Region-specific Manner Research Articles

Abstract 3434: Digital spatial profiling platform allows for spatially resolved, high-plex quantification of mRNA distribution and abundance on FFPE and fresh frozen tissue sections

Abstract Characterization of spatial mRNA expression across multiple targets in a single tissue section has proven difficult to accomplish. To address this unmet need, we have developed a novel imaging platform, Digital Spatial Profiling (DSP), designed to simultaneously analyze 10s to 100s of RNAs (or proteins) from discrete regions by detecting oligo-conjugated probes. We show that this system can resolve mRNA expression panels in a highly multiplexed and automated manner on human FFPE and fresh frozen tissues. DSP RNA probes consist of antisense target-recognition sequences and UV-cleavable tags that can be released in a precise manner by the DSP instrument and digitally counted using nCounter® reagents. For this, probes are first hybridized overnight in situ to human FFPE or fresh frozen tissue sections. After hybridization, tags are released by nondestructive exposure of UV light, automatically collected by the DSP Instrument, and quantified using nCounter® technology. The nCounter PlexSet™ reagents are used to enable the analysis of 96 regions of interest with a single nCounter run. To demonstrate the regional specificity of RNA detection on DSP, we profiled distinct regions in human tonsillitis tissues using a 48-plex of RNA target probes. Our results show that genes are detected in the expected region-specific manner. Using an orthogonal in situ hybridization (ISH) approach, we validate the RNA DSP results in tonsil across multiple targets. To determine the regionally specific RNA expression in tumor area and nontumor area in non-small cell lung cancer (NCSLC), we used a high-plex probe mix of relevant Immuno-oncology RNA targets. These results show that the DSP platform can be used to obtain high-plex, spatial RNA expression data on tissue sections. The protein profiling assay on DSP, presented elsewhere, depends upon FFPE-compatible antibodies, which can be difficult to generate for some targets. Utilizing spatial mRNA profiling provides an alternative for cases where the necessary antibodies do not exist. In all, simultaneous, high-plex RNA and protein detection with DSP on serial sections could provide a novel method that can bridge the gap between translational research discovery and clinical applications. Citation Format: Daniel Zollinger, Kristina Sorg, Jill McKay-Fleisch, Kristi Barker, Karen Nguyen, Chris Merritt, Joseph Beechem. Digital spatial profiling platform allows for spatially resolved, high-plex quantification of mRNA distribution and abundance on FFPE and fresh frozen tissue sections [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3434.

Methylenetetrahydrofolate Reductase Deficiency Deregulates Regional Brain Amyloid-β Protein Precursor Expression and Phosphorylation Levels.

Deregulation of the amyloid-β protein precursor (AβPP) plays a critical role in the neurodegenerative cascade of Alzheimer's disease (AD). Significantly, common functional polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene are a risk factor for the development of late-onset AD. Reduced MTHFR activity is associated with alterations in folate and homocysteine metabolism. Here, we first show that in young MTHFR knockout mice, mild and severe MTHFR deficiency markedly increase cortical and hippocampal AβPP phosphorylation at the regulatory Thr668 site. However, the hippocampus is especially vulnerable to the effects of aging and mild MTHFR deficiency. Notably, the effects of severe MTHFR deficiency in young mice are recapitulated by prolonged dietary folate deficiency in old mice, which leads to regional brain accumulation of cystathionine due to impaired methylation of homocysteine. The incremental AβPP phosphorylation at Thr668 mediated by severe genetic-or diet-induced impairment of the folate cycle correlates with enhanced accumulation of demethylated protein phosphatase 2A (PP2A), and activation of glycogen synthase kinase-3β (GSK-3β). Lastly, we show that severe disturbances in folate metabolism can also affect AβPP expression levels in a brain region specific manner. Together our findings identify a novel link between genetic MTHFR deficiency, activation of GSK-3β, demethylation of PP2A, and enhanced phosphorylation of AβPP at Thr668, which is known to critically influence neuronal AβPP function and pathological amyloidogenic processing. Deregulation of AβPP provides a novel mechanism by which common human MTHFR polymorphisms may interact with dietary folate deficiency to alter neuronal homeostasis and increase the risk for sporadic AD.

Integrative Analysis of Hippocampus Gene Expression Profiles Identifies Network Alterations in Aging and Alzheimer's Disease.

Alzheimer’s disease (AD) is a neurodegenerative disorder contributing to rapid decline in cognitive function and ultimately dementia. Most cases of AD occur in elderly and later years. There is a growing need for understanding the relationship between aging and AD to identify shared and unique hallmarks associated with the disease in a region and cell-type specific manner. Although genomic studies on AD have been performed extensively, the molecular mechanism of disease progression is still not clear. The major objective of our study is to obtain a higher-order network-level understanding of aging and AD, and their relationship using the hippocampal gene expression profiles of young (20–50 years), aging (70–99 years), and AD (70–99 years). The hippocampus is vulnerable to damage at early stages of AD and altered neurogenesis in the hippocampus is linked to the onset of AD. We combined the weighted gene co-expression network and weighted protein–protein interaction network-level approaches to study the transition from young to aging to AD. The network analysis revealed the organization of co-expression network into functional modules that are cell-type specific in aging and AD. We found that modules associated with astrocytes, endothelial cells and microglial cells are upregulated and significantly correlate with both aging and AD. The modules associated with neurons, mitochondria and endoplasmic reticulum are downregulated and significantly correlate with AD than aging. The oligodendrocytes module does not show significant correlation with neither aging nor disease. Further, we identified aging- and AD-specific interactions/subnetworks by integrating the gene expression with a human protein–protein interaction network. We found dysregulation of genes encoding protein kinases (FYN, SYK, SRC, PKC, MAPK1, ephrin receptors) and transcription factors (FOS, STAT3, CEBPB, MYC, NFKβ, and EGR1) in AD. Further, we found genes that encode proteins with neuroprotective function (14-3-3 proteins, PIN1, ATXN1, BDNF, VEGFA) to be part of the downregulated AD subnetwork. Our study highlights that simultaneously analyzing aging and AD will help to understand the pre-clinical and clinical phase of AD and aid in developing the treatment strategies.

Brain morphologic changes in early stages of psychosis: Implications for clinical application and early intervention.

To date, a large number of magnetic resonance imaging (MRI) studies have been conducted in schizophrenia, which generally demonstrate gray matter reduction, predominantly in the frontal and temporo-limbic regions, as well as gross brain abnormalities (e.g., a deviated sulcogyral pattern). Although the causes as well as timing and course of these findings remain elusive, these morphologic changes (especially gross brain abnormalities and medial temporal lobe atrophy) are likely present at illness onset, possibly reflecting early neurodevelopmental abnormalities. In addition, longitudinal MRI studies suggest that patients with schizophrenia and related psychoses also have progressive gray matter reduction during the transition period from prodrome to overt psychosis, as well as initial periods after psychosis onset, while such changes may become almost stable in the chronic stage. These active brain changes during the early phases seem to be relevant to the development of clinical symptoms in a region-specific manner (e.g., superior temporal gyrus atrophy and positive psychotic symptoms), but may be at least partly ameliorated by antipsychotic medication. Recently, increasing evidence from MRI findings in individuals at risk for developing psychosis has suggested that those who subsequently develop psychosis have baseline brain changes, which could be at least partly predictive of later transition into psychosis. In this article, we selectively review previous MRI findings during the course of psychosis and also refer to the possible clinical applicability of these neuroimaging research findings, especially in the diagnosis of schizophrenia and early intervention for psychosis.

Multiple roles of epithelial heparan sulfate in stomach morphogenesis.

Heparan sulfate proteoglycans (HSPGs) have been shown to regulate various developmental processes. However, the function of heparan sulfate (HS) during the development of mammalian stomach has not been characterized yet. Here, we investigate the role of epithelial HS in embryonic stomach by examining mice deficient in the glycosyltransferase gene Ext1 We show that HS exhibits a specific and dynamic expression pattern in mouse embryonic stomach. Depletion of the epithelial HS leads to stomach hypoplasia, with phenotypic differences in the gastric mucosa between the forestomach and hindstomach. In the posterior stomach, HS depletion disrupts glandular stomach patterning and cytodifferentiation via attenuation of Fgf signaling activity. Inhibition of Fgf signaling in vitro recapitulates the patterning defect. Ligand and carbohydrate engagement assay (LACE) reveals a diminished assembly of Fgf10 and Fgfr2b in the mutant. In the anterior stomach, loss of epithelial HS leads to stratification and differentiation defects of the multilayered squamous epithelium, along with reduced Hh and Bmp signaling activity. Our data demonstrate that epithelial HS plays multiple roles in regulating mammalian stomach morphogenesis in a regional-specific manner.

Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.

Circular RNA expression and regulatory network prediction in posterior cingulate astrocytes in elderly subjects

BackgroundCircular RNAs (circRNAs) are a novel class of endogenous, non-coding RNAs that form covalently closed continuous loops and that are both highly conserved and abundant in the mammalian brain. A role for circRNAs in sponging microRNAs (miRNAs) has been proposed, but the circRNA-miRNA-mRNA interaction networks in human brain cells have not been defined. Therefore, we identified circRNAs in RNA sequencing data previously generated from astrocytes microdissected from the posterior cingulate (PC) of Alzheimer’s disease (AD) patients (N = 10) and healthy elderly controls (N = 10) using four circRNA prediction algorithms - CIRI, CIRCexplorer, find_circ and KNIFE.ResultsOverall, utilizing these four tools, we identified a union of 4438 unique circRNAs across all samples, of which 70.3% were derived from exonic regions. Notably, the widely reported CDR1as circRNA was detected in all samples across both groups by find_circ. Given the putative miRNA regulatory function of circRNAs, we identified potential miRNA targets of circRNAs, and further, delineated circRNA-miRNA-mRNA networks using in silico methods. Pathway analysis of the genes regulated by these miRNAs identified significantly enriched immune response pathways, which is consistent with the known function of astrocytes as immune sensors in the brain.ConclusionsIn this study, we performed circRNA detection on cell-specific transcriptomic data and identified potential circRNA-miRNA-mRNA regulatory networks in PC astrocytes. Given the known function of astrocytes in cerebral innate immunity and our identification of significantly enriched immune response pathways, the circRNAs we identified may be associated with such key functions. While we did not detect recurrent differentially expressed circRNAs in the context of healthy controls or AD, we report for the first time circRNAs and their potential regulatory impact in a cell-specific and region-specific manner in aged subjects. These predicted regulatory network and pathway analyses may help provide new insights into transcriptional regulation in the brain.

GSK3β Regulates Brain Energy Metabolism

SUMMARYGSK3β is a serine threonine kinase implicated in the progression of Alzheimer’s disease. Although the role of GSK3β in growth and pathology has been extensively studied, little is known about the metabolic consequences of GSK3β manipulation, particularly in the brain. Here, we show that GSK3β regulates mitochondrial energy metabolism in human H4 neuroglioma cells and rat PC12-derived neuronal cells and that inhibition of GSK3β in mice in vivo alters metabolism in the hippocampus in a region-specific manner. We demonstrate that GSK3β inhibition increases mitochondrial respiration and membrane potential and alters NAD(P)H metabolism. These metabolic effects are associated with increased PGC-1α protein stabilization, enhanced nuclear localization, and increased transcriptional co-activation. In mice treated with the GSK3β inhibitor lithium carbonate, changes in hippocampal energy metabolism are linked to increased PGC-1α. These data highlight a metabolic role for brain GSK3β and suggest that the GSK3β/PGC-1α axis may be important in neuronal metabolic integrity.

Cocaine Self-administration Alters Transcriptome-wide Responses in the Brain’s Reward Circuitry

BackgroundGlobal changes in gene expression underlying circuit and behavioral dysregulation associated with cocaine addiction remain incompletely understood. Here, we show how a history of cocaine self-administration (SA) reprograms transcriptome-wide responses throughout the brain’s reward circuitry at baseline and in response to context and/or cocaine re-exposure after prolonged withdrawal (WD). MethodsWe assigned male mice to one of six groups: saline/cocaine SA + 24-hour WD or saline/cocaine SA + 30-day WD + an acute saline/cocaine challenge within the previous drug-paired context. RNA sequencing was conducted on six interconnected brain reward regions. Using pattern analysis of gene expression and factor analysis of behavior, we identified genes that are strongly associated with addiction-related behaviors and uniquely altered by a history of cocaine SA. We then identified potential upstream regulators of these genes. ResultsWe focused on three patterns of gene expression that reflect responses to 1) acute cocaine, 2) context re-exposure, and 3) drug + context re-exposure. These patterns revealed region-specific regulation of gene expression. Further analysis revealed that each of these gene expression patterns correlated with an addiction index—a composite score of several addiction-like behaviors during cocaine SA—in a region-specific manner. Cyclic adenosine monophosphate response element binding protein and nuclear receptor families were identified as key upstream regulators of genes associated with such behaviors. ConclusionsThis comprehensive picture of transcriptome-wide regulation in the brain’s reward circuitry by cocaine SA and prolonged WD provides new insight into the molecular basis of cocaine addiction, which will guide future studies of the key molecular pathways involved.

Pre-reproductive stress and fluoxetine treatment in rats affect offspring A-to-I RNA editing, gene expression and social behavior.

Adenosine to inosine RNA editing is an epigenetic process that entails site-specific modifications in double-stranded RNA molecules, catalyzed by adenosine deaminases acting on RNA (ADARs). Using the multiplex microfluidic PCR and deep sequencing technique, we recently showed that exposing adolescent female rats to chronic unpredictable stress before reproduction affects editing in the prefrontal cortex and amygdala of their newborn offspring, particularly at the serotonin receptor 5-HT2c (encoded by Htr2c). Here, we used the same technique to determine whether post-stress, pre-reproductive maternal treatment with fluoxetine (5 mg/kg, 7 days) reverses the effects of stress on editing. We also examined the mRNA expression of ADAR enzymes in these regions, and asked whether social behavior in adult offspring would be altered by maternal exposure to stress and/or fluoxetine. Maternal treatment with fluoxetine altered Htr2c editing in offspring amygdala at birth, enhanced the expression of Htr2c mRNA and RNA editing enzymes in the prefrontal cortex, and reversed the effects of pre-reproductive stress on Htr2c editing in this region. Furthermore, maternal fluoxetine treatment enhanced differences in editing of glutamate receptors between offspring of control and stress-exposed rats, and led to enhanced social preference in adult offspring. Our findings indicate that pre-gestational fluoxetine treatment affects patterns of RNA editing and editing enzyme expression in neonatal offspring brain in a region-specific manner, in interaction with pre-reproductive stress. Overall, these findings imply that fluoxetine treatment affects serotonergic signaling in offspring brain even when treatment is discontinued before gestation, and its effects may depend upon prior exposure to stress.

Region-specific Expression of NMDA Receptor GluN2C Subunit in Parvalbumin-Positive Neurons and Astrocytes: Analysis of GluN2C Expression using a Novel Reporter Model

Hypofunction of NMDA receptors in parvalbumin (PV)-positive interneurons has been proposed as a potential mechanism for cortical abnormalities and symptoms in schizophrenia. GluN2C-containing receptors have been linked to this hypothesis due to the higher affinity of psychotomimetic doses of ketamine for GluN1/2C receptors. However, the precise cell-type expression of GluN2C subunit remains unknown. We describe the expression of the GluN2C subunit using a novel EGFP reporter model. We observed EGFP(GluN2C) localization in PV-positive neurons in the nucleus reticularis of the thalamus, globus pallidus externa and interna, ventral pallidum and substantia nigra. In contrast, EGFP(GluN2C)-expressing cells did not co-localize with PV-positive neurons in the cortex, striatum, hippocampus or amygdala. Instead, EGFP(GluN2C) expression in these regions co-localized with an astrocytic marker. We confirmed functional expression of GluN2C-containing receptors in the PV-neurons in substantia nigra and cortical astrocytes using electrophysiology. GluN2C was found to be enriched in several first-order and higher order thalamic nuclei. Interestingly, we found that a previous GluN2C β-gal reporter model excluded expression from PV-neurons and certain thalamic nuclei but exhibited expression in the retrosplenial cortex. GluN2C’s unique distribution in neuronal and non-neuronal cells in a brain region-specific manner raises interesting questions regarding the role of GluN2C-containing receptors in the central nervous system.

Neuregulin 1 Deficiency Modulates Adolescent Stress-Induced Dendritic Spine Loss in a Brain Region-Specific Manner and Increases Complement 4 Expression in the Hippocampus.

One neuropathological feature of schizophrenia is a diminished number of dendritic spines in the prefrontal cortex and hippocampus. The neuregulin 1 (Nrg1) system is involved in the plasticity of dendritic spines, and chronic stress decreases dendritic spine densities in the prefrontal cortex and hippocampus. Here, we aimed to assess whether Nrg1 deficiency confers vulnerability to the effects of adolescent stress on dendritic spine plasticity. We also assessed other schizophrenia-relevant neurobiological changes such as microglial cell activation, loss of parvalbumin (PV) interneurons, and induction of complement factor 4 (C4). Adolescent male wild-type (WT) and Nrg1 heterozygous mice were subjected to chronic restraint stress before their brains underwent Golgi impregnation or immunofluorescent staining of PV interneurons, microglial cells, and C4. Stress in WT mice promoted dendritic spine loss and microglial cell activation in the prefrontal cortex and the hippocampus. However, Nrg1 deficiency rendered mice resilient to stress-induced dendritic spine loss in the infralimbic cortex and the CA3 region of the hippocampus without affecting stress-induced microglial cell activation in these brain regions. Nrg1 deficiency and adolescent stress combined to trigger increased dendritic spine densities in the prelimbic cortex. In the hippocampal CA1 region, Nrg1 deficiency accentuated stress-induced dendritic spine loss. Nrg1 deficiency increased C4 protein and decreased C4 mRNA expression in the hippocampus, and the number of PV interneurons in the basolateral amygdala. This study demonstrates that Nrg1 modulates the impact of stress on the adolescent brain in a region-specific manner. It also provides first evidence of a link between Nrg1 and C4 systems in the hippocampus.

11β-HSD Types 1 and 2 in the Songbird Brain.

Glucocorticoid (GC) hormones act on the brain to regulate diverse functions, from behavior and homeostasis to the activity of the hypothalamic–pituitary–adrenal axis. Local regeneration and metabolism of GCs can occur in target tissues through the actions of the 11β-hydroxysteroid dehydrogenases [11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and 11 beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2), respectively] to regulate access to GC receptors. Songbirds have become especially important model organisms for studies of stress hormone action; however, there has been little focus on neural GC metabolism. Therefore, we tested the hypothesis that 11β-HSD1 and 11β-HSD2 are expressed in GC-sensitive regions of the songbird brain. Localization of 11β-HSD expression in these regions could provide precise temporal and spatial control over GC actions. We quantified GC sensitivity in zebra finch (Taeniopygia guttata) brain by measuring glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) expression across six regions, followed by quantification of 11β-HSD1 and 11β-HSD2 expression. We detected GR, MR, and 11β-HSD2 mRNA expression throughout the adult brain. Whereas 11β-HSD1 expression was undetectable in the adult brain, we detected low levels of expression in the brain of developing finches. Across several adult brain regions, expression of 11β-HSD2 covaried with GR and MR, with the exception of the cerebellum and hippocampus. It is possible that receptors in these latter two regions require direct access to systemic GC levels. Overall, these results suggest that 11β-HSD2 expression protects the adult songbird brain by rapid metabolism of GCs in a context and region-specific manner.

Nuclear receptor agonist-driven modification of inflammation and amyloid pathology enhances and sustains cognitive improvements in a mouse model of Alzheimer\u2019s disease

BackgroundAlzheimer’s disease (AD) is a highly prevalent neurodegenerative disorder characterized by pathological hallmarks of beta-amyloid plaque deposits, tau pathology, inflammation, and cognitive decline. Treatment remains a clinical obstacle due to lack of effective therapeutics. Agonists targeting nuclear receptors, such as bexarotene, reversed cognitive deficits regardless of treatment duration and age in murine models of AD. While bexarotene demonstrated marked efficacy in decreasing plaque levels following short-term treatment, prolonged treatment did not modulate plaque burden. This suggested that plaques might reform in mice treated chronically with bexarotene and that cessation of bexarotene treatment before plaques reform might alter amyloid pathology, inflammation, and cognition in AD mice.MethodsWe utilized one-year-old APP/PS1 mice that were divided into two groups. We treated one group of mice for 2 weeks with bexarotene. The other group of mice was treated for 2 weeks with bexarotene followed by withdrawal of drug treatment for an additional 2 weeks. Cognition was evaluated using the novel-object recognition test either at the end of bexarotene treatment or the end of the withdrawal period. We then analyzed amyloid pathology and microgliosis at the conclusion of the study in both groups.ResultsBexarotene treatment enhanced cognition in APP/PS1 mice similar to previous findings. Strikingly, we observed sustained cognitive improvements in mice in which bexarotene treatment was discontinued for 2 weeks. We observed a sustained reduction in microgliosis and plaque burden following drug withdrawal exclusively in the hippocampus.ConclusionsOur findings demonstrate that bexarotene selectively modifies aspects of neuroinflammation in a region-specific manner to reverse hippocampal-dependent cognitive deficits in AD mice and may provide insight to inform future studies with nuclear receptor agonists.

Expression of aggrecan components in perineuronal nets in the mouse cerebral cortex

Specific regions of the cerebral cortex are highly plastic in an organism’s lifetime. It is thought that perineuronal nets (PNNs) regulate plasticity, but labeling for Wisteria floribunda agglutinin (WFA), which is widely used to detect PNNs, is observed throughout the cortex. The aggrecan molecule—a PNN component—may regulate plasticity, and may also be involved in determining region-specific vulnerability to stress. To clarify cortical region-specific plasticity and vulnerability, we qualitatively analyzed aggrecan-positive and glycosylated aggrecan-positive PNNs in the mature mouse cerebral cortex. Our findings revealed the selective expression of both aggrecan-positive and glycosylated aggrecan-positive PNNs in the cortex. WFA-positive PNNs expressed aggrecan in a region-specific manner in the cortex. Furthermore, we observed variable distributions of PNNs containing WFA- and aggrecan-positive molecules. Together, our findings suggest that PNN components and their function differ depending on the cortical region, and that aggrecan molecules may be involved in determining region-specific plasticity and vulnerability in the cortex.

Avian coronary endothelium is a mosaic of sinus venosus- and ventricle-derived endothelial cells in a region-specific manner.

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein-Tol2 system and fluorescent dye labeling, four types of quail-chick chimeras were made and quail-specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium-derived inner cells and the other was surface mesothelium-derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region-specific manner, and the sources of ECs were basically the same as those observed in mice.

Microglial dynamics during brain development.

Microglial dynamics during brain development.

Optogenetics and Chemogenetics.

Optogenetics and chemogenetics provide the ability to modulate neurons in a type- and region-specific manner. These powerful techniques are useful to test hypotheses regarding the neural circuit mechanisms of general anesthetic end points such as hypnosis and analgesia. With both techniques, a genetic strategy is used to target expression of light-sensitive ion channels (opsins) or designer receptors exclusively activated by designer drugs in specific neurons. Optogenetics provides precise temporal control of neuronal firing with light pulses, whereas chemogenetics provides the ability to modulate neuronal firing for several hours with the single administration of a designer drug. This chapter provides an overview of neuronal targeting and experimental strategies and highlights the important advantages and disadvantages of each technique.

Multiparametric MRI and Computational Modelling in the Assessment of Human Articular Cartilage Properties: A Comprehensive Approach.

Quantitative magnetic resonance imaging (qMRI) is a promising approach to detect early cartilage degeneration. However, there is no consensus on which cartilage component contributes to the tissue's qMRI signal properties. T1, T1ρ, and T2⁎ maps of cartilage samples (n = 8) were generated on a clinical 3.0-T MRI system. All samples underwent histological assessment to ensure structural integrity. For cross-referencing, a discretized numerical model capturing distinct compositional and structural tissue properties, that is, fluid fraction (FF), proteoglycan (PG) and collagen (CO) content and collagen fiber orientation (CFO), was implemented. In a pixel-wise and region-specific manner (central versus peripheral region), qMRI parameter values and modelled tissue parameters were correlated and quantified in terms of Spearman's correlation coefficient ρs. Significant correlations were found between modelled compositional parameters and T1 and T2⁎, in particular in the central region (T1: ρs ≥ 0.7 [FF, CFO], ρs ≤ −0.8 [CO, PG]; T2⁎: ρs ≥ 0.67 [FF, CFO], ρs ≤ −0.71 [CO, PG]). For T1ρ, correlations were considerably weaker and fewer (0.16 ≤ ρs ≤ −0.15). QMRI parameters are characterized in their biophysical properties and their sensitivity and specificity profiles in a basic scientific context. Although none of these is specific towards any particular cartilage constituent, T1 and T2⁎ reflect actual tissue compositional features more closely than T1ρ.

The impact of early-life stress on the expression of HPA-associated genes in the adult murine brain

Abstract Early life is an important period for the development of the nervous system and for the programming of behavioural phenotypes in adulthood. In our study, two types of early-life stress were used: prolonged separation of pups from their mothers (for 3 h/day, maternal separation (MS)) and brief separation (for 15 min/day, handling (HD)). We analysed the effects of early-life stress on behaviour and the expression of HPA-associated genes in the hypothalamus, hippocampus, and frontal cortex of male mice. Adult mice in the MS group demonstrated reduced locomotor activity and deficiencies in spatial long-term memory, while the HD showed no significant changes. Additionally, early-life MS resulted in reduced hippocampal Crhr1 mRNA, increased MR/GR mRNA in the hippocampus and hypothalamus. Both groups, HD and MS, showed increased Avp mRNA in the hypothalamus. Thus, prolonged maternal separation but not brief leads to adverse behavioural changes and influences the expression of HPA-associated genes in a brain region-specific manner.