Laser Capture Microdissection: Combining LCM with IHC to investigate NMDA receptor subunit composition

Abstract

The Neurokinin 1 receptor is a member of the tachykinin receptor sub‐family. It is distributed throughout the CNS, although to a lesser extent than its natural agonist, Substance P.There is much evidence to implicate the NK1 receptor within the physiology of acute pain, such as the anatomical localisation of Substance P and NK1 receptors within lamina I of the spinal cord, as well as correlative increases in Substance P production and NK1 receptor internalisation in response to increasing noxious stimuli. Reproduction of in vivo efficacy in the clinic has, however, been unsuccessful.Selective NR2 subunit mediated NMDA antagonism has also shown in vivo efficacy, expression of this subunit within the superficial lamina of the spinal cord adding anatomical evidence to support this mechanism. However, co‐expression of the NK1 receptor and the NMDA receptor has not been reported.We have developed a methodology to allow the isolation of high quality RNA, via Laser Capture Microdissection, following the immunolabelling of specific proteins of interest.Using this technology, we validated methodology via PCR of dissected samples, as well as looking at differential expression of the NR2A and NR2B receptor subunits on NK1 +ve and NK1 –ve samples taken from the rat striatum.