Analysis of NK1r and NMDAr expression in dorsal root ganglia neurons in the presence of nerve growth factor (NGF) and prostaglandin E2 (PGE2)
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2012COAR
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El laboratorio del departamento de Anestesiología de la Universidad de New York, investiga como la SustanciaP (SP) regula el aumento de la producción de NMDA relacionado con la concentración de Calcio citoplasmático presente en las neuronas primarias de los Ganglios de la Raíz Dorsal (GRD), como posible mecanismo donde ellas pueden ser sobre-excitadas después de sufrir daño. Estudios preliminares mostraron que neuronas GRD requieren de Factor de Crecimiento Neural (FCN) y prostaglandina E2 (PGE2) para tal fin. Mi proyecto consistió en estudiar si las neuronas de GRD producen respuesta al estímulo de NMDA aumentando sus receptores y/o los receptores de neuro-kinin 1 (NK1) Los cultivos primarios de neuronas de GRD fueron tratados así: control (solo medio de cultivo), PGE2 y NGF independientes y simultáneos en el mismo medio. Resultados: el tratamiento con FCN+PGE2 mostraron aumento en la expresión de NK1r comparados con grupo de control. En las subunidades de NMDAr, la NR1 no mostró ningún aumento significativo en su expresión bajo ninguno de los tratamientos. La subunidad NR2B mostró un aumento en la expresión en neuronas de la proteína en los receptores de NMDA ni de NK1r.
Abstract
The Anesthesiology Laboratory from The New York University, Langone Medical Center
investigates the role of Substance P (SP) in regulating the NMDA-evoked increase in cytoplasmic Ca2+ ([Ca2+]cyt) present in primary sensory Dorsal Root Ganglia (DRG)
neurons, as a possible mechanism by which DRG neurons may become hyperexcitable
following injury. Preliminary studies showed that in tissue culture, DRG
neurons require both nerve growth factor (NGF) and prostaglandin E2 (PGE2) in order to
get a NMDA-evoked increase in [Ca2+]cyt . Hence, my project consisted in investigating
whether the ability of these neurons become NMDA responsive was due to an increase
in NMDA receptors and/or due to neurokinin receptors. NGF and PGE2 have been previously shown to increase during injury and to contribute in neuronal sensitization. In order to evaluate changes in the expression of NMDA and Neurokinin receptors in cultured adult rat DRG neuron, we used two different methods: Immunostaining and Western Blotting.
For the immunostaining: we prepared single cell cultures of DRG neurons exposed to
three different treatments: the control, which is the culture media; the addition of PGE2 and NGF independently and simultaneously. We used specific primary antibodies against the Substance P (SP) receptor, the NK1r, and for the subunits NR1 and NR2B in N-methyl D-aspartate (NMDA) receptor. We found that treatment with both factors NGF+PGE2, showed a slight increase in the expression of the NK1r as compared to the control group. About NMDAr’s subunits, the NR1 does not show any significant
increase in its expression under any of the treatments. The NR2B subunit shows an
increase in expression in the medium size DRG neurons in all the treated groups
compared to control. We used Western blots to measure levels of protein expression from the receptors presented in NK1r, and NMDAr NR2B subunit using as a source the whole protein from the cell culture. Western blot analysis did not showed any increase in the expression of NK1r or NR2B subunit in any of the treatments. Therefore, the observed increase in NMDA-responsiveness gotten in the laboratory does not reflect a change in the expression of NMDAr or in the neurokinin receptor.
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