Scripps Florida Scientists Awarded $3 Million to Develop New, More Effective Pain Treatments

We profiled Dr. Laura Bohn research in one of our news stories. We are excited to share the news.Dr. Laura Bohn

JUPITER, FL, February 29, 2012 – Scripps Florida scientists have been awarded $3.1 million by the National Institute on Drug Abuse, part of the National Institutes of Health, to study and develop several new compounds that could prove to be effective in controlling pain without the unwanted side effects common with opiate drugs, such as morphine, Oxycontin®, and Vicoden®.

Laura Bohn, an associate professor in the Department of Molecular Therapeutics and Neuroscience at Scripps Research, and Thomas Bannister, an assistant professor in the Department of Chemistry and associate scientific director in the Translational Research Institute at Scripps Research, will serve as joint principal investigators for the new five-year study.

Their study will focus on four new classes of compounds that appear to differ fundamentally from opiates inthe side effects that they can produce.

“Once we more fully understand how these compounds work, we expect to optimize and develop them as novel drugs,”said Bohn. “We hope to produce potent pain relievers without the problems associated with current treatments.” Full article: http://www.scripps.edu/news/press/20120229bohn-bannister.html

We wish her great success in her research aimed at discovering improved solution for managing pain.

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.

Opioid-Induced Hyperalgesia and CaMKII alpha

Many of my backstories have featured Pain Researchers.  In several, I have featured use of our our i-Fect ™ Transfection Kit for enhancing the delivery of siRNA in vitro and in vivo to study the expression of genes invovled in Neuropathic and Nociceptive Pain.

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…

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.

Knockdown of rSNSR1 in vivo

I have featured successes with delivering siRNA in vivo in this blog. These included stories on Dr. Philipe Serrat and his team at the University of Sherbrooke and Dr. Mark Behlke’s work at Integrated DNA and Dicerna.

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.

The Quest for Better Pain Therapies

G- protein coupled receptor (GPCR) and Drug Responsiveness

About Dr. Laura Bohn 

bohn1

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).

gpcr_regulation 

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.

Featuring Dr. Laura Bohn

Dr. Laura Bohn

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.

She, her team and callaborators are focused on an interesting and important aspect of pain therapies discovery…finding ways to de-couple the benefits of opiate based pain medications from their current side-effects including constipation, respiratory suppression and addiction.

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

The Sensory and Autonomic Side of Spinal Cord Injury

About Dr. Matt Ramer

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.

ACIC3 Receptors Knockdown in vivo

Researchers using siRNA complexed with our i-Fect ™ transfection regent have successfully knocked down ASIC3 Receptors in vivo. This publication joins the growing parade (starting with Luo et al, 2005) that reference successful modulation of receptors involved in pain using siRNA complexes. These studies all share animal behavior studies showing a marked change in response to pain stimuli after treatment.

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.