Optimization of electron microscopy for human brains with long-term fixation and fixed-frozen sections.

BackgroundAbnormal connectivity across brain regions underlies many neurological disorders including multiple sclerosis, schizophrenia and autism, possibly due to atypical axonal organization within white matter. Attempts at investigating axonal organization on post-mortem human brains have been hindered by the availability of high-quality, morphologically preserved tissue, particularly for neurodevelopmental disorders such as autism. Brains are generally stored in a fixative for long periods of time (often greater than 10 years) and in many cases, already frozen and sectioned on a microtome for histology and immunohistochemistry. Here we present a method to assess the quality and quantity of axons from long-term fixed and frozen-sectioned human brain samples to demonstrate their use for electron microscopy (EM) measures of axonal ultrastructure.ResultsSix samples were collected from white matter below the superior temporal cortex of three typically developing human brains and prepared for EM analyses. Five samples were stored in fixative for over 10 years, two of which were also flash frozen and sectioned on a freezing microtome, and one additional case was fixed for 3 years and sectioned on a freezing microtome. In all six samples, ultrastructural qualitative and quantitative analyses demonstrate that myelinated axons can be identified and counted on the EM images. Although axon density differed between brains, axonal ultrastructure and density was well preserved and did not differ within cases for fixed and frozen tissue. There was no significant difference between cases in axon myelin sheath thickness (g-ratio) or axon diameter; approximately 70% of axons were in the small (0.25 μm) to medium (0.75 μm) range. Axon diameter and g-ratio were positively correlated, indicating that larger axons may have thinner myelin sheaths.ConclusionThe current study demonstrates that long term formalin fixed and frozen-sectioned human brain tissue can be used for ultrastructural analyses. Axon integrity is well preserved and can be quantified using the methods presented here. The ability to carry out EM on frozen sections allows for investigation of axonal organization in conjunction with other cellular and histological methods, such as immunohistochemistry and stereology, within the same brain and even within the same frozen cut section

Atypical miRNA expression in temporal cortex associated with dysregulation of immune, cell cycle, and other pathways in autism spectrum disorders.

BackgroundAutism spectrum disorders (ASDs) likely involve dysregulation of multiple genes related to brain function and development. Abnormalities in individual regulatory small non-coding RNA (sncRNA), including microRNA (miRNA), could have profound effects upon multiple functional pathways. We assessed whether a brain region associated with core social impairments in ASD, the superior temporal sulcus (STS), would evidence greater transcriptional dysregulation of sncRNA than adjacent, yet functionally distinct, primary auditory cortex (PAC).MethodsWe measured sncRNA expression levels in 34 samples of postmortem brain from STS and PAC to find differentially expressed sncRNA in ASD compared with control cases. For differentially expressed miRNA, we further analyzed their predicted mRNA targets and carried out functional over-representation analysis of KEGG pathways to examine their functional significance and to compare our findings to reported alterations in ASD gene expression.ResultsTwo mature miRNAs (miR-4753-5p and miR-1) were differentially expressed in ASD relative to control in STS and four (miR-664-3p, miR-4709-3p, miR-4742-3p, and miR-297) in PAC. In both regions, miRNA were functionally related to various nervous system, cell cycle, and canonical signaling pathways, including PI3K-Akt signaling, previously implicated in ASD. Immune pathways were only disrupted in STS. snoRNA and pre-miRNA were also differentially expressed in ASD brain.ConclusionsAlterations in sncRNA may underlie dysregulation of molecular pathways implicated in autism. sncRNA transcriptional abnormalities in ASD were apparent in STS and in PAC, a brain region not directly associated with core behavioral impairments. Disruption of miRNA in immune pathways, frequently implicated in ASD, was unique to STS

Possible sexually dimorphic role of miRNA and other sncRNA in ASD brain

BackgroundAutism spectrum disorder (ASD) is sexually dimorphic in brain structure, genetics, and behaviors. In studies of brain tissue, the age of the population is clearly a factor in interpreting study outcome, yet sex is rarely considered. To begin to address this issue, we extend our previously published microarray analyses to examine expression of small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), in ASD and in the control temporal cortex in males and females. Predicted miRNA targets were identified as well as the pathways they overpopulate.FindingsAfter considering age, sexual dimorphism in ASD sncRNA expression persists in the temporal cortex and in the patterning that distinguishes regions. Among the sexually dimorphic miRNAs are miR-219 and miR-338, which promote oligodendrocyte differentiation, miR-125, implicated in neuronal differentiation, and miR-488, implicated in anxiety. Putative miRNA targets are significantly over-represented in immune and nervous system pathways in both sexes, consistent with previous mRNA studies. Even for common pathways, the specific target mRNAs are often sexually dimorphic. For example, both male and female target genes significantly populate the Axonal Guidance Signaling pathway, yet less than a third of the targets are common to both sexes.ConclusionsOur findings of sexual dimorphism in sncRNA levels underscore the importance of considering sex, in addition to age, when interpreting molecular findings on ASD brain

Neuron numbers increase in the human amygdala from birth to adulthood, but not in autism.

Remarkably little is known about the postnatal cellular development of the human amygdala. It plays a central role in mediating emotional behavior and has an unusually protracted development well into adulthood, increasing in size by 40% from youth to adulthood. Variation from this typical neurodevelopmental trajectory could have profound implications on normal emotional development. We report the results of a stereological analysis of the number of neurons in amygdala nuclei of 52 human brains ranging from 2 to 48 years of age [24 neurotypical and 28 autism spectrum disorder (ASD)]. In neurotypical development, the number of mature neurons in the basal and accessory basal nuclei increases from childhood to adulthood, coinciding with a decrease of immature neurons within the paralaminar nucleus. Individuals with ASD, in contrast, show an initial excess of amygdala neurons during childhood, followed by a reduction in adulthood across nuclei. We propose that there is a long-term contribution of mature neurons from the paralaminar nucleus to other nuclei of the neurotypical human amygdala and that this growth trajectory may be altered in ASD, potentially underlying the volumetric changes detected in ASD and other neurodevelopmental or neuropsychiatric disorders

Preliminary evidence of increased striatal dopamine in a nonhuman primate model of maternal immune activation.

Women exposed to a variety of viral and bacterial infections during pregnancy have an increased risk of giving birth to a child with autism, schizophrenia or other neurodevelopmental disorders. Preclinical maternal immune activation (MIA) models are powerful translational tools to investigate mechanisms underlying epidemiological links between infection during pregnancy and offspring neurodevelopmental disorders. Our previous studies documenting the emergence of aberrant behavior in rhesus monkey offspring born to MIA-treated dams extends the rodent MIA model into a species more closely related to humans. Here we present novel neuroimaging data from these animals to further explore the translational potential of the nonhuman primate MIA model. Nine male MIA-treated offspring and 4 controls from our original cohort underwent in vivo positron emission tomography (PET) scanning at approximately 3.5-years of age using [18F] fluoro-l-m-tyrosine (FMT) to measure presynaptic dopamine levels in the striatum, which are consistently elevated in individuals with schizophrenia. Analysis of [18F]FMT signal in the striatum of these nonhuman primates showed that MIA animals had significantly higher [18F]FMT index of influx compared to control animals. In spite of the modest sample size, this group difference reflects a large effect size (Cohen's d = 0.998). Nonhuman primates born to MIA-treated dams exhibited increased striatal dopamine in late adolescence-a hallmark molecular biomarker of schizophrenia. These results validate the MIA model in a species more closely related to humans and open up new avenues for understanding the neurodevelopmental biology of schizophrenia and other neurodevelopmental disorders associated with prenatal immune challenge

Neuroprotective efficacy of P7C3 compounds in primate hippocampus.

There is a critical need for translating basic science discoveries into new therapeutics for patients suffering from difficult to treat neuropsychiatric and neurodegenerative conditions. Previously, a target-agnostic in vivo screen in mice identified P7C3 aminopropyl carbazole as capable of enhancing the net magnitude of postnatal neurogenesis by protecting young neurons from death. Subsequently, neuroprotective efficacy of P7C3 compounds in a broad spectrum of preclinical rodent models has also been observed. An important next step in translating this work to patients is to determine whether P7C3 compounds exhibit similar efficacy in primates. Adult male rhesus monkeys received daily oral P7C3-A20 or vehicle for 38 weeks. During weeks 2-11, monkeys received weekly injection of 5'-bromo-2-deoxyuridine (BrdU) to label newborn cells, the majority of which would normally die over the following 27 weeks. BrdU+ cells were quantified using unbiased stereology. Separately in mice, the proneurogenic efficacy of P7C3-A20 was compared to that of NSI-189, a proneurogenic drug currently in clinical trials for patients with major depression. Orally-administered P7C3-A20 provided sustained plasma exposure, was well-tolerated, and elevated the survival of hippocampal BrdU+ cells in nonhuman primates without adverse central or peripheral tissue effects. In mice, NSI-189 was shown to be pro-proliferative, and P7C3-A20 elevated the net magnitude of hippocampal neurogenesis to a greater degree than NSI-189 through its distinct mechanism of promoting neuronal survival. This pilot study provides evidence that P7C3-A20 safely protects neurons in nonhuman primates, suggesting that the neuroprotective efficacy of P7C3 compounds is likely to translate to humans as well

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

Preliminary evidence of neuropathology in nonhuman primates prenatally exposed to maternal immune activation

Maternal infection during pregnancy increases the risk for neurodevelopmental disorders in offspring. Rodent models have played a critical role in establishing maternal immune activation (MIA) as a causal factor for altered brain and behavioral development in offspring. We recently extended these findings to a species more closely related to humans by demonstrating that rhesus monkeys (Macaca mulatta) prenatally exposed to MIA also develop abnormal behaviors. Here, for the first time, we present initial evidence of underlying brain pathology in this novel nonhuman primate MIA model. Pregnant rhesus monkeys were injected with a modified form of the viral mimic polyI:C (poly ICLC) or saline at the end of the first trimester. Brain tissue was collected from the offspring at 3.5 years and blocks of dorsolateral prefrontal cortex (BA46) were used to analyze neuronal dendritic morphology and spine density using the Golgi-Cox impregnation method. For each case, 10 layer III pyramidal cells were traced in their entirety, including all apical, oblique and basal dendrites, and their spines. We further analyzed somal size and apical dendrite trunk morphology in 30 cells per case over a 30 μm section located 100 ± 10 μm from the soma. Compared to controls, apical dendrites of MIA-treated offspring were smaller in diameter and exhibited a greater number of oblique dendrites. These data provide the first evidence that prenatal exposure to MIA alters dendritic morphology in a nonhuman primate MIA model, which may have profound implications for revealing the underlying neuropathology of neurodevelopmental disorders related to maternal infection

The hippocampi of children with chromosome 22q11.2 deletion syndrome have localized anterior alterations that predict severity of anxiety

BACKGROUND: Individuals with 22q11.2 deletion syndrome (22q11.2DS) have an elevated risk for schizophrenia, which increases with history of childhood anxiety. Altered hippocampal morphology is a common neuroanatomical feature of 22q11.2DS and idiopathic schizophrenia. Relating hippocampal structure in children with 22q11.2DS to anxiety and impaired cognitive ability could lead to hippocampus-based characterization of psychosis-proneness in this at-risk population. METHODS: We measured hippocampal volume using a semiautomated approach on MRIs collected from typically developing children and children with 22q11.2DS. We then analyzed hippocampal morphology with Localized Components Analysis. We tested the modulating roles of diagnostic group, hippocampal volume, sex and age on local hippocampal shape components. Lastly, volume and shape components were tested as covariates of IQ and anxiety. RESULTS: We included 48 typically developing children and 69 children with 22q11.2DS in our study. Hippocampal volume was reduced bilaterally in children with 22q11.2DS, and these children showed greater variation in the shape of the anterior hippocampus than typically developing children. Children with 22q11.2DS had greater inward deformation of the anterior hippocampus than typically developing children. Greater inward deformation of the anterior hippocampus was associated with greater severity of anxiety, specifically fear of physical injury, within the 22q11.2DS group. LIMITATIONS: Shape alterations are not specific to hippocampal subfields. CONCLUSION: Alterations in the structure of the anterior hippocampus likely affect function and may impact limbic circuitry. We suggest these alterations potentially contribute to anxiety symptoms in individuals with 22q11.2DS through modulatory pathways. Altered hippocampal morphology may be uniquely linked to anxiety risk factors for schizophrenia, which could be a powerful neuroanatomical marker of schizophrenia risk and hence protection