A Rapid and Reliable Method of Counting Neurons and Other Cells in Brain Tissue: A Comparison of Flow Cytometry and Manual Counting Methods

It is of critical importance to understand the numbers and distributions of neurons and non-neurons in the cerebral cortex because cell numbers are reduced with normal aging and by diseases of the CNS. The isotropic fractionator method provides a faster way of estimating numbers of total cells and neurons in whole brains and dissected brain parts. Several comparative studies have illustrated the accuracy and utility of the isotropic fractionator method, yet it is a relatively new methodology, and there is opportunity to adjust procedures to optimize its efficiency and minimize error. In the present study, we use 142 samples from a dissected baboon cortical hemisphere to evaluate if isotropic fractionator counts using a Neubauer counting chamber and fluorescence microscopy could be accurately reproduced using flow cytometry methods. We find greater repeatability in flow cytometry counts, and no evidence of constant or proportional bias when comparing microscopy to flow cytometry counts. We conclude that cell number estimation using a flow cytometer is more efficient and more precise than comparable counts using a Neubauer chamber on a fluorescence microscope. This method for higher throughput, precise estimation of cell numbers has the potential to rapidly advance research in post-mortem human brains and vastly improve our understanding of cortical and subcortical structures in normal, injured, aged, and diseased brains

Variation in the cortical area map of C57BL/6J and DBA/2J inbred mice predicts strain identity

BACKGROUND: Recent discoveries suggest that arealization of the mammalian cortical sheet develops in a manner consonant with principles established for embryonic patterning of the body. Signaling centers release morphogens that determine regional growth and tissue identity by regulating regional expression of transcription factors. Research on mouse cortex has identified several candidate morphogens that affect anteroposterior or mediolateral cortical regionalization as well as mitogenesis. Inbred strains of laboratory mice can be exploited to study cortical area map formation if there are significant phenotypic differences with which to correlate gene polymorphism or expression data. Here we describe differences in the cortical area map of two commonly used inbred strains of laboratory mice, C57BL/6J and DBA/2J. Complete cortical hemispheres from adult mice were dissected and stained for the cytochrome oxidase enzyme in order to measure histochemically defined cortical areas. RESULTS: C57BL/6J has the larger neocortex, relatively larger primary visual cortex (V1), but relatively smaller posterior medial barrel subfield of the primary somatosensory cortex (PMBSF). The sample of C57BL/6J and DBA/2J mice can be discriminated with 90% accuracy on the basis of these three size dimensions. CONCLUSION: C57BL/6J and DBA/2J have markedly different cortical area maps, suggesting that inbred strains harbor enough phenotypic variation to encourage a forward genetic approach to understanding cortical development, complementing other approaches

Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor

Using gene expression databases for classical trait QTL candidate gene discovery in the BXD recombinant inbred genetic reference population: Mouse forebrain weight

<p>Abstract</p> <p>Background</p> <p>Successful strategies for QTL gene identification benefit from combined experimental and bioinformatic approaches. Unique design aspects of the BXD recombinant inbred line mapping panel allow use of archived gene microarray expression data to filter likely from unlikely candidates. This prompted us to propose a simple five-filter protocol for candidate nomination. To filter more likely from less likely candidates, we required candidate genes near to the QTL to have mRNA abundance that correlated with the phenotype among the BXD lines as well as differed between the parental lines C57BL/6J and DBA/2J. We also required verification of mRNA abundance by an independent method, and finally we required either differences in protein levels or confirmed DNA sequence differences.</p> <p>Results</p> <p>QTL mapping of mouse forebrain weight in 34 BXD RI lines found significant association on chromosomes 1 and 11, with each C57BL/6J allele increasing weight by more than half a standard deviation. The intersection of gene lists that were within ± 10 Mb of the strongest associated location, that had forebrain mRNA abundance correlated with forebrain weight among the BXD, and that had forebrain mRNA abundance differing between C57BL/6J and DBA/2J, produced two candidates, <it>Tnni1 </it>(troponin 1) and <it>Asb3 </it>(ankyrin repeat and SOCS box-containing protein 3). Quantitative RT-PCR confirmed the direction of an increased expression in C57BL/6J genotype over the DBA/2J genotype for both genes, a difference that translated to a 2-fold difference in Asb3 protein. Although Tnni1 protein differences could not be confirmed, a 273 bp indel polymorphism was discovered 1 Kb upstream of the transcription start site.</p> <p>Conclusion</p> <p>Delivery of well supported candidate genes following a single quantitative trait locus mapping experiment is difficult. However, by combining available gene expression data with QTL mapping, we illustrated a five-filter protocol that nominated <it>Asb3 </it>and <it>Tnni1 </it>as candidates affecting increased mouse forebrain weight. We recommend our approach when (1) investigators are working with phenotypic differences between C57BL/6J and DBA/2J, and (2) gene expression data are available on <url>http://www.genenetwork.org</url> that relate to the phenotype of interest. Under these circumstances, measurement of the phenotype in the BXD lines will likely also deliver excellent candidate genes.</p

Geometric morphometrics defines shape differences in the cortical area map of C57BL/6J and DBA/2J inbred mice

BACKGROUND: We previously described planar areal differences in adult mouse visual, somatosensory, and neocortex that collectively discriminated C57BL/6J and DBA/2J inbred strain identity. Here we use a novel application of established methods of two-dimensional geometric morphometrics to examine shape differences in the cortical area maps of these inbred strains. RESULTS: We used Procrustes superimposition to align a reliable set of landmarks in the plane of the cortical sheet from tangential sections stained for the cytochrome oxidase enzyme. Procrustes superimposition translates landmark configurations to a common origin, scales them to a common size, and rotates them to minimize an estimate of error. Remaining variation represents shape differences. We compared the variation in shape between C57BL/6J and DBA/2J relative to that within each strain using a permutation test of Goodall's F statistic. Significant differences in shape in the posterior medial barrel subfield (PMBSF), as well as differences in shape across primary sensory areas, characterize the cortical area maps of these common inbred, isogenic strains. CONCLUSION: C57BL/6J and DBA/2J have markedly different cortical area maps, in both size and shape. These differences suggest polymorphism in genetic factors underlying cortical specification, even between common isogenic strains. Comparing cortical phenotypes between normally varying inbred mice or between genetically modified mice can identify genetic contributions to cortical specification. Geometric morphometric analysis of shape represents an additional quantitative tool for the study of cortical development, regardless of whether it is studied from phenotype to gene or gene to phenotype

Genetics of the Hippocampal Transcriptome in Mouse: A Systematic Survey and Online Neurogenomics Resource

Differences in gene expression in the CNS influence behavior and disease susceptibility. To systematically explore the role of normal variation in expression on hippocampal structure and function, we generated an online microarray database for a diverse panel of strains of mice, including most common inbred strains and numerous recombinant inbred lines (www.genenetwork.org). Using this resource, coexpression networks for families of genes can be generated rapidly to test causal models related to function. The data set is optimized for quantitative trait locus (QTL) mapping and was used to identify over 5500 QTLs that modulate mRNA levels. We describe a wide variety of analyses and novel synthetic approaches that take advantage of this resource, and demonstrate how both the data and associated tools can be applied to the study of gene regulation in the hippocampus and relations to structure and function

A Rapid New Assay to Detect RNA Editing Reveals Antipsychotic-Induced Changes in Serotonin-2C Transcripts

ABSTRACT We report the development of a new assay as an alternative to direct DNA sequencing to measure RNA-edited variation in tissue. The new assay has been validated and is accurate, cheaper, more rapid, and less labor-intensive than DNA sequencing. We also outline the statistical modeling required for analyses of the hierarchical, clustered RNA-editing data generated in these studies. Using the new technique, we analyzed the effects of long-term antipsychotic medication on serotonin-2C receptor (5-HT 2C R) RNA editing in rat brain. Our hypothesis that a drug with high affinity for 5-HT 2C R, such as clozapine, would alter its RNA-editing profile was not confirmed. Whereas haloperidol, a typical antipsychotic drug that is primarily a dopamine receptor antagonist, reduced 5-HT 2C VNV isoform frequency and the level of RNA editing at the D site, risperidone and not the prototype atypical antipsychotic drug clozapine increased the frequency of 5-HT 2C VNV and D-site editing. Our data emphasize that caution is required in the interpretation of RNA-editing data in studies of psychiatric disorders, because these studies usually include subjects who received long-term exposure to medication. This newly established method will facilitate high-throughput investigations of RNA editing in disease pathology and in the pharmacological activity of drugs. The revelation that the human genome comprises between 30,000 and 40,000 protein-coding genes was unexpected, because such few genes are unlikely to explain the functional diversity between humans and less complex organisms. These interspecies differences could arise from post-transcriptional processes such as RNA editing and alternative splicing, which allow a single gene to generate several protein variants. The current study focuses on RNA editing. The recent detection of 1637 potential new substrates for RNA editing Because of their relatively recent discovery, substrates of RNA editing have not been extensively characterized. Adenine-to-inosine RNA editing in mammalian brain has been identified in ionotropic receptors (such as the GluR2 subunit of the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor) and in the serotonin (5-HT)-2C receptor (R), which is coupled to GTP-binding protein. Discrepancies between genomic DNA and cDNA sequences led to the discovery of nucleotide changes caused by the activity of ADAR enzymes. In the fully edited 5-HT 2C R, three amino acid codons in the pre-mRNA are changed so that the sequence coding for IRNPI becomes VRGP

Cross-Sectional Exploration of Plasma Biomarkers of Alzheimer\u27s Disease in Down Syndrome: Early Data from the Longitudinal Investigation for Enhancing Down Syndrome Research (LIFE-DSR) Study

With improved healthcare, the Down syndrome (DS) population is both growing and aging rapidly. However, with longevity comes a very high risk of Alzheimer’s disease (AD). The LIFE-DSR study (NCT04149197) is a longitudinal natural history study recruiting 270 adults with DS over the age of 25. The study is designed to characterize trajectories of change in DS-associated AD (DS-AD). The current study reports its cross-sectional analysis of the first 90 subjects enrolled. Plasma biomarkers phosphorylated tau protein (p-tau), neurofilament light chain (NfL), amyloid β peptides (Aβ1-40, Aβ1-42), and glial fibrillary acidic protein (GFAP) were undertaken with previously published methods. The clinical data from the baseline visit include demographics as well as the cognitive measures under the Severe Impairment Battery (SIB) and Down Syndrome Mental Status Examination (DS-MSE). Biomarker distributions are described with strong statistical associations observed with participant age. The biomarker data contributes to understanding DS-AD across the spectrum of disease. Collectively, the biomarker data show evidence of DS-AD progression beginning at approximately 40 years of age. Exploring these data across the full LIFE-DSR longitudinal study population will be an important resource in understanding the onset, progression, and clinical profiles of DS-AD pathophysiology

Pollutants Increase Song Complexity and the Volume of the Brain Area HVC in a Songbird

Environmental pollutants which alter endocrine function are now known to decrease vertebrate reproductive success. There is considerable evidence for endocrine disruption from aquatic ecosystems, but knowledge is lacking with regard to the interface between terrestrial and aquatic ecosystems. Here, we show for the first time that birds foraging on invertebrates contaminated with environmental pollutants, show marked changes in both brain and behaviour. We found that male European starlings (Sturnus vulgaris) exposed to environmentally relevant levels of synthetic and natural estrogen mimics developed longer and more complex songs compared to control males, a sexually selected trait important in attracting females for reproduction. Moreover, females preferred the song of males which had higher pollutant exposure, despite the fact that experimentally dosed males showed reduced immune function. We also show that the key brain area controlling male song complexity (HVC) is significantly enlarged in the contaminated birds. This is the first evidence that environmental pollutants not only affect, but paradoxically enhance a signal of male quality such as song. Our data suggest that female starlings would bias their choice towards exposed males, with possible consequences at the population level. As the starling is a migratory species, our results suggest that transglobal effects of pollutants on terrestrial vertebrate physiology and reproduction could occur in birds