Network vs Individual Bursting Neurons

Motor Neurons and MEA
Dysregulated bursting is at the root of many motor neuron/neuromuscular junction disease. ArunA Biomedical teaming with Axion Biosystems have generated relevant bursting data from our Mouse Motor Neurons cultured on Axion-Bioystem’s Maestro MEA.

Figure: Mouse Motor Neuron Network Modulation by Bicuculline-ckeck out the entire presentation to learn more: GFP+ Motor Neurons: Development and in-vitro Functional Assessment on Microelectrode Arrays

Protocol User’s Guide for Culturing Motor Neuron on MEA(pdf – 679Kb)

Name Catalog # Type Species Applications Size Price
Motor Neurons-GFP+ Quick Start Kit mMN7205.QS Primary Neurons M Cell Assays 750,000 $349
Motor Neurons-GFP+ HTS Kit mMN7205-HTS Primary Neurons M Cell Assays 4 X 750,000 $989
GDNF (Human, Mouse) PR27022-2 Protein H; M 2 ug 10 ug $108 $205
AB2™ Basal Neural Medium AB27011.3 Cell Growth Media H; M Cell Assays 500 ml $69

We will continue providing you content we believe important. Should you have questions, do not hesitate to contact us. Thank you and we stand ready to serve you and your team.

Pete Shuster-CEO and Owner, Neuromics, 612-801-1007, pshuster@neuromics.com

The Importance of in vivo Like Astroglial-Neuron Co-Cultures

The Power of Neuromics’-Aruna Biomedical’s hAstroPro™-NeuroNet™ Co-Cultures. We are pleased to be the first to market with our in vivo like co-cultures. Here are key points of what make these cultures unique:

  • Pure, Potent and Proven

  • Proof That These areTrue Co-Cultures

  •  Co-Cultures vs Neuron Only Cultures

What have we learned? The mix of Astroglia vs Neurons can have a dramatic impact on your data end points. This can lead to wrong conclusion being drawn from your tox and compound/small molecule neurodegenerative disease assays. We stand ready to serve you and your team. Questions? Don not hesitate to call 612-801-1007 or e-mail me pshuster@neuromics.com. Pete Shuster, CEO and Owner, Neuromics

More on STEMEZ hN2 Primary Human Neurons

My company’s STEMEZTM hN2 Primary Human Neuron Discovery Kits have been a frequent topic on “News Behind the Neuroscience News”. My friends at Aruna Biomedical continue to broaden the capabilities of these Kits based on customer feedback.

I am seeing increasing demand for these cells as these capabilities are published. Here’s the latest:

A. Young, D.W. Machacek, S.K. Dhara, P.R. MacLeish, M. Benveniste, M.C. Dodla, C.D. Sturkie and S.L. Stice. Ion channels and ionotrophic receptors in a human embryonic stem cell derived neural progenitors. doi:10.1016/j.neuroscience.2011.04.039. Markers used:…mouse nonoclonal anti nestin (neuromics), mouse monoclonal anti tuj-1 (neuromics)…

Abstract: Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 h after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted. Fourteen days after the removal of basic fibroblast growth factor, most cells expressed microtubule-associated protein 2 and TUJ1, markers characterizing a post-mitotic neuronal phenotype as well as neural developmental markers Cdh2 and Gbx2. Real-time PCR was performed to determine the ionotrophic receptor subunit expression profile. Differentiated neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the hNP cells tested under whole-cell voltage clamp exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin-sensitive, voltage-dependent sodium channel current. Action potentials could also be elicited by current injection under whole-cell current clamp in a minority of cells. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and has the capability to produce excitable cells that can generate action potentials, a landmark characteristic of a neuronal phenotype. This is the first report of an efficient and simple means of generating human neuronal cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

STEMEZ hN2 Cells-Electrophysiology Data

STEMEZ hN2 Cells-Electrophysiology Data

 

 

 

 

 

I will continue to post updates here.

Ion Channels and Neuromics’ STEMEZ Cells

In my conversation with neuro-drug discover researchers, I am frequently being asked about the potential of using our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kits for studying ion channels. How effective are these cells as a source for studying neurodegenerative diseases and for drug screening assays?  There is good news from Dr. Steve Stice and my friends from ArunA and UGA.

When differentiated, these  neural progenitors express subunits of glutamatergic,  GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium  and calcium channel subunits were also expressed. Functionally, virtually all the NP cells exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited  tetrodotoxin sensitive, voltage-dependent sodium channel current under whole-cell voltage clamp and action potentials could be elicited by current injection under whole-cell current clamp.  These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and also results in the capability to produce excitable cells that can generate action potentials. This is the first data demonstrating capabilitiesof these cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.
hNP1_Gene_Expression

Images: Glutamate receptor expression in hNP cells and differentiated hNP cells The expression of ionotropic glutamate receptors might also be an indicator of neuronal maturation. These receptors are composed of three distinct families: NMDA, kainate and AMPA receptors. The hNP cells and differentiated hNP cells cultured in the absence of bFGF for 2 weeks were analyzed for mRNA expression of subunits of each glutamate receptor subtype relative to hESCs. Significant increases (p<0.05) in Grin2b were seen in hNP cells (20 fold) and differentiated hNP cells (25 fold) relative to hESCs (Figure 3A). Additionally, Grin1 and Grin2d were significantly increased (p<0.05) only in differentiated hNP cells relative to hESCs, but not in undifferentiated hNP cells (Figure 3A). Of the kainate receptors, Grik4 and Grik5 were significantly (p<0.05) increased only in undifferentiated hNP cells relative to hESCs (Figure 3B); whereas, Grik2 was significantly (p<0.05) increased only in hNP cells where bFGF had been removed (Figure 3B). AMPA receptor subunits were also examined. Gria1 and Gria4 were up regulated in hNP cells relative to hESCs (Figure 3C). Two week differentiated hNP cells showed significant (p<0.05) up regulation of Gria2 and Gira4 relative to hESCs (Figure 3C). To determine if functional glutamate channels exist in differentiated hNP cells, calcium influx in response to AMPA, kainic acid or NMDA application was measured on hNP cells, 14 days after the removal of bFGF. Figure 3G indicates that NMDA could not depolarize differentiated or undifferentiated hNP cells enough to cause significant calcium influx above background. In contrast, AMPA and kainic acid can cause calcium influx which can be potentiated by AMPA receptor specific modulator, cyclothiazide (50 μM, Figure 3G).Calcium influx was detected in the presence of cyclothiazide in calcium activity as measured (Figure 3H).

hNP1_Electrophysiology

Images: Sodium channel activity in differentiated hNP cells was measured using whole cell voltage clamp. 81 total hNP cells cultured in the absence of bFGF from 4 to 27 days were analyzed. Of these, 34 exhibited no fast inward currents in response to a step depolarization indicating the 348 absence of functional voltage gated sodium channels (Figure 4G). The remaining cells yielded between 0.04 – 1.5 nA of inward current in response to the step depolarization (Figures 4B and 4G). These currents inactivated rapidly in all cases (Figures 4B and 4C) and could be abolished with the addition of 1 μM TTX (n = 3 cells; Figure 4C). Voltage-dependent steady state inactivation (n = 11 cells; Figure 4D) and recovery from fast inactivation (n = 5 cells; Figure 4E) were also observed on several positive cells. A subset of these cells was subjected to current clamp and action potentials were elicited by current injection (n = 8 cells, Figure 4F). In support of this, increasing concentrations of a sodium channel activator veratridine in a FLIPR assay on differentiated hNP cells show an increasing calcium response (Figure 4H). This probably resulted from voltage-gated sodium channel depolarization of cells that subsequently allowed calcium influx through calcium channels. These data indicate that differentiation of hNP cells by removal of bFGF can lead to a neuronal cell that can generate action potentials and depolarize the cell. The 58% hit rate for voltage-gated sodium channel function (Figure 4G), does not reflect the true proportion of sodium channel positive cells in our differentiated hNP cells, but rather our ability to morphologically distinguish these cells from negative cells by eye. An example of the morphology of a sodium channel positive cell is shown in Figure 4A. The positive cells were phase bright with a few long processes.

Featuring Dr. Richard Rogers

Obesity Energy, Thermogenesis and Appetite

Dr. Richard Rogers

Dr. Richard Rogers

 
Obesity and its evil twin, diabetes, are rapidly becoming our #1 health epidemic. Today 10% of all medical costs in the U.S. are dueto an overweight population, and this percentage is growing rapidly. Today, the breakdown is about $1500 per year in medical costs for obese versus normal weight individuals. This translates into more than $145 billion spent annually.
Given the size of the epidemic, a growing focus for my company is providing reagents to researchers who study bioprocesses involved in energy metabolism. This includes researchers studying what happens to energy expenditure when the “fuel tank” is full and also empty. Both states could give clues as to why we overeat.

In my routine follow up with researchers using our reagents, I started to get an appreciation on how these complex energy pathways are being unraveled and better understood. That appreciation forms the roots of this “News Behind the Neuroscience News” story. It is a story that has the hormones Leptin and TRH playing a starring role supported by hindbrain neuro/glial-circuitry and brown adipose tissue (BAT).

The Rogers Lab

I became aware of the Richard Rogers Lab in my follow up with Montina Van Meter checking on our  LepRb/OBRb antibody. She shared that it was giving them results better than most others they has used. She then gave me an overview of her research involved with  the control of feeding behavior and energy utilization including thermogenesis (“heat generation”) catalyzed in BAT.  Cool…this was a lab we wanted to make sure we served and served well.

Tina not only kept me informed on how our reagents were working, but also generously alerted to me publications referencing use of our reagents:
·        Maria J. Barnes, Richard C. Rogers, Montina J. Van Meter and Gerlinda E. Hermann. Co-localization of TRHR1 and LepRb
receptors on neurons in the hindbrain of the rat.
 doi:10.1016/j.brainres.2010.07.094…Included are excellent images of stained LepRb (OB-Rb)-(Dilution 1:500) and  GAD1-Dilution (2ug/ml) expressing neurons localized in loose clusters of cells in the DMN, NST, and the VLM…
·        Hung Hsuchou, Yi He, Abba J. Kastin, Hong Tu, Emily N. Markadakis, Richard C. Rogers, Paul B. Fossier, and Weihong Pan. Obesity induces functional astrocytic leptin receptors in hypothalamus. Brain, Mar 2009; doi:10.1093/brain/awp029…unique sequence of ObRb at its cytoplasmic tail (CH14104, Neuromics, Edina, MN, USA). This antibody was raised
against rat ObRb…

I found this research to be unique and intriguing. This led me to an interview with Dr. Rogers. Here is his backstory.

Beginnings

Dr. Rogers credits serendipity as a driving force in his interest in Neuroscience. It started with a bike ride and chance introduction with a ham radio operator when he was a youngster. This catalyzed his interest in electronics and circuitry.

This interest morphed to a passion for Neuroscience (circuits and signaling). He entered the first college program devoted to Neuroscience studies at UCLA.  He received his Ph. D. in 1979. His post-doc focused on digestive regulation. Here, he  investigated the neural circuitry involved in the normal control of gastric function.

Evolutions

In collaboration with his wife Dr. Gerlinda Hermann, his work evolved to solving the mystery of why we don’t eat (abnormal gastric function). What causes gastro-intestinal shutdown?  The breakthrough was their ability to show cross-talk between the immune system and nervous system. This research is a foundation for the discovery of therapies for sufferers of appetite shut down cause by cancer therapies and certain immune related pathologies.

The main culprit is TNF-α. The blood level of this peptide is elevated as a consequence of immune activation caused by infection, cancer, radiation exposure and chronic autoimmune disease. The breakthrough was showing that  TNF-α receptors are on neurons in the brainstem that control gastric functions, including emesis.  Neurons in the nucleus of the solitary tract (NST) respond to TNF-α greatly increasing the sensitivity of gastric vagal control circuitry.  This causes emptying of the gut to dramatically slow,l leading to nausea, vomiting and cessation of appetite.

Currently, they are delving into the complexities of  TNF-α signaling processes. This includes the role of astrocytes and glia.

See: Gerlinda E Hermann and Richard C Rogers.  TNF activates astrocytes and catecholaminergic neurons in the solitary nucleus: implications for autonomic control. doi: 10.1016/j.brainres.2009.03.059.

 Energy,Obesity and Thermogenesis-Active Astrocytes

Recently, the lab took another road less traveled. Dr. Gerlinda Hermann discovered interesting aspects of leptin and thyrotropin releasing hormone (TRH) signaling.  This research looks at signaling in thermogenesis and feeding behavior. A most interesting aspect includes conclusions concerning the role of astrocytes.  Their colleague Dr. Weihong Pan  showed that adult obese mice, (2 months after being placed on a high-fat diet) showed a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology (doi: 10.1093/brain/awp029).

Non-shivering thermogenesis usually occurs in BAT. It uncouples the ATP energy producing process by generating heat rather than driving the conversion of ADP to ATP. This creates an ingenious way to untangle complex processes related to Leptin signaling. What happens when there is sufficient energy for the thermogenic process? Conversely, what happens when there is insufficient energy?

 Leptin and TRH act synergistically in the hindbrain to drive thermogenesis. However, in a starving condition there is a subsequent drop in Leptin and thermogenesis. Behind these simple facts are complex processes that occur in the hindbrain. The team is providing important insights including location of events and relevant signaling molecules. (doi:10.1016/j.brainres.2010.07.094).

The Future-Caged Compounds and Live Cell Signaling

Caged compounds are bioactive molecules attached to photolabile groups, that release the active component on contact with photons of the right energy level – the process of photolysis. Photolysis is now widely applied in biology to induce neurotransmitter and otherm ligand-receptor interactions in conditions that are otherwise subject to poor diffusional access and receptor desensitization, as well as for labile ligands.

This novel technology affords Dr. Rogers and his team the capability do live cell imaging of calcium signaling. From these they will help us gain a deeper understanding of what is happing and where. Specifically, we will more exactly learn the role that astrocytes and glia play in controlling the role of   Leptin and related signaling molecules in controlling energy, metabolism and feeding behavior. This could lead to important target for future therapies.

On Deck-Dr. Richard Rogers

I am exciting to be profiling Dr. Richard Rogers in my upcoming Neuroscience Backstory feature. This is an important feature because it focuses on the timely topic of modulation of the brain-gut axis by cytokines, hormones and CNS pathways involved in the control of feeding behavior and energy utilization. Given the acceleration in the growth of obesity in the US and related pathologies, his research is becoming increasingly important.  We will also be featuring related research on what drives lack of appetite in cancer patients. This is a key intersection as the signaling pathways involved in insatiable and cessation of appetite are related.

I also wanted to share a recent article on yet another intersection which focuses on thermogensis which occures in brown adipose tissue (BAT): Maria J. Barnes, Richard C. Rogers, Montina J. Van Meter and Gerlinda E. Hermann. Co-localization of TRHR1 and LepRb receptors on neurons in the hindbrain of the rat. doi:10.1016/j.brainres.2010.07.094.

LepOBRB-TRHR3

Example images: Distribution of LepRb+ fibers in hindbrain. LepRb-ir (red) fibers and varicosities are seen among TRHR1-ir (green) cells and fibers. These red and green fibers are adjacent and co-mingle but do not show co-localization of receptors. This pattern is seen in (A) fascicles of the solitary tract (ST); (B) raphe pallidus (RP), and (C) raphe obscurrus (RO). (D) Border between the medial solitary nucleus (NST) and the area postrema (AP; white dashed line) showing an abundance of LepRb-ir (red) fibers and
 neurons (white arrows for selected neurons) in the NST but not the AP. (E) LepRb-ir staining is suppressed by pretreatment of tissue with LepRb epitope blocking peptide. (F) TRHR1-ir staining is suppressed by treatment with excess TRHR1. Scale bar A–D=100 microns; E, F=300 microns. cc=central canal. Note: this pus references use of our LepRb (OB-Rb) and GAD1 antibodies.

Featuring Dr. Pat Carr

Amyotrophic Lateral Sclerosis (ALS)-New Twists on Root Causes

Teacher, Mentor and Friend    Dr. Pat Carr has been a key figure in helping shape the direction of my company. He has a gift for communicating the nuances of his research and coaching me on how to best serve labs like his. Based on these interactions, it came as no surprise to learn of his being Recognized for Excellence in Teaching, Research and Service at University of North Dakota.

“Dr. Carr has a magic way of teaching,” said second-year medical student, Tyson Bolinske. “He is able to take the most difficult topics and, through detailed notes, logically break down the material.

From a recent dialog, I learned of his growing work on the Ventral Horn and search for root causes of Amyotrophic Lateral Sclerosis (ALS).   I wanted to learn more! I would like to thank Pat for agreeing to share his story and giving me the opportunity to feature highlights in  “News Behind the Neuroscience News”.

 Information on ALS

ALS is an insidious disease.  It is a progressive neurodenerative disease that is always fatal. Approximately 5600 new cases are diagnosed each year. Average survival is typically 3-5 years from onset. The most common form of ALS in the United States is “sporadic” ALS. It can happen to anyone at anytime.  The other is the inherited form named “Familial” ALS (FALS). Only about 5 to 10% of all ALS patients appear to have FALS. As the disease progresses the symptons become more acute. Paralysis spreads through the body affecting  speech, swallowing, chewing and breathing. Ventilator support is need in late stages

 Pat’s Journey

Pat took the “road less traveled”.  He was a passionate hockey player in Canada. He  concluded in his late teens that he was not at a level to take this road to wealth and fame.

Pat Carr

Pat Carr

06/04–present Associate Professor, Department of Anatomy & Cell Biology, School of Medicine and Health Sciences, University of North Dakota 

1996–98 Research Associate/Adjunct Assistant Professor/Auxilliary Assistant Professor, Department of Anatomy;Wright State University

 07/98–06/04 Assistant Professor, Department of Anatomy & Cell Biology, School of Medicine and Health Sciences, University of North Dakota

Postdoc, National Institutes of Health, Neuroscience, 1994-96

Postdoc, University of Manitoba, Neuroscience, 1992-1994    

Ph.D., University of Manitoba, Physiology, 1992

Next was a stint as an automechanic in Brandon, Canada. The discipline and logic involved in fixing cars catalyzed an interest in Science which led to him going to Brandon University to study Geology. When the oil market collapsed in 1983, he decided to change his studies to Zoology and earned a BS in 1984.

A passion was sparked when he did field research in the Canadien Rockies studying parasites in Columbian Ground  Squirrels. He loved it, but recognized the limited value of continuing thsese studies. This lead to the wide open field of Neuroscience and the opportunity to study and solve problems that could benefit mankind. His graduate work at University of Manitoba and focusing on Neuropathic Pain and the Dorsal Horn. He then moved on to studying Ventral Horn and Motor Control Function for his Post Doc at Wright State.

From Pain to ALS

It was Pat’s work in Pain at the University of North Dakota that brought me into initial contact with him. He generously put some of our key Pain/Inflammation and  Neurotransmission Research Antibodies through their paces. These included some of our Neuropeptide and Neuropeptide Receptors , P2X Receptors and TRPV1s (Vanilloids).

His previous work in studying the Ventral Horn combined with a colleagues mouse model of ALS combined to create a prefect opportunity to advance the understanding of ALS.  Pat cautioned me with this insight:  ”sometimes it is  not what you want to study; it is what you can study.  The model is  SOD1 (superoxide dismutase 1) which is core to FALS.(occurs in only about 10% of the ALS cases).

Pat is broadening the play field by looking at what else is happening in sporadic ALS vs FALS. Specifically, he is looking at modulation of alpha Motor Neurons and how the activity of adjacent Renshaw Cells impact signaling and modulation.  Renshaw Cells act as a “governor” on the activity of these alpha Motor Neurons. 

He is drilling down by studying the signaling of ChAT (Choline Acetyltransferase), VAChT (Vesicular acetylcholine transporter) and related molecules. By gaining a deeper understanding of how Renshaw Cells signaling changes the activity of alpha Motor Neurons in ALS,  Pat and his team are taking steps towards discovering roots causes.

As these root causes are further illuminated, I will be reporting specifics in my blog.

Delivering 27mer DsiRNAs to Mice DRGs

I have been a proponent of using 27mer DsiRNAs (Dicer Substrate Small Interfering RNAs) with our i-Fect kits to deliver siRNA to the CNS for gene expression analysis. The potency of this platform was highlighted in my profile of Dr. Mark Behlke.

It was further confirmed  in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’  i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:

Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.

IT Delivery of siRNA in vivo supplement

March Profile-Dr. Evanna Gleason

Coming soon…evanna_gleasonNow for something completely different…Synaptic Transmission Research.

I am pleased to be featuring Dr. Evanna Gleason.  I selected her because She and her team’s research is a basic component of most areas  Neuroscience Research including pain, neurodegeneration, vision, TBI, SCI, drug addiction, neuro-disorders and more…This base component is how neurons communicate with other cells at synapses. 

She focuses on synaptic transmission in the vertebrate retina. Retinal neurons have distinctive anatomical and physiological properties that suggest they employ unique synaptic mechanisms. The long term objective her research is to understand how retinal synapses are specialized to transmit visual information.

More to come in March!