Gene Expression Analyses of Neurons, Astrocytes, and Oligodendrocytes Isolated by Laser Capture Microdissection From Human Brain: Detrimental Effects of Laboratory Humidity

Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM\u27s application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain

Transcription Factor Phox2 Upregulates Expression of Norepinephrine Transporter and Dopamine β-Hydroxylase in Adult Rat Brains

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons

Low Gene Expression of Bone Morphogenetic Protein 7 in Brainstem Astrocytes in Major Depression

The noradrenergic locus coeruleus (LC) is the principal source of brain norepinephrine, a neurotransmitter thought to play a major role in the pathology of major depressive disorder (MDD) and in the therapeutic action of many antidepressant drugs. The goal of this study was to identify potential mediators of brain noradrenergic dysfunction in MDD. Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β superfamily, is a critical mediator of noradrenergic neuron differentiation during development and has neurotrophic and neuroprotective effects on mature catecholaminergic neurons. Real-time PCR of reversed transcribed RNA isolated from homogenates of LC tissue from 12 matched pairs of MDD subjects and psychiatrically normal control subjects revealed low levels of BMP7 gene expression in MDD. No differences in gene expression levels of other members of the BMP family were observed in the LC, and BMP7 gene expression was normal in the prefrontal cortex and amygdala in MDD subjects. Laser capture microdissection of noradrenergic neurons, astrocytes, and oligodendrocytes from the LC revealed that BMP7 gene expression was highest in LC astrocytes relative to the other cell types, and that the MDD-associated reduction in BMP7 gene expression was limited to astrocytes. Rats exposed to chronic social defeat exhibited a similar reduction in BMP7 gene expression in the LC. BMP7 has unique developmental and trophic actions on catecholamine neurons and these findings suggest that reduced astrocyte support for pontine LC neurons may contribute to pathology of brain noradrenergic neurons in MDD

Laser Capture Microdissection and RT- PCR Analyses of Specific Cell Types in Locus Coeruleus From Postmortem Human Brain

Morphological studies have shown pathology of neurons and glia in many brain disorders, including psychiatric disorders such as major depression. However, most biochemical characterizations of postmortem human brain tissue have not made a distinction between neurons and glia. Laser capture microdissection (LCM) to isolate specific cell types has the potential to advance our understanding of human brain pathologies. Here, RT-PCR was used to evaluate the utility of LCM in the capture of noradrenergic neurons, astrocytes and oligodendrocytes from the locus coeruleus (LC) of postmortem human brain. The 3 LC cell types were individually identified using modifications of established histological and morphological methods. LCM settings were optimized for each cell type and captured cell bodies were those having no nearby cell body of a different phenotype. LC neurons (200), astrocytes (500), and oligodendrocytes (500) were captured within the LC from 3 postmortem brains. RNA was isolated, reversed transcribed, and markers for neurons (tyrosine hydroxylase [TH], dopamine beta-hydroxylase [DBH]), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (myelin oligodendrocyte glycoprotein [MOG]), along with 3 references (actin, GAPDH, ubiquitin C) were PCR amplified and quantified by standardized end-point PCR. RNA quality as assessed by RIN was not altered by LCM as compared to RNA isolated from homogenized tissue. TH gene expression was found only in neurons in 2 of the 3 brains. DBH gene expression was ~5-fold greater in neurons than in astrocytes and oligodendrocytes. GFAP gene expression in astrocytes was 7- and 5-fold greater than that in neurons and oligodendrocytes, respectively. MOG gene expression was only detected in oligodendrocytes. Different expression ratios of marker genes between neurons and glia suggest that simple cross contamination of mRNA is unlikely. Glial cells may contain DBH mRNA. Alternatively, DBH, but not TH, mRNA may occur in neuronal dendrites or axons in close association with glial cells that become captured with glia during LCM. GFAP may be expressed in low levels in neurons and oligodendrocytes, or alternatively, GFAP mRNA may be located in astrocytic processes in close association with neuronal and oligodendrocyte cell bodies. Use of a single marker to identify a cell type may be insufficient; other cell types for comparison or additional markers may be required. Multiple well-characterized markers can be used to evaluate clarity of cell capture for each sample. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of specific cell types in postmortem human brain

Localization of Cholinergic Innervation and Neurturin Receptors in Adult Mouse Heart and Expression of the Neurturin Gene

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals

Glutamate Receptor Gene Expression in Human Noradrenergic Neurons: Evidence of Altered Glutamate-Noradrenergic Signaling in Depression and Suicide

There is abundant evidence that both glutamatergic and noradrenergic transmission are disrupted in stress-related disorders such as major depressive disorder (MDD). Glutamate provides a major excitatory input to the noradrenergic locus coeruleus (LC), the primary source of norepinephrine in the brain. Stress increases glutamate activation of noradrenergic neurons in the LC. Glutamate signaling in the LC is mediated by several glutamate receptors expressed in the LC. Previous work from this laboratory has demonstrated elevated protein levels of the NMDA receptor subunit NR2C in the LC from MDD subjects compared to normal control subjects. Here, laser capture microdissection and quantitative gene expression analyses were used to evaluate the gene expression of selected glutamate receptors in noradrenergic neurons from the LC in postmortem brains from MDD subjects (n=6 males; 42±7 y) and psychiatrically normal control subjects (n=6 males; 42±7 y). No significant differences in brain pH, postmortem intervals, or RNA quality (estimated by RIN values) were observed between the two groups. None of the subjects had a diagnosis of a substance abuse disorder and none had a positive toxicological finding of antidepressant medication. Five of the 6 MDD subjects died by suicide. Gene expression levels were normalized using 3 reference genes and using cell number. The quality of the capture of pure populations of noradrenergic neurons was confirmed by examining gene expression of cell-type specific markers, including markers for glia. Gene expression levels of the NR2B and NR2C subunits of the NMDA receptor were robustly and significantly elevated in noradrenergic neurons collected from MDD subjects compared to control subjects. In contrast, gene expression levels of the AMPA receptor subunit GluR1, the metabotropic glutamate receptor mGluR5, as well as the reference genes actin, ubiquitin C, and GAPDH were not significantly different comparing MDD to control subjects. Altered glutamate receptor gene expression in noradrenergic neurons from MDD subjects provides evidence of abnormal glutamatergic control of noradrenergic neurotransmission in MDD

Herpes Simplex Virus Co-Infection-Induced Chlamydia Trachomatis Persistence Is Not Mediated by Any Known Persistence Inducer or Anti-Chlamydial Pathway

Several inducers of chlamydial persistence have been described, including interferon-γ (IFN-γ), IFN-α, IFN-β, and tumour necrosis factor-α (TNF-α) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-γ, IFN-α, and TNF-α are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-β, IFN-γ, indoleamine 2,3-dioxygenase, lymphotoxin-α and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C6-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development

Team-Taught Grand Rounds Promote Horizontal and Vertical Integration in a Discipline-Based Medical Curriculum

Discipline‐based medical curricula face the challenges of promoting horizontal (across course) and vertical (across years) integration, as well as providing opportunities for students to build the skills needed to become “residents‐as‐teachers”. To address these issues, we developed an Integrated Grand Rounds (IGR) series in which cases are co‐presented to M1/M2 students by clinical and basic science faculty. Sub‐topics relevant to the case are expanded upon by means of live patient interviews and small group sessions led by M3/M4 students. IGR effectiveness is measured by comparison of pre‐/post‐test scores and student attitude questionnaires. Overall, student post‐test scores improved by 23% and \u3e; 95% of all students felt that this activity was an effective way to both integrate information across courses and highlight clinical applications of basic science material. Additionally, all M3/M4 students polled felt that the IGR provided a valuable opportunity to review important basic science concepts and practice clinical teaching skills. The IGR series has proven to be a highly successful tool for cross‐course and longitudinal integration and is enthusiastically supported by both faculty and students. Notably, the IGR provides an efficient and cost‐effective vehicle to expand interdisciplinary connections and enhance integration. As a result, we are in the process of expanding its use in our curriculum