Building Muscle-TRIM32

What causes Muscular Dystrophies like Limb girdle muscular dystrophy type 2H (LGMD2H)? Here researchers demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, they show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly, they show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. These studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H: Nicklas S , Otto A , Wu X , Miller P , Stelzer S , et al. 2012 TRIM32 Regulates Skeletal Muscle Stem Cell Differentiation and Is Necessary for Normal Adult Muscle Regeneration. PLoS ONE 7(1): e30445. doi:10.1371/journal.pone.0030445.

Stem Cell Markers  like PAX7 are important for types of studies. PAX7 is a marker for muscle progenitors or stem cells. If these cells fail to differentiate the level of PAX7 expression will remain high. MyoD is an indicator for successfully differentiating cells. As a consequence, MyoD expression levels increase within the cell and the ratio of Pax7:MyoD is reduced, thus inhibiting proliferation and inducing differentiation through Myogenin expression. In the absence of TRIM32, this balance of Pax7:MyoD is dysregulated, because degradation of c-Myc is not initiated, MyoD levels do not increase and Myogenin expression is not activated.

Figure . Differentiation is strongly reduced in satellite cell progeny on TRIM32−/− myofibers. (a), (c) and (e) Immunostainings of satellite cells on freshly isolated myofibers from wild type (+/+) and TRIM32−/− (−/−) mice cultured for 24 h (a), 48 h (c) and 72 h (e) and labelled with the indicated markers (upper grey boxes). (b), (d) and (f) Diagrams showing the relative frequency of Pax7+/MyoD− cells on fibers cultured for 24 h (b), Pax7+/Myogenin− cells on fibers cultured for 48 h (d) and Pax7−/Myogenin+ cells on fibers cultured for 72 h (f). In all cases cells on myofibers from wild type mice and TRIM32−/− mice are compared. (mean ± std; *P<0.001 compared to wild type).


Check out our more publications referencing use of our PAX7.

Neurotrophins-Growth Factors Update

Our Neurotrophins and Growth Factor Antibodies and Recombinant Proteins help support a wide span of research areas. These areas include: neuroscience, immunology, cardiac disease research and cancer.

I would like to update you on recent publications highlighting use of some of these reagents: Aiko Sada, Kazuteru Hasegawa, Pui Han Pin, Yumiko Saga. NANOS2 Acts Downstream of Glial Cell Line-Derived Neurotrophic Factor Signaling to Suppress Differentiation of Spermatogonial Stem Cells. DOI: 10.1002/stem.790. Copyright © 2011 AlphaMed Press...anti-GFRA1 (1:200, Neuromics, Edina, MN)...

Images: Ngn3-Cre targets GFRA1-negative cells. (A-F): At P7, Rosa-YFP; Ngn3-Cre double transgenic testes were immunostained with the indicated markers. Most of YFP-positive spermatogonia (Ngn3-lineage cells) were not stained with GFRA1. Scale bar, 100 μm. (G): Quantification of YFP-positive cells per GFRA1-positive cells at P7 and 6 weeks (N=3).

Karine Bédard, Stéphanie Segura, Stéphanie Mahaut, Catherine Tardivel, Guylaine Ferland, Bruno Lebrun, Pierrette Gaudreau. Effects of aging and caloric restriction on brainstem satiety center signals in rats. Mechanisms of Ageing and Development. dx.doi.org/10.1016/j.mad.2012.01.004...goat serum and 0.3% Triton-X-100) (Sigma–Aldrich), overnight at 4 °C with chicken anti-rat BDNF antibody (Neuromics, Edina, MN; diluted 1/200 in blocking buffer), rinsed 3× 10 min in fresh PBS and incubated for 2 h, in the dark...

Tali Kobilo, Qing-Rong Liu, Kriti Gandhi, Mohammed Mughal, Yavin Shaham and Henriette van Praag. Running is the neurogenic and neurotrophic stimulus in environmental enrichment. doi: 10.1101/lm.2283011. Learn. Mem. 2011. 18: 605-609... human recombinant BDNF (0.1 µg) monomer (Neuromics)...

Masamichi Shinoda, Masatake Asano, Daisuke Omagari, Kuniya Honda, Suzuro Hitomi, Ayano Katagiri, and Koichi Iwata. Nerve Growth Factor Contribution via Transient Receptor Potential Vanilloid 1 to Ectopic Orofacial Pain. The Journal of Neuroscience, 11 May 2011, 31(19):7145-7155; doi:10.1523/JNEUROSCI.0481-11.2011....expression immunohistochemically in TG- anti-p75 goat antibody (1:200; Neuromics)...



Images: Mandibular nerve fibers were labeled by β-NGF which was administrated into the lower lip with CFA (A) or saline (F). On day 1 after β-NGF administration into the lower lip with CFA or saline, β-NGF-positive whisker pad or lower lip TG neurons defined by FG or DiI, respectively. B, G, β-NGF-positive TG neurons. C, H, DiI-labeled TG neurons. D, I, FG-labeled TG neurons. E, J, DiI- and FG-labeled β-NGF-positive TG neurons. Mandibular nerve fibers (K) and TG neurons (L) on day 1 after labeled BSA administration into the lower lip with CFA. Open arrow, β-NGF-positive nerve fibers. Arrow, DiI-labeled β-NGF-positive TG neurons. Arrowhead, FG-labeled β-NGF-positive TG neurons. Scale bar, 50 μm. M, Frequency of β-NGF-positive neurons in FG- or DiI-labeled TG neurons after CFA or saline injection into the lower lip (n = 5 in CFA-injected group; n = 4 in saline-injected group; Student's t test).

F. R. Carreño, J. D. Walch, M. Dutta, T. P. Nedungadi, J. T. Cunningham. Brain-Derived Neurotrophic Factor-Tyrosine Kinase B Pathway Mediates NMDA Receptor NR2B Subunit Phosphorylation in the Supraoptic Nuclei Following Progressive Dehydration. Journal of Neuroendocrinology. Volume 23, Issue 10, pages 894–905, October 2011. DOI: 10.1111/j.1365-2826.2011.02209.x...TrkB receptor (Neuromics, Edina, MN, USA)...

Jie Shen, Yoko Ishii, Guihua Xu, Thanh Chung Dang, Takeru Hamashima, Takako Matsushima, Seiji Yamamoto, Yuichi Hattori, Yusuke Takatsuru, Junichi Nabekura and Masakiyo Sasahara. PDGFR-β as a positive regulator of tissue repair in a mouse model of focal cerebral ischemia. Journal of Cerebral Blood Flow & Metabolism , (28 September 2011). doi:10.1038/jcbfm.2011.136...goat polyclonal anti-PDGFR-β antibody (1:100; Neuromics, Edina)...

Thomas Karsten Kilvaer, Andrej Valkov, Sveinung W. Sorbye, Tom Donnem, Eivind Smeland, Roy Martin Bremnes,and Lill-Tove Busund. Platelet-Derived Growth Factors in Non-GIST Soft-Tissue Sarcomas Identify a Subgroup of Patients with Wide Resection Margins and Poor Disease-Specific Survival. Volume 2010 (2010), Article ID 751304, 10 pages doi:10.1155/2010/751304...PDGF-CC (goat polyclonal; GT15151; Neuromics; 1 : 80)...

I will keep you posted on new developments.

Aging-Caloric Restriction and BDNF-Leptin

As we age, selective BDNF receptors increase in the the dorsal vagal complex (DVC), the brainstem center mediating the satiety reflex. Aging also affects the suppressor of cytokine signaling-3. This results in abnormal responses to BDNF and Leptin Signaling.

In this study, the investigators found that by restricting calories in rats, DVC BDNF immunoreactive concentrations were elevated and resulting in satiety threshold stability.  This indicates functional desensitization of the DVC to these signals: Karine Bédard, Stéphanie Segura, Stéphanie Mahaut, Catherine Tardivel, Guylaine Ferland, Bruno Lebrun, Pierrette Gaudreau. Effects of aging and caloric restriction on brainstem satiety center signals in rats. Mechanisms of Ageing and Development. dx.doi.org/10.1016/j.mad.2012.01.004.

The authors used our BDNF Antibody to determine expression in the DVC.....goat serum and 0.3% Triton-X-100) (Sigma–Aldrich), overnight at 4 °C with chicken anti-rat BDNF antibody (Neuromics, Edina, MN; diluted 1/200 in blocking buffer), rinsed 3× 10 min in fresh PBS and incubated for 2 h, in the dark...

These findings could lead to potential therapies based on modulating BDNF expression in the DVC.

Lineage Selection-Neural Stem Cells for SC Grafts

Our Neural Progenitor Markers keep moving up in the "hit parade". These markers are important for lineage selection. This selection is essential to circumvent the possibility of tumor formation and facilitate the safe translation of ES-based therapies to humans.

Here's a recent pub referencing use of several of our markers for selecting or confirming lineage: J. Simon Lunn, Crystal Pacut, Emily Stern, Stacey A. Sakowski, J. Matthew Velkey, Sue O'Shea, Eva Feldman. Intraspinal transplantation of neurogenin-expressing stem cells generates spinal cord neural progenitors. dx.doi.org/10.1016/j.nbd.2011.12.044...Tuj1 (Neuromics, 1:1000), Nestin (Neuromics, 1:500)...
Highlights: Expression of appropriate transcription factors is one approach to direct the differentiation of ES cells towards a specific lineage and stop proliferation. Neural differentiation can be initiated in ES cells by expression of Neurogenin1 (Ngn1). In this study we investigate the effects of controlled Ngn1 expression on mouse ES (mES) cell differentiation in vitro and following grafting into the rat spinal cord. In vitro, Ngn1 expression in mES cells leads to rapid and specific neural differentiation, and a concurrent decrease in proliferation. Similarly transplantation of Ngn1-expressing mES cells into the spinal cord lead to in situ differentiation and spinal precursor formation. These data demonstrate that Ngn1 expression in mES cells is sufficient promote neural differentiation and inhibit proliferation, thus establishing an approach to safely graft ES cells into the spinal cord.

Image: Neural progenitors were labeled with anti-rat Nestin polyclonal antibody (Cat#:GT15114) and stained with conjugated donkey anti-goat secondary anti-body (green). Differentiated neurons were labeled with neuron-specific mouse anti-β-III tubulin/ Tuj1-(Cat#MO15013 monoclonal antibody (red). Nuclei were stained with DAPI (blue).

I will continue to post updates on the application of Neuromics' Stem Cell Markers

Early Diagnosis of Diabetic Retinopathy

The earlier the diagnosis the better the outcome. This is especially true with autoimmune diseases like Diabetic Retinopathy (DR). DR is the leading cause of blindness among persons of working age in the industrialized world. Here I feature a publication that shows axoglial alterations at the distal portion of the optic nerve could be the first structural change in the diabetic visual pathway. This could prove good news for discovering better therapies thus preventing blindness: Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018

Oligodendrocytes are responsible for insulating axons. Disruptions in the formation of oligodendrocytes could initiate the domino effect that leads to decreasing and eventual total loss of vision. The authors, for example, discovered that in diabetic rats, oligodendrocyte lineage (OL) cells showed hypertrophic somas and a high number of processes.

Images/Data: OL linage evaluation. Immature OL (O1+ cells) and OL precursor (PDGFR-α+ cells) were evaluated by immunostaining and measured as optical density (OD) per section. In the distal ON from animals that were diabetic for 6 weeks, significantly increased O1 and PDGFR-α immunostaining was observed, with the presence of disorganized and hypertrophic cells. Data are mean ± SEM (n = 5 animals per group); *P < 0.01 versus age-matched controls, by Student′s t-test. Scale bar = 50 μm.

At the ultrastructural level, alterations and loss of larger axons were observed in the distal ON from animals that were diabetic for 6 weeks. In these fibers, myelin was highly disorganized, and frequent lamellar membranous bodies were observed.

I will track new develops in this research and post relevant results here.

Primary Neuron Assays for Studying Neurodegeneration

Our goal is to provide our customers and collaborators the tools they need to insure success. This is defined by having the specific Primary Neurons, Growth Factor plus the Markers to meet unique research needs.

The proof is in the results. Here are some highlights.

Images/Data: FIGURE 5. Microglial p38α MAPK-dependent TNFα is involved in LPS-induced neurite degeneration. (A) Photomicrographs of MAP-2 immunocytochemistry show the morphology of neurons after 72h of co-culture with microglia. The arrow points to the appearance of neurites that have been damaged by LPS-activated WT microglia. In contrast, the arrowhead points to the morphological appearance of healthy, undamaged neurites. (B) Diagram of the Sholl method for quantifying the total number of healthy neurites that intersect the concentric circles. (C) Quantification of healthy neurites by the Sholl analysis demonstrates that LPS stimulation of p38α WT microglia in co-culture causes neurite degeneration as seen by a significant reduction in the number of intersections by healthy neurites in the LPS-stimulated group compared to the unstimulated group (white bars). This degeneration can be attenuated by the addition of a blocking antibody to TNFα (5μg/ml), while the non-immune IgG control was not protective (gray bars). Microglia from p38α KO mice stimulated with LPS (black bar) also have significantly less neurite degeneration than the LPS-stimulated p38α WT microglia (white bar). However, by adding TNFα back to the p38α KO microglia co-culture, there is a significant decrease in the healthy neurite arborization compared to the p38α KO microglia stimulated with LPS alone (black bars). (***p<0.005; Bonferroni’s multiple comparison test). Data represents 2 independent experiments. Scale bar equals 25μm. Molecular Neurodegeneration 2011, 6:84 doi:10.1186/1750-1326-6-84

hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to Neurofilament light or low molecular weight chain NF-L, a marker of neurons. Many of the differentiating cells show strong cytoplasmic and clearly fibrillar staining for NF-L. Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture.

We will continue to post relevant images and data that demonstrate our capabilities.

TRPV1s in Action

Our TRPV1s continue to be widely used and published. This recent publication features use out TRPV1-C guinea pig polyclonal for immunohistochemistry and TRPV1-mouse specific for Western Blotting: Sarah E. Canetta, Edlira Luca, Elyse Pertot, Lorna W. Role, David A. Talmage. Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation. PLoS ONE 6(9): e25108. doi:10.1371/journal.pone.0025108...IHC: TRPV1 (guinea pig, 1:1000, GP14100 Neuromics); WB: TRPV1 (rabbit, 1:1000, RA14113 Neuromics).

Figure. Sensory axons, but not soma, from Type III Nrg1+/− mice show reduced capsaicin responsiveness compared to axons from WT mice. (A) Representative traces of intracellular calcium along sensory axons in response to 1 µM capsaicin or 56 mM KCl. The change in intracellular calcium from baseline over time ([(F−F0)/F0]*100) is shown for WT (left) and Type III Nrg1+/− (right) axons. Hatched diagonal lines indicate where the time course was non-continuous. (B) Quantification of the maximum change in intracellular calcium in response to application of 1 µM capsaicin or 56 mM KCl by genotype. Averages of 5 animals per genotype were compared using a Student's t-test. Type III Nrg1+/− axons showed a significantly decreased response to capsaicin (p<0.05), but not to KCl, relative to WTs. Graph shows mean±SEM. (C) Type III Nrg1+/− sensory soma show normal response to capsaicin. Quantification of maximal change in fluorescence from baseline ([(F−F0)/F0]*100) in WT or Type III Nrg1+/− sensory neuron soma in response to 1 µM capsaicin or 56 mM KCl. Average responses from 4 WT and 4 Type III Nrg1+/− animals to application of capsaicin or KCl were compared by genotype using a Student's t-test. There was no statistically significant difference between genotypes. Graphs show mean±SEM. (D) Type III Nrg1 (green) and TRPV1 (red) are co-expressed along P21 WT cultured sensory neuron axons identified with a pan-axonal (PA) marker (blue). White arrows indicate examples where Type III Nrg1 and TRPV1 are in close proximity. Scale bar equals 10 µm. (E) P21 WT and Type III Nrg1+/− sensory neuron cultures have equivalent levels of total TRPV1 protein. Total TRPV1 protein measurement by immunoblot. The 95 kD TRPV1 band and the 35 kD GAPDH band are shown from a representative experiment comparing protein from P21 WT and Type III Nrg1+/− cultures. Quantification of fold change in intensity of TRPV1:GAPDH normalized to WT average. There was no statistically significant change in the ratio of TRPV1 to GAPDH between genotypes (WT, Type III Nrg1+/−, n = 3 animals). Genotype comparisons were made using a Student's t-test. Graph shows mean±SEM. doi:10.1371/journal.pone.0025108.g004
All TRPV1 Publications.

Primary Neurons vs PC12 cells for Compound Testing

This publication compares PC12 Cells vs E18 Primary Cortical Neurons. The cells showed permeability to some key compounds where the Neurons did not. This demonstrates the importance of including primary neurons in compound testing assays for Neuro-disease research: Wei Zhang , Radhia Benmohamed, Anthony C. Arvanites, Richard I. Morimoto, Robert J. Ferrante, Donald R. Kirsch, Richard B. Silverman. Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells. Bioorganic & Medicinal Chemistry. Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.11.039.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2–3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC50 of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood–brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cell potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73)
were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar
potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel
therapeutic candidates for ALS.

see: Bioorg. Med. Chem. 2011, 19, 613. and J. Med. Chem. 2012, in press

Related Links: Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Image: E18 hippocampal neurons stained with Tau (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites (yellow) Axons (low doublecortin content) are red. Blue staining is the nuclear DNA.

Salivary Neuropeptides Y2s and Satiety

A possible new slant for combating obesity

The researchers in this study acheived a sustained increased PYY_3-36 (a neuropeptide) expression via viral vector-mediated gene delivery targeting salivary glands and the good news: this increase resulted in a significant long-term reduction in food intake (FI) and body weight (BW). This is evidence for new functions of the previously characterized gut peptide PYY_3-36 suggesting a potential simple and efficient alternative therapeutic approach for the treatment of obesity: Andres Acosta1, Maria D. Hurtado1, Oleg Gorbatyuk, Michael La Sala, David Duncan, George Aslanidi, Martha Campbell-Thompson, Lei Zhang, Herbert Herzog, Antonis Voutetakis, Bruce J. Baum, Sergei Zolotukhin. Salivary PYY: A Putative Bypass to Satiety. PLoS ONE 6(10): e26137. doi:10.1371/journal.pone.0026137. Received: July 22, 2011; Accepted: September 20, 2011; Published: October 10, 2011...Rabbit anti-Y2R (Dilution: 1:3000) using TSA...

Images: A) Immunolocalization of Y2R-positive cells in the hippocampus of C57Bl/6J mouse (WT), a (+) control. (B) Immunolocalization of Y2R in the tongue epithelia of Y2R KO mouse, a (-) control. VEG – von Ebner's gland. (C) Immunolocalization of Y2R-positive cells in the CV area of the tongue of a C57Bl/6J mouse. (D) close-up of (C). (E), and (F) close ups of (D), top and bottom rectangles, respectively. doi:10.1371/journal.pone.0026137.g003

These findings could prove a first step in identifying new targets for obesity fighting therapies. Increasing PYY salivary output would provide satiety with less food intake. This would give a whole new perspective on dieting. If we are satsified with less food intake is this really dieting? Stay tuned.

Opioid Addiction During Pregnancy-Implications for Neuro-development

I would like to thank Dr. Carmen Sato-Bigbee, Virginia Commonwealth University School of Medicine, for kindly sharing this important study. The publication also references use of our Mu Opioid and Nociceptin/Orphanin FQ Receptor Antibodies: Andrew C. Eschenroeder, Allison A. Vestal-Laborde, Emilse S. Sanchez, Susan E. Robinson, Carmen Sato-Bigbee. Oligodendrocyte responses to buprenorphine uncover novel and opposing roles of μ-opioid- and nociceptin/orphanin FQ receptors in cell development: Implications for drug addiction treatment during pregnancy. Glia Volume 60, Issue 1, pages 125–136, January 2012.

Highlights: Oligodendrocytes are responsible for making myelin in the CNS. The authors have shown We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. In this study, perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the l-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is the first study showing the potential role of the NOP receptor in myelination.

High levels of opiate exposure could negatively disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination. Understanding this pathway, could help researchers find ways to favorably modulate myelination and protect neuro-development of fetuses exposed to high levels of opiates during pregnancy.

Related Data:

Direct treatment of immature oligodendrocytes with buprenorphine alters MBP expression in a dose-specific manner. Cells isolated from 9-day-old rat brains were incubated for 4 days in CDM with or without 0.25, 0.5, 1.0, and 3.0 lM buprenorphine. MBP levels were determined by western blotting using b-actin levels as loading controls. Figures correspond to representative experiments. Results in the bar graph are expressed as percentage of controls (0 lM buprenorphine) 6 SEM from five experiments and correspond to the combined scanning of the four major MBP isoforms. **P <0.005 and ***P<0.0001.

Pre-oligodendrocytes express both MOR and the NOP receptor. Cells isolated from 9-day-old rat brain were allowed to fully attachon the culture plates by overnight incubation and stained by double
immunocytochemistry with O4 (green) together with anti-MOR or anti-NOP receptor antibodies (red). Scale bar: 20 lm. The western blot shows MOR and NOP receptor expression in two different samples of developing oligodendrocytes directly isolated from 9-day-old rat brains.

I will be posting future studies investigating the molecular mechanisms by which buprenorphine and methadone affect myelination and neuron-glial interactions. These should provide deeper understanding into these developmental processes and new and better strategies for the managing of both pregnant addicts and drug addiction in adolescence.

IF Staining of Human Primary Neurons

Primary Neurons are inputs or raw materials for cell based assays. When cells do not work as promised, there are multiple costs including lost time and potentially flawed data. Neuromics strives to provide easy to culture, potent and cost effective cells. Proving these capabilities is an ongoing activity for us. This includes testing these cells using our markers.

I wanted to share new immunofluorescence images. Here is a link to the protocol: staining primary neurons.

hN2 cells stained with our chicken polyclonal antibody to Vimentin, in red. Islands of Hn2 cells form after 4 days in culture forming beautiful flower like structures. Vimentin is a well established marker of early differentiating neuronal lineage cells. Taken with a 10X objective lens. Blue staining is the nuclear DNA.

hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to MAP2, a marker of neurons. Differentiating cells show strong cytoplasmic staining for MAP2 . Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture.

hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to Neurofilament light or low molecular weight chain NF-L, a marker of neurons. Many of the differentiating cells show strong cytoplasmic and clearly fibrillar staining for NF-L. Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture.

Fear Changes Hippocampus Neuropns

Fear in mice catalyzes rapid accumalation of EphrinB2 pyramidal neurons of the CA1 area.

This study suggests that rapid accumulation of EphrinB2 in hippocampal CA1 neurons is involved in the behavioural and cellular modifications induced by contextual fear conditioning. A similar mechanism does not appear to occur in lateral amygdala neurons, in spite of the robust behavioural and cellular modifications induced in such structure by cued fear conditioning: Antonio Trabalzaa, Sandra Colazingaria, Carmelo Sgobiob, Arturo Bevilacqua. Contextual learning increases dendrite complexity and EphrinB2 levels in hippocampal mouse neurons. Behavioural Brain Research. doi:10.1016/j.bbr.2011.11.008.

Diabetic retinopathy blindness-root causes

Diabetic retinopathy is a leading cause of acquired blindness. This publication from our friends at University of Buenos Aires touches on potential root causes: Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018
" In animals that had been diabetic for 6 weeks, a large increase in astrocyte reactivity occurred in the distal (but not the intraorbital) portion, which coincided with significant axon loss. Moreover, profound myelin alterations and altered morphologic features of oligodendrocyte lineage were observed at the distal (but not the proximal) optic nerve portion. The present results suggest that axoglial alterations at the distal portion of the optic nerve could be the first structural change in the diabetic visual pathway."
The authors used our PDGFR Alpha/CD140A Marker to Study the change in Oligodendrocyte Lineage precursor cells. Expression of the protein was increased in these cells with the presence of disorganized and hypertrophic cells. This could disrupt formation of myelin resulting the pathological alteration at the distal portion.

Opioid Induced Itch

Our widely used and frequently published Opioid Receptors Antibodies are used for a spectrum of pain research. Here is an interesting study on the root causes of opioid induced itch.

Xian-Yu Liu, Zhong-Chun Liu, Yan-Gang Sun, Michael Ross1, Seungil Kim, Feng-Fang Tsai, Qi-Fang Li, Joseph Jeffry, Ji-Young Kim, Horace H. Loh, Zhou-Feng Chen. Unidirectional Cross-Activation of GRPR by MOR1D Uncouples Itch and Analgesia Induced by Opioids. Cell, Volume 147, Issue 2, 14 October 2011, Pages 261-262.

Root Causes: Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Mouse epiSCs Into Myelinating Cells

This study published recently in Nature Methods hit my radar scope becaused it referenced use of our widely used and frequently published stem cell marker Tuj 1 (Neuron-specific class III beta-tubulin): Fadi J Najm, Anita Zaremba, Andrew V Caprariello, Shreya Nayak, Eric C Freundt, Peter C Scacheri, Robert H Miller & Paul J Tesar. Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells. Nature Methods (2011) doi:10.1038/nmeth.1712.

Dr. Paul Tesar and his team at Case Western University demonstrated the ability to convert pluripotent epiblast stem cells into pure populations of myelinating cells, called oligodendrocyte progenitor cells (OPCs). First, stem cells in a petri dish are treated with molecules to direct them to become the most primitive cells in the nervous system. To produce OPCs, these primitive cells are treated with a defined set of proteins. The cells were cultured on laminin and treated withh apporopriate growth factors. The OPCs were nearly homogenous and could be multiplied to obtain more than a trillion cells.

The OPCs were treated with thyroid hormone, which is key to regulating the transition of the OPCs to oligodendrocytes. The result was the OPCs stopped proliferating and turned into oligodendrocytes within four days.

These methods could used to potentially produce stable and pure populations of human OPCs in a significant enough number to treat patients with demyelinating diseases such as multiple sclerosis and cerebral palsy.

Immunostaining Neurons and Glia

I would like to thank Dr. Gerry Shaw, University of Florida for his excellent work with our Primary Neurons and Astrocytes and Neuronal-Glial Markers. Here's an example image with many more to follow:

Image: E18 hippocampal neurons stained with MAPT (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites, but doublecortin is more abundant in the growth cones and periphery. As a result, the periphery appears green while the more proximal regions of the cells are yellow. The single longer process of this cell, presumably an axon, has a low doublecortin content and so appears red. Blue staining is the nuclear DNA. Protocol on datasheet.

BDNF and Exercise Study-Running Mice

Get fit and get smart. There is increasing evidence that vigorous exercise increases secretion of Brain-Derived Neurotrophic Factor (BDNF) Protein.  BDNF is a catalyst of processes that increase growth of neurons especially in the hippocampus.

In this study researchers show new cell proliferation, survival, neuron number, and neurotrophin levels were enhanced only when running was accessible to mice. They conclude that exercise is the critical factor mediating increased BDNF levels and adult hippocampal neurogenesis: Tali Kobilo, Qing-Rong Liu, Kriti Gandhi, Mohammed Mughal, Yavin Shaham and Henriette van Praag. Running is the neurogenic and neurotrophic stimulus in environmental enrichment. doi: 10.1101/lm.2283011. Learn. Mem. 2011. 18: 605-609... human recombinant BDNF (0.1 µg) monomer (Neuromics)...
BDNF, CF Recombinant Protein
Related Reagents:
Neuron-Glial Expressed-Includes Neurotrophin Proteins
Neurotrophins and Growth Factor Antibodies
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Keep your brain healthy.

Immune-Inflammatory Response and Pain Research

Our Pain and Inflammation Related Research Antibodies are increasingly being used to study the root causes of immune/inflammatory related pain induction. Here're related publications: Lintao Qu, Pu Zhang, Robert H. LaMotte, Chao Ma. Neuronal Fc-gamma receptor I mediated excitatory effects of IgG immune complex on rat dorsal root ganglion neurons. Brain, Behavior, and Immunity. Volume 25, Issue 7, October 2011, Pages 1399-1407......rabbit-anti-TRPV1, 1:1000, Neuromics...

Highlights: Pain often accompanies antigen-specific immune-related disorders though little is known of the underlying neural mechanisms. A common feature among these disorders is the elevated level of antigen-specific immunoglobulin (Ig) G in the serum and the presence of IgG immune complex (IC) in the affected tissue. We hypothesize that IC may directly activate the Fc-gamma receptor type I (FcγRI) expressed in nociceptive dorsal root ganglion (DRG) neurons and increase neuronal excitability thus potentially contributing to pain. Immunofluorescent labeling indicated that FcγRI, but not FcγRIIB or FcγRIII, was expressed in a subpopulation of rat DRG neurons including those expressing nociceptive markers. Calcium imaging revealed that the IC, but neither of the antibody (IgG) or antigen alone, produced an increase in intracellular calcium. This effect was abolished by the removal of the IgG Fc portion in the IC or the application of an anti-FcγRI antibody, suggesting a key role of the FcγRI receptor. Removal of extracellular calcium or depletion of intracellular calcium stores prevented the IC-induced calcium response. In whole-cell current-clamp recordings, IC depolarized the resting membrane potential, decreased the rheobase, and increased the number of action potentials evoked by a depolarizing current at 2× rheobase. In about half of the responsive neurons, IC evoked action potential discharges. These results suggest that a subpopulation of nociceptive neurons expresses functional FcγRI and that the activation of this receptor by IC increases neuronal excitability.

B. Huanga, X. Zhaoc, L.-B. Zhengb, L. Zhanga, B. Nia. Different expression of tissue inhibitor of metalloproteinase family members in rat dorsal root ganglia and their changes after peripheral nerve injury. Neuroscience, Volume 193, 13 October 2011, Pages 421-428....anti-P2X3 (rabbit, Neuromics, MN, USA)...

Xona Microfluidics and Neurons

I am impressed with these Video from the Jeon Lab at UC Irvine. It represents a novel method for neuro-drug discovery:
Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device.

This technology represents a way to separate axon from cell bodies.

hNP1™ Human Neural Progenitors Video Protocols

Video Protocols designed to insure your success with our hNP1^TM Human Neural Progenitors

hNP1™ Human Neural Progenitors Video Protocols

Thawing, Culturing and Transfer-Sub Culturing Protocols.