TRPV1 Expression and Temporal Lobe Epilepsy

Dr Bret. Smith and his lab at Tulane have demontrated a link between Temporal Lobe Epilepsy (TLE) and VR1 N-Terminus (TRPV1) expression.

In this excellent study they showed an increase in expression in TLE mice vs controls.

Muthu D. Bhaskaran and Bret N. Smith. Effects of TRPV1 activation on synaptic excitation in the dentate gyrus of a mouse model of temporal lobe epilepsy. doi:10.1016/j.expneurol.2010.01.021
...with polyclonal VR1 N-terminus (1:2500) (Neuromics, Edina)..

Abstract: Temporal lobe epilepsy (TLE) is a condition characterized by an imbalance between excitation and inhibition in the temporal lobe. Hallmarks of this change are axon sprouting and accompanying synaptic reorganization in the temporal lobe. Synthetic and endogenous cannabinoids have variable therapeutic potential in treating intractable temporal lobe epilepsy, in part because cannabinoid ligands can bind multiple receptor types. This study utilized in vitro electrophysiological methods to examine the effect of transient receptor potential vanilloid type 1 (TRPV1) activation in dentate gyrus granule cells in a murine model of TLE. Capsaicin, a selective TRPV1 agonist had no measurable effect on overall synaptic input to granule cells in control animals, but significantly enhanced spontaneous and miniature EPSC frequency in mice with TLE. Exogenous application of anandamide, an endogenous cannabinoid that acts at both TRPV1 and cannabinoid type 1 receptors (CB1R), also enhanced glutamate release in the presence of a CB1R antagonist. Anandamide reduced the EPSC frequency when TRPV1 were blocked with capsazepine. Western blot analysis of TRPV1 receptor indicated protein expression was significantly greater in the dentate gyrus of mice with TLE compared with control mice. This study indicates that a prominent cannabinoid agonist can increase excitatory circuit activity in the synaptically reorganized dentate gyrus of mice with TLE by activating TRPV1 receptors, and suggests caution in designing anticonvulsant therapy based on modulating the endocannabinoid system.

Images: Western blot detection of TRPV1 receptor expression in the dentate gyrus. A. Diagram of dentate gyrus showing the microdissected area (box). B. Western blot showing TRPV1 receptor expression in two untreated mice and in two pilocarpine-treated mice that survived SE. Actin was used as the loading control which did not change significantly. C. Graph showing a significant (p less than 0.05; n=4) TRPV1 expression in epileptic mice.

Related Reagents:

TRPV (Vanilloid); TRPM; TRPA and TRPCs

Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels, Biogenic Amines and more

Primary Neurons and Astrocytes- Primary human, rat and mouse neurons and astrocytes by Category.

More Success with TRPV1

Dr. Irmgard Tegeder and team have been using our VR1 N-Terminus (TRPV1) as a marker to study how B kinase (IKK) plays a role in sensory neuron excitability and nociception.

The antibody was used for both Western Blot and Immunohistochemistry. Here's a recent publication of their results:

Vanessa Bockhart, Cristina Elena Constantin, Annett Häussler, Nina Wijnvoord, Maike Kanngiesser, Thekla Myrczek, Geethanjali Pickert,Laura Popp, Jürgen-Markus Sobotzik, Manolis Pasparakis, Rohini Kuner, Gerd Geisslinger, Christian Schultz, Michaela Kress, and Irmgard Tegeder. Inhibitor B Kinase β Deficiency in Primary Nociceptive Neurons Increases TRP Channel Sensitivity. The Journal of Neuroscience, October 14, 2009, 29(41):12919-12929; doi:10.1523/JNEUROSCI.1496-09.2009

Related Reagents:
All TRPV (Vanilloid); TRPM; TRPA and TRPC Antibodies
Pain and Inflammation Research Antibodies

Excellent TRPV1-N IHC and WB

Kudos to Dr. Federica MF van Dissel-Emiliani and her team for the excellent Immunohistochemistry and Western Blot results using our TRPV1-N Antibody(Catalog #: RA10110) . The antibody was in their study demonstrating the sensitivity of spermatogenesis to capsaicin.

Here's the related publication:

Sefika C Mizrak, Bart M Gadella, Hatice Erdost, Aytekin Ozer, Ana MM van Pelt, Federica MF van Dissel-Emiliani. Spermatogonial stem cell sensitivity to capsaicin: An in vitro study. Reproductive Biology and Endocrinology 2008, 6:52 doi:10.1186/1477-7827-6-52.

Anti TRPV1 antibody staining: Bouin's fixed, paraffin embedded 5 um-thick rat testis sections were deparaffinized and boiled in a microwave oven (700 Watt) 3x10 min in sodium citrate buffer (0.1 mM, pH=6) for antigen retrieval. All subsequent incubations were performed for 1 hour at room temperature. The slides were then blocked with 5 % goat serum in 1 % BSA/PBS and incubated with the rabbit anti human - VR1 antibody (Neuromics, Edina, MN, USA; 1:500 in 1% BSA/PBS). Biotinilated goat anti-rabbit secondary antibody (BA-1000, Vector Labs; 1:200 in 1% BSA/PBS) was then applied. The ABC kit was finally used according to the manufacturer's instructions. Antibody reactivity was finally detected by diaminobenzidine staining (DAB, Sigma, St. Louis, MO, USA). Sections were counterstained with hematoxylin, dehydrated, mounted with Pertex and studied. Goat serum was applied on control sections.

Image: Photomicrograph of a section through an adult rat testis showing TRPV1 labelling of premeiotic germ cells, at stage II of the seminiferous epithelium. Arrow, undifferentiated spermatogonia; arrow head, early pachytene spermatocytes; asterisk, Sertoli cells.

SDS-PAGE and Western blotting: Protein lysates from the cell lines Gc-5spg and Gc-6spg and the control glioma cell line (A10-85) were prepared in RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) including 1 mM phenylmethylsulfonylfluoride. Of each sample, 50 μg were separated on a 12% SDS-polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). Western blots were blocked using Blotto-A, containing 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline (10 mM Tris; 150 mM NaCl, pH 7.6), including 0.05% Tween-20. Rabbit polyclonal anti-VR1 antibody (Neuromics) was diluted 1:1000 in Blotto-A and incubated for 1 h at room temperature. Blots were washed with Tris-buffered saline with 0.05% Tween-20. After incubation with goat anti-rabbit-HRP (P-0260 Dako Cytomation, 1:5000 inBlotto-A) secondary antibody for 1 h, blots were incubated with the electrochemiluminescence kit (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK)and exposed to an x-ray film (RX-omat, Kodak, Chalone / Saone, France).

TRPV1 Staining of Mouse DRG

This excellent staining comes to us courtesy of Katharina Zimmermann (Childrens Hospital Boston, Clapham Lab). This is some of the best staining we've seen using our VR1 C-Terminus (TRPV1) - mouse specific antibody.

Images: TRPV1 staining of C57BL/6 mouse dorrsal root ganglia. Tissues were stained using Alexa Fluor© 488 (Green) and counterstained with DAPI (blue).

cryosections, 10 microns thickness