Publications by authors named "Holly Richendrfer"

15 Publications

  • Page 1 of 1

Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood-Brain Barrier after Traumatic Brain Injury.

J Neurotrauma 2021 Feb 19;38(4):385-398. Epub 2020 Oct 19.

Neurotrauma and Brain Barriers Research Laboratory, Alpert Medical School of Brown University, Providence, Rhode Island, USA.

Neuroinflammation and dysfunction of the blood-brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI.
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http://dx.doi.org/10.1089/neu.2020.7229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875610PMC
February 2021

Recombinant Human Proteoglycan-4 Mediates Interleukin-6 Response in Both Human and Mouse Endothelial Cells Induced Into a Sepsis Phenotype.

Crit Care Explor 2020 Jun 10;2(6):e0126. Epub 2020 Jun 10.

Department of Emergency Medicine, Warren Alpert School of Medicine, Brown University, Providence, RI.

Objectives: Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro.

Design: Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression.

Setting: Research laboratory.

Subjects: Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (/), (), (), and double knockout () lung microvascular endothelial cells.

Interventions: Cells were treated with 100 or 250 ng/mL lipopolysaccharide- K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ-Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma.

Measurements And Main Results: Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60-73% in human lung microvascular endothelial cells. In genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls.

Interpretation And Conclusions: Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.
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http://dx.doi.org/10.1097/CCE.0000000000000126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314356PMC
June 2020

Proteoglycan-4 regulates fibroblast to myofibroblast transition and expression of fibrotic genes in the synovium.

Arthritis Res Ther 2020 05 13;22(1):113. Epub 2020 May 13.

Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Rinker Health Sciences Campus, 9401 Jeronimo Road, Irvine, CA, 92618, USA.

Background: Synovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and is a major component of synovial fluid. PRG4 is a ligand of the CD44 receptor. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo.

Methods: OA synoviocytes were treated with TGF-β ± PRG4 for 24 h and α-SMA content was determined using immunofluorescence. Rhodamine-labeled rhPRG4 was incubated with OA synoviocytes ± anti-CD44 or isotype control antibodies and cellular uptake of rhPRG4 was determined following a 30-min incubation and α-SMA expression following a 24-h incubation. HEK-TGF-β cells were treated with TGF-β ± rhPRG4 and Smad3 phosphorylation was determined using immunofluorescence and TGF-β/Smad pathway activation was determined colorimetrically. We probed for stress fibers and focal adhesions (FAs) in TGF-β-treated murine fibroblasts and fibroblast migration was quantified ± rhPRG4. Synovial expression of fibrotic markers: α-SMA, collagen type-I, and PLOD2 in Prg4 gene-trap (Prg4) and recombined Prg4 animals were studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-month-old Prg4&Cd44 and Prg4&Cd44 animals.

Results: PRG4 reduced α-SMA content in OA synoviocytes (p < 0.001). rhPRG4 was internalized by OA synoviocytes via CD44 and CD44 neutralization attenuated rhPRG4's antifibrotic effect (p < 0.05). rhPRG4 reduced pSmad3 signal in HEK-TGF-β cells (p < 0.001) and TGF-β/Smad pathway activation (p < 0.001). rhPRG4 reduced the number of stress fiber-positive myofibroblasts, FAs mean size, and cell migration in TGF-β-treated NIH3T3 fibroblasts (p < 0.05). rhPRG4 inhibited fibroblast migration in a macrophage and fibroblast co-culture model without altering active or total TGF-β levels. Synovial tissues of 9-month-old Prg4 animals had higher α-SMA, collagen type-I, and PLOD2 (p < 0.001) content and Prg4 re-expression reduced these markers (p < 0.01). Prg4 re-expression also reduced α-SMA and PLOD2 staining in CD44-deficient mice.

Conclusion: PRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is partially mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo.
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http://dx.doi.org/10.1186/s13075-020-02207-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222325PMC
May 2020

Lubricin as a Therapeutic and Potential Biomarker in Sepsis.

Crit Care Clin 2020 Jan 21;36(1):55-67. Epub 2019 Oct 21.

Department of Emergency Medicine, Alpert Medical School, Brown University, 222 Richmond Street, Providence, RI 02903, USA; Department of Emergency Medicine, Research Laboratory, Rhode Island Hospital, 1 Hoppin Street, CORO West, Room 4.303, Providence, RI 02903, USA. Electronic address:

Proteoglycan 4 (or lubricin), a mucin-like glycoprotein, was originally classified as a lubricating substance within diarthrodial joints. More recently, lubricin has been found in other tissues and has been implicated in 2 inflammatory pathways within the cell, via the Toll-like receptors (TLRs) and CD44. Lubricin is an antagonist of TLR2 and TLR4, and appears to enter cells via the CD44 receptor. Because of lubricin's action on these receptors, downstream processes of inflammation are halted, thereby preventing release of cytokines (a hallmark of inflammation and sepsis) from the cell, indicating lubricin's role as a biomarker and possible therapeutic for sepsis.
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http://dx.doi.org/10.1016/j.ccc.2019.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069234PMC
January 2020

Cluster analysis profiling of behaviors in zebrafish larvae treated with antidepressants and pesticides.

Neurotoxicol Teratol 2018 Sep - Oct;69:54-62. Epub 2017 Oct 31.

Brown University, Department of Molecular and Cellular Biology and Biochemistry, Providence, RI 02912, United States.

Antidepressants are used by a substantial number of women in their childbearing years. Treatment may continue during pregnancy, since untreated depression poses a risk to the mother and child. However, many antidepressants readily pass through the placental barrier to reach the fetus or may be ingested by the newborn via breastmilk. Little is known about the effects of antidepressants on brain development and subsequent behavior in young children. In the current study, we used zebrafish as a model system to examine the neurodevelopmental effects of three commonly prescribed antidepressants, sertraline, duloxetine and bupropion. Zebrafish were exposed to these antidepressants during development and were examined for changes in larval avoidance behavior, activity, social behaviors, and anxiety-related behaviors. The results show that antidepressants commonly affect larval swim speeds and resting, and differentially affect other behaviors depending upon the exposure period. Using cluster analysis profiling, we compared the obtained results to previous reports on behavioral defects induced by organophosphate pesticides. We found that the behavioral profiles induced by antidepressants and pesticides overlap, indicating a common mechanism of action. We conclude that developmental antidepressant exposures lead to specific behavioral changes in zebrafish larvae. At present, it is not known if antidepressants have similar effects in human development.
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http://dx.doi.org/10.1016/j.ntt.2017.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930167PMC
October 2019

An asymptomatic mutation complicating severe chemotherapy-induced peripheral neuropathy (CIPN): a case for personalised medicine and a zebrafish model of CIPN.

NPJ Genom Med 2016 8;1:16016. Epub 2016 Jun 8.

Department of Pediatrics, Division of Pediatric Hematology-Oncology, Hasbro Children's Hospital and The Warren Alpert Medical School at Brown University, Providence, RI, USA.

Targeted next-generation sequencing (NGS) identified a novel loss of function mutation in , a gene linked to Charcot-Marie-Tooth disease (CMT), in a paediatric acute lymphoblastic leukaemia patient with severe chemotherapy-induced peripheral neuropathy (CIPN) due to vincristine. The patient was clinically asymptomatic, and lacked a family history of neuropathy. The effect of the mutation was modelled in a zebrafish knockdown system that recapitulated the symptoms of the patient both prior to and after treatment with vincristine. Confocal microscopy of pre- and post-synaptic markers revealed that the GARS knockdown results in changes to peripheral motor neurons, acetylcholine receptors and their co-localisation in neuromuscular junctions (NMJs), whereas a sensitive and reproducible stimulus-response assay demonstrated that the changes correlating with the GARS mutation in themselves fail to produce peripheral neuropathy symptoms. However, with vincristine treatment the GARS knockdown exacerbates decreased stimulus response and NMJ lesions. We propose that there is substantial benefit in the use of a targeted NGS screen of cancer patients who are to be treated with microtubule targeting agents for deleterious mutations in CMT linked genes, and for the screening in zebrafish of reagents that might inhibit CIPN.
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http://dx.doi.org/10.1038/npjgenmed.2016.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685301PMC
June 2016

Chlorpyrifos and malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity.

Neurotoxicology 2015 Jul 14;49:50-8. Epub 2015 May 14.

Brown University, Department of Molecular and Cellular Biology and Biochemistry, Providence, RI 02912, United States.

Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size.
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http://dx.doi.org/10.1016/j.neuro.2015.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523399PMC
July 2015

Effects of embryonic cyclosporine exposures on brain development and behavior.

Behav Brain Res 2015 Apr 13;282:117-24. Epub 2015 Jan 13.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Electronic address:

Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures.
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http://dx.doi.org/10.1016/j.bbr.2015.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323675PMC
April 2015

High-throughput analysis of behavior in zebrafish larvae: effects of feeding.

Zebrafish 2014 Oct 25;11(5):455-61. Epub 2014 Aug 25.

1 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University , Providence, Rhode Island.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. Identification of these factors is facilitated by high-throughput analyses of behavior in zebrafish larvae, which can be imaged in multiwell or multilane plates. However, the nutritional needs of zebrafish larvae during the behavioral experiments are not fully understood. Zebrafish larvae begin feeding between 4 and 5 days postfertilization (dpf), but can live solely on nutrients derived from the yolk until at least 7 dpf. To examine whether feeding affects behavior, we measured a broad range of behaviors with and without feeding at 5, 6, and 7 dpf. We found that feeding did not have a significant effect on behavior in 5-day-old larvae. In contrast, fed 6- and 7-day-old larvae displayed increased avoidance responses to visual stimuli, increased swim speeds, and decreased resting in comparison to unfed larvae. In addition, the fed 7-day-old larvae displayed a decrease in thigmotaxis and a decrease in the distance between larvae in the presence of visual stimuli. Thus, feeding affects a range of behaviors in 6- and 7-day-old larvae. We conclude that 5-day-old larvae are well-suited for high-throughput analyses of behavior, since effects of feeding can be avoided at this time. For high-throughput analyses of behavior in older larvae, standard feeding protocols need to be developed.
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http://dx.doi.org/10.1089/zeb.2014.0989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172468PMC
October 2014

Automated high-throughput behavioral analyses in zebrafish larvae.

J Vis Exp 2013 Jul 4(77):e50622. Epub 2013 Jul 4.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.
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http://dx.doi.org/10.3791/50622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731428PMC
July 2013

Exposure to female pheromones stimulates a specific type of neuronal population in the male but not female magnocellular division of the medial preoptic nucleus (MPN mag) of the Syrian hamster.

Horm Behav 2013 Aug 15;64(3):421-9. Epub 2013 Jun 15.

Lehigh University, Dept of Biological Sciences, United States. Electronic address:

The magnocellular division of the medial preoptic area (MPN mag) integrates pheromonal and hormonal signals to play a critical role in the expression of male typical sex behavior. The MPN mag contains two morphologically distinct neuronal populations; the percentage of each type within the nucleus is sex specific. Males have more neurons with a single nucleolus whereas females have more with multiple nucleoli. To determine which neuronal subtype mediates pheromonal induction of copulation, tissue from male and female hamsters exposed to female pheromones was immunolabeled for the immediate early protein (EGR-1). Subsequently the tissue was counterstained and the number of ERG-1 neurons with one or two nuclei was determined. The results indicate that pheromones stimulate neurons with single nucleoli in males but fail to stimulate either neuronal subtype in females suggesting that synaptic input to the MPN mag is sexually differentiated.
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http://dx.doi.org/10.1016/j.yhbeh.2013.06.004DOI Listing
August 2013

Developmental sub-chronic exposure to chlorpyrifos reduces anxiety-related behavior in zebrafish larvae.

Neurotoxicol Teratol 2012 Jul 8;34(4):458-65. Epub 2012 May 8.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.

Neurobehavioral disorders such as anxiety, autism, and attention deficit hyperactivity disorders are typically influenced by genetic and environmental factors. Although several genetic risk factors have been identified in recent years, little is known about the environmental factors that either cause neurobehavioral disorders or contribute to their progression in genetically predisposed individuals. One environmental factor that has raised concerns is chlorpyrifos, an organophosphate pesticide that is widely used in agriculture and is found ubiquitously in the environment. In the present study, we examined the effects of sub-chronic chlorpyrifos exposure on anxiety-related behavior during development using zebrafish larvae. We found that sub-chronic exposure to 0.01 or 0.1 μM chlorpyrifos during development induces specific behavioral defects in 7-day-old zebrafish larvae. The larvae displayed decreases in swim speed and thigmotaxis, yet no changes in avoidance behavior were seen. Exposure to 0.001 μM chlorpyrifos did not affect swimming, thigmotaxis, or avoidance behavior and exposure to 1 μM chlorpyrifos induced behavioral defects, but also induced defects in larval morphology. Since thigmotaxis, a preference for the edge, is an anxiety-related behavior in zebrafish larvae, we propose that sub-chronic chlorpyrifos exposure interferes with the development of anxiety-related behaviors. The results of this study provide a good starting point for examination of the molecular, cellular, developmental, and neural mechanisms that are affected by environmentally relevant concentrations of organophosphate pesticides. A more detailed understanding of these mechanisms is important for the development of predictive models and refined health policies to prevent toxicant-induced neurobehavioral disorders.
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http://dx.doi.org/10.1016/j.ntt.2012.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573706PMC
July 2012

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

Behav Brain Res 2011 Sep 28;223(1):135-44. Epub 2011 Apr 28.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders.
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http://dx.doi.org/10.1016/j.bbr.2011.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111907PMC
September 2011

Neuronal composition of the magnocellular division of the medial preoptic nucleus (MPN mag) is sex specific in the Syrian hamster (Mesocricetus auratus).

Brain Res 2010 Sep 23;1351:97-103. Epub 2010 Jul 23.

Lehigh University, Department of Biological Sciences, Bethlehem, PA, USA. Electronic address:

The magnocellular division of the medial Preoptic nucleus (MPN mag) plays a critical role in the regulation of male sexual behavior in the hamster. Results from previous studies indicated that the number of neurons in the MPN mag is greater in males than females but failed to find significant differences in the volume of the nucleus suggesting that other elements in the nucleus may be greater in the female. The results of the present study, using NeuN to identify neurons, are in line with this hypothesis. The data show that (1) neurons in the MPN mag display two distinct phenotypes, those with a single nucleolus and those with multiple nucleoli; (2) the percentage of each phenotype is sex specific, differing over the course of development and (3) there is no sex difference in the number of glial cells at any age. Sex differences in the numbers of each type are correlated with developmental milestones and suggest that morphological changes are influenced by changes in circulating gonadal steroids during development.
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http://dx.doi.org/10.1016/j.brainres.2010.06.068DOI Listing
September 2010

Temperature, peroxide concentration, and immunohistochemical staining method affects staining intensity, distribution, and background.

Appl Immunohistochem Mol Morphol 2009 Dec;17(6):543-6

Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.

The techniques used to label neural tissue for specific antigens can vary significantly. Some immunostaining methods use free-floating tissue sections, whereas others use tissue sections mounted on slides. Mounting sections on glass slides before labeling the tissue with antigens is preferred method for neonatal tissue; processing young tissue by free-floating methods often destroy it. Surprisingly optimal temperature for storing tissue can vary with age. This study describes parameters developed to obtain robust staining of both young and old tissue. Our results show the most robust staining was found in tissue that was (1) stored at very low temperatures (-20 degrees C and -80 degrees C), (2) pretreated with 0.01% peroxide, and (3) entirely immersed in the staining solutions during immunohistochemistry.
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http://dx.doi.org/10.1097/PAI.0b013e3181a91595DOI Listing
December 2009