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

I will continue to post updates here.

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.

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).

I am excited to present a recent publication that includes use of this kit to study Opioid-Induced Hyperalgesia. In this study Dr. Zaijie Jim Wang and his team at University of Illiniois Chicago down regulate CaMKII alpa expression. Their data implicates, for the first time, an essential role of CaMKII alpha as a cellular mechanism leading to and maintaining opioid-induced hyperalgesia.

Yan Chen, Cheng Yang, and Zaijie Jim Wang. Ca2+/Calmodulin-Dependent Protein Kinase II Is Required for the Initiation and Maintenance of Opioid-Induced Hyperalgesia. The Journal of Neuroscience, January 6, 2010, 30(1):38-46; doi:10.1523/JNEUROSCI.4346-09.2010.

…KN93 and KN92 were administered intrathecally by percutaneous puncture through the L5-L6 intervertebral space, as described previously (Hylden and Wilcox, 1980; Chen et al., 2009). A lateral tail flick was considered as success of the intrathecal injection. To inhibit CaMKII, CaMKII was targeted by small interfering RNA (siRNA). Four days after morphine pellet implantation, mice were treated with CaMKII siRNA (5′-CACCACCAUUGAGGACGAAdTdT-3′, 3′-dTdTGUGGUGGUAACUCCUGCUU-5′) (Zayzafoon et al., 2005) or Stealth RNAi negative control (Invitrogen) (2 µg, i.t., twice per day for 3 consecutive days). These oligos were mixed with the transfection reagent i-Fect (Neuromics), in a ratio of 1:5 (w/v) (Luo et al., 2005). Mechanical and thermal sensitivity tests were performed daily…

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 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.

I am pleased to report the parade of success with use our i-FectTM in vivo grows. 

Here’s the most recent study:

Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell, Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
…For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul…

Related Data:

Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.

About Dr. Laura Bohn  Dr. Laura Bohn  Background: Spring 2009-Associate Professor (tenured) at The Scripps Research Institute, Department of Molecular Therapeutics, Jupiter, FL. 10/2007- Associate Professor (tenured), The Ohio State University College of Medicine, Departments of Pharmacology and Psychiatry, Program in Pharmacogenomics 8/2003-9/2007 Assistant Professor, The Ohio State University College of Medicine, 1/1999–8/2003 Post-Doc/Assistant Research Professor. Duke University Medical Center, Department of Cell Biology. Durham, NC.

My company’s foundation is built on serving pain researchers. As a result, I have the good fortune of working with customers and collaborators who openly share the subtleties of their research and the future impact it could have on improving pain therapies.

Pain is complex. Today, pain therapies often fall short and are rife with unwelcome side effects. This undesrcores why I am pleased to feature Dr. Laura Bohn. She and her team are probing ways to improve  response effectiveness and reduce side effects.

 Beginnings

The story starts with Laura’s Post Doc work in Dr. Marc Caron’s lab at Duke University.  Marc in Collaboration with Dr. Dr. Robert Lefkowitz genetically engineered mice that lacked a protein switched called  “beta-arrestin 2.”  This switch is part of the opioid pathway that regulates how we perceive pain. The GPCR, muOpioid (mOR) is the primary target for narcotic pain killers, like morphine.

In her initial work, Laura found that morphine treated mice lacking the beta-arrestin2 switch swere able to tolerate  mild pain stimuli up to 3X longer than normal mice.  These mice had a higher level of sensitivity to morphine both in magnitude and duration. 

Bingo. This path for Laura’s excellent journey is now lit…understanding how the molecular regulation of G protein coupled receptors (GPCR) can translate to overall drug responsiveness in vivo.  Getting better response from lower dose is all good.

Current Work

As a researcher at Ohio State University, Laura and her team have continued to broaden and deepen their understanding of  GPCR signaling and beta-arrestin desensitivation (figure 1).

She is currently doing research with mice that have genetic deletions of GRKs (GRK3, GRK4, GRK5, and GRK6; heterozygotes for GRK2) and barrestin-2.

This expands the playing field. This expansion includes  studying other GCPR related pathways. Serotonin 2A receptors (5-HT2ARs), for example, are molecular targets for drug-induced hallucinations:

Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn. Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo. Published online on January 14, 2008, 10.1073/pnas.0708862105.

The conclusion: 5-HT2AR–β-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.

Future Considerations

I look for Laura and her team to continue the quest of doing more for less when it comes to novel pain and other therapies. Further success would provide the foundation for the development of therapies that would require less dosing, better response and reduced side effects.

Laura mentioned to me that further directions could involve the use of gene silencing tools like siRNA. The effects of silencing GPCR-beta-Arrestin receptors in-vivo would be an important study as it would enable she and her team to study  impact of  desensitivation on the repsonse to morphine and other drugs by normal mice.

Dr. Laura Bohn

January’s Story: Decoupling GPCR Pain Therapies from Destructive Side Effects. 

We are pleased to have Dr. Laura Bohn as our “coming soon” featured researcher. 

She caught my attention when she referenced one of our  Opioid Receptor Antibodies in the publication: C. E. Groer, K. Tidgewell, R. A. Moyer, W. W. Harding, R. B. Rothman, T. E. Prisinzano, and L. M. Bohn. An Opioid Agonist that Does Not Induce µ-Opioid Receptor—Arrestin Interactions or Receptor Internalization. DOI: 10.1124/mol.106.028258.

I am looking forward to drilling into the specifics of this important research.

About Dr. Matt Ramer

Matt Ramer

• 2001-Present-Associate Professor-University of British Columbia and ICORD
• Post Doc-King’s College London
• PhD.-Physiology-Queen’s College Kingston, Ontario

Matt Ramer Website

Awards and Funding

Email: ramer@icord.org

Lab Members: A. Gaudet, J. Inskip, A. Scott, L. Soril

Finding Fixes for Injured Nerves

I first became aware of Matt’s research in early 2005. This was catalyzed when he kindly shared excellent IHC images his lab generated using our BDNF and NT-3 antibodies. I was impressed with him and his team’s data and related publications. I did not understand the context of his work and the potential future impact on people suffering peripheral nerve and spinal cord injury (SCI) and wanted to learn more.

He has generously taken the time to open up my view on spinal cord injury (SCI) and what are the challenges in finding therapies and cures. I had equated success soley with restoring mobility. I knew little of the bigger complexites and problems faced by sufferers of SCI.

More than 300,000 people in the United States and Canada suffer from SCI. The ecomonic cost is in the 10s of billions. One of the horrors of SCI is lost mobility.

People with SCI also suffer from a host of problems related to loss of senory and autonomic functions. Sensory and autonomic nerves in the periperal nervous systems (PNS) connect to the spinal cord dorsally. This is different and separate from those controlling movement and motor function. A little know fact is autonomic dysfunctions represent the primary causes of morbidity and mortality following SCI.

So what are the functions that are most important to SCI patients and how should they be prioritized for basic research and drug discovery? Here are two publications that provide insight:

Kim D. Anderson, Ph.D. Targeting Recovery: Priorities of the Spinal Cord-Injured Population. October 1, 2004, 21(10): 1371-1383. doi:10.1089/neu.2004.21.1371.

J A Inskip, L M Ramer, M S Ramer and A V Krassioukov. Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord advances online publication 10 June 2008; doi: 10.1038/sc.2008.61

The Funding Gap

If we look through the eyes of those suffering from SCI, we know that there are a mryiad of health issues that are outside the problem of lost mobility. I fear the public including those responsible for funding define cure as “the paralyzed can walk”. This is evidenced by a gap between motor vs. sensory/autonomic research and priorities. This gap needs to be closed. The work of Matt and his colleagues represents progress and needs to be supported with funding growth. This backstory highlights how research could feed the discovery of therapies that would answer the recovery priorities of SCI pateints. They, after all, know best.

The Backstory

The story starts in 2000 and highlights Matt’s research at King’s College London. This research was done in collaboration with Dr. Stephen McMahon and Dr. John Priestly.

They showed that regeneration in damaged rat sensory neurons was possible. Injured dorsal roots, treated with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure:

Matt S. Ramer, John V. Priestley & Stephen B. McMahon. Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312-316 (20 January 2000) | doi:10.1038/35002084.

This is a tight rope act. While there is opportunity for regeneration, there are also inhibitors to nerve growth at work. Regeneration becomes more problematic as a function of time. Of the neurotrophins that promote regeneration, NT-3 appears to best at combating the competing inhibitory effects of proteins like NOGO-A. These inhibitory proteins are suspected to be secreted by astrocytes and microglia:

Matt S. Ramer, Ishwari Duraisingam, John V. Priestley, and Stephen B. McMahon. Two-Tiered Inhibition of Axon Regeneration at the Dorsal Root Entry Zone. The Journal of Neuroscience, April 15, 2001, 21(8):2651-2660.

Images: Axon growth 2 weeks after rhizotomy plus immediate NT-3 treatment. A, In intact animals, CTB-labeled terminals are present in lamina I and III, but absent from lamina II. B, Regenerating axons grow along the pial surface of the cord and in the superficial laminae of the gray matter, avoiding the degenerating cuneate fasciculus. C, Dark-field micrograph of B. Scale bar: B, 100 µm. D, Dark-field parasaggital section from a 2 week rhizotomized and NT-3-treated rat. E, Same section as in D, immunostained for CTB. CTB-labeled axons can be seen on the pial surface (arrowheads) and within the cord. Many axons have turned to grow in a rostrocaudal direction but appear to do so in the superficial laminae of the gray matter rather than the white matter. Some individual axons can be traced for up to 2 mm. F, In zones in which the density of regenerated axons is greatest, they form a longitudinal bundle in the gray matter, with few axons in the more superficial white matter (arrows). G, Many axons possess terminal swellings that may be growth cones or termination bulbs. Scale bar: E, 300 µm. The Journal of Neuroscience, April 15, 2001, 21(8):2651-2660.

Through the Looking Glass

Matt and his colleagues continue to gain understanding and refine methods for nerve regeneration. They are also studying plasticity and how these neurons connect to sensory and autonomic neurons in the PNS. This is analogous to re-wiring what was once severed. This would enable restoring of functions important to sufferers of SCI. The related good news is that even partial reconnection enable restoration of these lost functions.

Stepping through the looking glass involves understanding the specific role of these neurons. His recent works include:

Matt Ramer. Anatomical and functional characterization of neuropil in the gracile fasciculus. The Journal of Comparative Neurology. 10.1002/cne.21785.

• Neurokinin-1 (NK 1) Receptor-Detects a band at 80-90 kDa on Western blots of membranes prepared from cells transfected with the rat substance P receptor (Vigna et al., 1994); stainingin rat spinal cord was blocked by preabsorbing the antiserum with the immunizing peptide (Mantyh et al., 1995)-Dilution 1:2,000
• Substance P-The distribution of immunoreactivity in rat spinal cord is identical to that described previously (Hunt et al.,1981); in dual-labeling experiments, it labels the same structures as a polyclonal rabbit anti-SP (1:1,000; Peninsula/Bachem; T-4107; data not shown).

Here Matt and his team report on the morphology, inputs, projections, and functional properties of these neurons. Small fusiform and larger lentiform neurons are most abundant in the gracile fasciculus of the cervical and lumbar enlargements and are absent from the cuneate fasciculus and corticospinal tract. Many have dendrites that run along the dorsal pia, and, although in transverse sections these neurons appear isolated from the gray matter, they are also connected to area X by varicose and sometimes loosely fasciculated dendrites. These neurons receive neurochemically diverse, compartmentalized synaptic inputs (primary afferent, intrinsic and descending), half express the substance P receptor, and some project supraspinally. Unlike substantia gelatinosa neurons, they do not express protein kinase C gamma. Functionally, they have small receptive fields, which are somatotopically appropriate with respect to their anterior-posterior position along the neuraxis. They respond to innocuous and/or noxious mechanical stimulation of the distal extremities, and some are prone to central sensitization or windup. Morphologically, neurochemically, and functionally, therefore, these cells most closely resemble neurons in laminae III-VI in the dorsal horn.

Closing Thoughts

There is hope for SCI patients. It is clear that related research and funding needs to expand dramatically beyond the current narrow focus on restored motor function and mobility. The priorities are documented and understood. The story continues. Real progress will be marked by answering these priorities with restored function. Sensing pain, pressure, temperature, etc. where today there is only nothingness. Controlling autonomic functions that pose such a risk to SCI sufferers. I will continue to report the progress of Dr. Matt Ramer and his colleagues. Godspeed to them.

In this study, Dr. Eric Lingueglia and his team found Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Emmanuel Deval, Jacques Noël, Nadège Lay, Abdelkrim Alloui, Sylvie Diochot, Valérie Friend, Martine Jodar, Michel Lazdunski and Eric Lingueglia. ASIC3, a sensor of acidic and primary inflammatory pain. The EMBO Journal advance online publication 16 October 2008; doi: 10.1038/emboj.2008.213

 Cy3-labelled siRNA no. 1121 and its corresponding scramble (no. 1121S; GCTCACACTACGCAGAGAT) synthesized by MWG Biotech (Germany) were injected in rats by intrathecal bolus to the lumbar region of the spinal cord once a day for 3 days before the induction of inflammation with CFA. Each 10-ml injection corresponded to 2 mg of siRNA complexed with i-Fect siRNA transfection reagent (Neuromics) at a ratio of 1:4 (w:v) (Luo et al, 2005), following the supplier’s suggested protocol. siRNA uptake in lumbar DRGs
was monitored by fluorescence microscopy on cryostat sections 24 h after a single intrathecal injection.

Here’s a synopsis of results:

Inflammation was produced by CFA injection, which led to primary heat hyperalgesia, and this hyperalgesia was drastically reduced by the ASIC3 blocker APETx2 injected subcutaneously, which only access cutaneous nociceptors. It was also drastically reduced when, before triggering the inflammation state, intrathecal
injections of an siRNA against ASIC3 had induced a knockdown of ASIC3 expression in lumbar DRGs.

I will continue to publish updates.

I asked Dr. Nicolas Beaudet, a Sarret lab member, why he joined the lab. He said, “ Philippe is a great communicator. He has the ability to articulate his complex research in a way that is easy to understand, visionary and exciting”. The aspect that Nicolas finds most intriguing is the systems approach that Philippe and the team take in understanding the mechanisms of pain. This enables them to work at them molecular level up to the whole animal. This is a key step in finding potential new pain therapies.

Drilling Down

Philippe and his team centered their efforts on G Protein Coupled Receptors (GPCRs) such as apelin, chemokines and neurotensin. As a common point, they were all recently identified in the central nervous system to provide a potential role in pain modulation.

Lately, the focus has been on the roles of Neurotensin Receptor 1 (NTS1) and Neurotensin Receptor 2 (NTS2). Recent studies have highlighted the role of these receptors in pain modulation and more is to come…:

• Geneviève Roussy, Marc-André Dansereau , Louis Doré-Savard, Karine Belleville, Nicolas Beaudet, Elliott Richelson and Philippe Sarret. Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model.Journal of Neurochemistry 105: 1100 – 1114
• Sarret, P, Perron, A, Stroh, T and Beaudet, A (2003). Immunohistochemical distributionmof NTS2 neurotensin receptors in the rat central nervous system. J Comp Neurol 461: 520–538.

• Sarret, P, Esdaile, MJ, Perron, A, Martinez, J, Stroh, T and Beaudet, A (2005). Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors. J Neurosci 25: 8188–8196.
• Dobner, PR (2006). Neurotensin and pain modulation. Peptides 27: 2405–2414.
• Maeno, H, Yamada, K, Santo-Yamada, Y, Aoki, K, Sun, YJ, Sato, E et al. (2004). Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception. Brain Res998: 122–129.      

The wow factor for me was the clever way Philippe and his team used a new technology of 27mer NTS2 Dicer Duplex siRNA (DsiRNA) delivery in vivo as a proof for the potential of DisRNAs-based pain therapies.

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.

What Happened

Using an acute pain model, anti-nociceptive effects of NTS2, induced by a selective agonist, were significantly reduced following NTS2 silencing This resulted in rats showing an increased sensitivity to pain. By day four, the knockdown effects showed a decrease with the NTS2 function returning to normal.

What ‘s next

So we have a great start. We know that agonists binding to NTS2 in the CNS lead to analgesia. We know that DsiRNA can be used to alter the expression of this gene in vivo. We have provided a key step in learning how the NTS2 receptors can be manipulated to block pain. However, now we need to unravel the underlying mechanisms explaining these spinal analgesic properties.

It is my hope that Philippe and his team are appropriately funded. This would catalyze further discoveries in how expression of G Protein Coupled Receptors like NTS1, NTS2, APJ, CCR2 can be targeted to modulate pain. By using rodents, the team can develop tools like DsiRNA to increase the potency and duration of pain blockade. Moreover, potential toxicity and side effects need to be addressed in order to move forward towards clinical studies. These pre-clinical models prove invaluable in taking the step to studies in humans.These therapies hold the promise of providing relief for chronic pain (neuropathic, arthritic, diabetic, cancer pain, etc.) sufferers without the current side effects. Stay tuned as I will be reporting the good news as it unfolds.