A postmortem stereological study of the amygdala in Williams syndrome.

Perturbations to the amygdala have been observed in neurological disorders characterized by abnormalities in social behavior, such as autism and schizophrenia. Here, we quantitatively examined the amygdala in the postmortem human brains of male and female individuals diagnosed with Williams Syndrome (WS), a neurodevelopmental disorder caused by a well-defined deletion of ~ 26 genes, and accompanied by a consistent behavioral profile that includes profound hypersociability. Using unbiased stereological sampling, we estimated nucleus volume, number of neurons, neuron density, and neuron soma area in four major amygdaloid nuclei- the lateral nucleus, basal nucleus, accessory basal nucleus, and central nucleus- in a sample of five adult and two infant WS brains and seven age-, sex- and hemisphere-matched typically developing control (TD) brains. Boundaries of the four nuclei examined were drawn on Nissl-stained coronal sections as four separate regions of interest for data collection. We found that the lateral nucleus contains significantly more neurons in WS compared to TD. WS and TD do not demonstrate significant differences in neuron number in the basal, accessory basal, or central nuclei, and there are no significant differences between WS and TD in nuclei volume, neuron density, and neuron soma area in any of the four nuclei. A similarly designed study reported a decrease in lateral nucleus neuron number in autism, mirroring the opposing extremes of the two disorders in the social domain. These results suggest that the number of neurons in the lateral nucleus may contribute to pathological disturbances in amygdala function and sociobehavioral phenotype.

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.

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.

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.

Neuronal populations in the basolateral nuclei of the amygdala are differentially increased in humans compared with apes: a stereological study.

In human and nonhuman primates, the amygdala is known to play critical roles in emotional and social behavior. Anatomically, individual amygdaloid nuclei are connected with many neural systems that are either differentially expanded or conserved over the course of primate evolution. To address amygdala evolution in humans and our closest living relatives, the apes, we used design-based stereological methods to obtain neuron counts for the amygdala and each of four major amygdaloid nuclei (the lateral, basal, accessory basal, and central nuclei) in humans, all great ape species, lesser apes, and one monkey species. Our goal was to determine whether there were significant differences in the number or percent of neurons distributed to individual nuclei among species. Additionally, regression analyses were performed on independent contrast data to determine whether any individual species deviated from allometric trends. There were two major findings. In humans, the lateral nucleus contained the highest number of neurons in the amygdala, whereas in apes the basal nucleus contained the highest number of neurons. Additionally, the human lateral nucleus contained 59% more neurons than predicted by allometric regressions on nonhuman primate data. Based on the largest sample ever analyzed in a comparative study of the hominoid amygdala, our findings suggest that an emphasis on the lateral nucleus is the main characteristic of amygdala specialization over the course of human evolution.

A comparative volumetric analysis of the amygdaloid complex and basolateral division in the human and ape brain.

The amygdaloid complex functions to facilitate effective appraisal of the social environment and is an essential component of the neural systems subserving social behavior. Despite its critical role in mediating social interaction, the amygdaloid complex has not attracted the same attention as the isocortex in most evolutionary analyses. We performed a comparative analysis of the amygdaloid complex in the hominoids to address the lack of comparative information available for this structure in the hominoid brain. We demarcated the amygdaloid complex and the three nuclei constituting its basolateral division, the lateral, basal, and accessory basal nuclei, in 12 histological series representing all six hominoid species. The volumes obtained for these areas were subjected to allometric analyses to determine whether any species deviated from expected values based on the other hominoids. Differences between groups were addressed using nonparametric comparisons of means. The human lateral nucleus was larger than predicted for an ape of human brain size and occupied the majority of the basolateral division, whereas the basal nucleus was the largest of the basolateral nuclei in all ape species. In orangutans the amygdala and basolateral division were smaller than in the African apes. While the gorilla had a smaller than predicted lateral nucleus, its basal and accessory basal nuclei were larger than predicted. These differences may reflect volumetric changes occurring in interconnected cortical areas, specifically the temporal lobe and orbitofrontal cortex, which also subserve social behavior and cognition, suggesting that this system may be acted upon in hominoid and hominid evolution.

Bright light suppresses hyperactivity induced by excitotoxic dorsal hippocampus lesions in the rat.

The hippocampus has been implicated in anxiety, novelty detection, spatial- contextual processing, and hyperactivity. Accordingly, the authors contrasted the role of the dorsal hippocampus (DH) and the basolateral amygdala complex (BLA) in an open field task that presents the onset and termination of a bright light gradient. In the dark, DH rats demonstrated impaired habituation of locomotion behavior and hyperactivity, whereas in bright light their behaviors were normal. DH rats responded differentially to the onset and termination of the light stimulus, which indicates they have normal novelty detection. BLA lesion rats responded normally to bright light. These results demonstrate that a mild fear stimulus, such as bright light, can suppress DH lesion-evoked hyperactivity, and this hyperactivity results from impaired contextual processing.

Selective neurotoxic amygdala lesions in monkeys disrupt reactivity to food and object stimuli and have limited effects on memory.

Monkeys with bilateral neurotoxic amygdala lesions and normal monkeys were administered tests of emotional reactivity, recognition memory, and reward association memory. There were 3 main findings. First, monkeys with amygdala lesions performed differently than normal monkeys on initial administrations of the emotional reactivity tests and on retests that were given 21-23 months after surgery. Second, they performed like normal monkeys on tests of recognition memory. Third, they were initially impaired on a test of reward association memory, but they were not impaired on a retest that was given 16 months after surgery. These findings underscore the role of the amygdala in aspects of emotional reactivity and reward association memory, but not in recognition memory. In addition, at least some of the behavioral effects of amygdala damage can be long lasting.

Some observations on cortical inputs to the macaque monkey amygdala: an anterograde tracing study.

We have previously described the origins of neocortical inputs to the lateral nucleus of the macaque monkey amygdala based on retrograde tracing studies. Here we report results from studies that have attempted to confirm the projections from several candidate afferent regions using (3)H-amino acid autoradiography as an anterograde tracer. We have charted, based on the results of 33 separate injections, the topographic distribution of cortical projections throughout the amygdala. Areas TE and TEO of the inferotemporal cortex, portions of the superior temporal gyrus, and the granular region of the insula project primarily to the lateral nucleus, with little or no innervation of other amygdaloid nuclei. In contrast, orbitofrontal, medial prefrontal, and anterior cingulate regions project primarily to the basal and accessory basal nuclei and provide little innervation to the lateral nucleus. The orbitofrontal and medial prefrontal cortices, but not the anterior cingulate cortex, project to medially situated amygdaloid areas such as the cortical and medial nuclei and to the periamygdaloid cortex. The agranular and dysgranular insula, the parainsula, and rostral portions of the superior temporal gyrus project both to the lateral, basal, and accessory basal nuclei and to the medially situated nuclei. Projections to the central nucleus are particularly prominent from these regions. These data are discussed in relation to the hierarchical processing of sensory information that occurs within the amygdaloid complex.