects within the Masquelet technique.

Segmental bone defects, often clinically treated with nondegradable poly(methylmethacrylate) (PMMA) in multistage surgeries, present a significant clinical challenge. Our study investigated the efficacy of 3D printed biodegradable polycaprolactone fumarate (PCLF)/PCL spacers in a one-stage surgical intervention for these defects, focusing on early bone regeneration influenced by spacer porosities. We compared nonporous PCLF/PCL and PMMA spacers, conventionally molded into cylinders, with porous PCLF/PCL spacers, 3D printed to structurally mimic segmental defects in rat femurs for a 4-week implantation study. Histological analysis, including tissue staining and immunohistochemistry with bone-specific antibodies, was conducted for histomorphometry evaluation. The PCLF/PCL spacers demonstrated compressive properties within 6 ± 0.5 MPa (strength) and 140 ± 15 MPa (modulus). Both porous PCLF/PCL and Nonporous PMMA formed collagen-rich membranes (PCLF/PCL: 92% ± 1.3%, PMMA: 86% ± 1.5%) similar to those induced in the Masquelet technique, indicating PCLF/PCL's potential for one-stage healing. Immunohistochemistry confirmed biomarkers for tissue regeneration, underscoring PCLF/PCL's regenerative capabilities. This research highlights PCLF/PCL scaffolds' ability to induce membrane formation in critical-sized segmental bone defects, supporting their use in one-stage surgery. Both solid and porous PCLF/PCL spacers showed adequate compressive properties, with the porous variants exhibiting BMP-2 expression and woven bone formation, akin to clinical standard PMMA. Notably, the early ossification of the membrane into the pores of porous scaffolds suggests potential for bone interlocking and regeneration, potentially eliminating the need for a second surgery required for PMMA spacers. The biocompatibility and biodegradability of PCLF/PCL make them promising alternatives for treating critical bone defects, especially in vulnerable patient groups.

Impact of Maternal Immune Activation on Nonhuman Primate Prefrontal Cortex Development: Insights for Schizophrenia.

Late adolescence is a period of dynamic change in the brain as humans learn to navigate increasingly complex environments. In particular, prefrontal cortical (PFC) regions undergo extensive remodeling as the brain is fine-tuned to orchestrate cognitive control over attention, reasoning, and emotions. Late adolescence also presents a uniquely vulnerable period as neurodevelopmental illnesses, such as schizophrenia, become evident and worsen into young adulthood. Challenges in early development, including prenatal exposure to infection, may set the stage for a cascade of maladaptive events that ultimately result in aberrant PFC connectivity and function before symptoms emerge. A growing body of research suggests that activation of the mother's immune system during pregnancy may act as a disease primer, in combination with other environmental and genetic factors, contributing to an increased risk of neurodevelopmental disorders, including schizophrenia. Animal models provide an invaluable opportunity to examine the course of brain and behavioral changes in offspring exposed to maternal immune activation (MIA). Although the vast majority of MIA research has been carried out in rodents, here we highlight the translational utility of the nonhuman primate (NHP) as a model species more closely related to humans in PFC structure and function. In this review, we consider the protracted period of brain and behavioral maturation in the NHP, describe emerging findings from MIA NHP offspring in the context of rodent preclinical models, and lastly explore the translational relevance of the NHP MIA model to expand understanding of the etiology and developmental course of PFC pathology in schizophrenia.

The inhibitory effects of vancomycin on rat bone marrow-derived mesenchymal stem cell differentiation.

OBJECTIVE: The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS: BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM ß-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS: Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%-60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS: Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

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.

Social cues influence foraging in dwarf hamsters (Phodopus campbelli).

In 2 experiments, dwarf hamsters (Phodopus campbelli) were trained to find palatable foods in an open field. The location of each food patch remained the same throughout each experiment, and only 1 food was available per day. Once subjects had been trained to find each food in its unique location, they progressed to a testing phase in which subjects' mates were allowed to eat and hoard the food that was available in the open field each day. The foods that subjects' mates brought back to the home cages then served as discriminative stimuli signaling which food could be obtained in the open field. Subjects generally approached the patch containing the food hoarded by their mates, suggesting that dwarf hamster burrows could function as information centers.

Decreased Neuron Density and Increased Glia Density in the Ventromedial Prefrontal Cortex (Brodmann Area 25) in Williams Syndrome.

Williams Syndrome (WS) is a neurodevelopmental disorder caused by a deletion of 25⁻28 genes on chromosome 7 and characterized by a specific behavioral phenotype, which includes hypersociability and anxiety. Here, we examined the density of neurons and glia in fourteen human brains in Brodmann area 25 (BA 25), in the ventromedial prefrontal cortex (vmPFC), using a postmortem sample of five adult and two infant WS brains and seven age-, sex- and hemisphere-matched typically developing control (TD) brains. We found decreased neuron density, which reached statistical significance in the supragranular layers, and increased glia density and glia to neuron ratio, which reached statistical significance in both supra- and infragranular layers. Combined with our previous findings in the amygdala, caudate nucleus and frontal pole (BA 10), these results in the vmPFC suggest that abnormalities in frontostriatal and frontoamygdala circuitry may contribute to the anxiety and atypical social behavior observed in WS.

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.

Evidence for evolutionary specialization in human limbic structures.

Increasingly, functional and evolutionary research has highlighted the important contribution emotion processing makes to complex human social cognition. As such, it may be asked whether neural structures involved in emotion processing, commonly referred to as limbic structures, have been impacted in human brain evolution. To address this question, we performed an extensive evolutionary analysis of multiple limbic structures using modern phylogenetic tools. For this analysis, we combined new volumetric data for the hominoid (human and ape) amygdala and 4 amygdaloid nuclei, hippocampus, and striatum, collected using stereological methods in complete histological series, with previously published datasets on the amygdala, orbital and medial frontal cortex, and insula, as well as a non-limbic structure, the dorsal frontal cortex, for contrast. We performed a parallel analysis using large published datasets including many anthropoid species (human, ape, and monkey), but fewer hominoids, for the amygdala and 2 amygdaloid subdivisions, hippocampus, schizocortex, striatum, and septal nuclei. To address evolutionary change, we compared observed human values to values predicted from regressions run through (a) non-human hominoids and (b) non-human anthropoids, assessing phylogenetic influence using phylogenetic generalized least squares regression. Compared with other hominoids, the volumes of the hippocampus, the lateral nucleus of the amygdala, and the orbital frontal cortex were, respectively, 50, 37, and 11% greater in humans than predicted for an ape of human hemisphere volume, while the medial and dorsal frontal cortex were, respectively, 26 and 29% significantly smaller. Compared with other anthropoids, only human values for the striatum fell significantly below predicted values. Overall, the data present support for the idea that regions involved in emotion processing are not necessarily conserved or regressive, but may even be enhanced in recent human evolution.

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.