A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter.
The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and beta-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K(+) (10 microM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar(9),Met (O(2))(11)]-substance P (10 microM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity.
Hilton
Kathryn J
KJ
School of Biomedical Sciences, University of Leeds, Clarendon Way, Leeds, LS2 9JT, UK.
Bateson
Alan N
AN
King
Anne E
AE
D000818:Animals; D000831:Animals, Newborn; D019470:Biolistics / Q000295:instrumentation / Q000379:methods*; D015415:Biomarkers; D002470:Cell Survival / Q000235:genetics; D005576:Colforsin / Q000031:analogs & derivatives; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics; D008954:Models, Biological; D009473:Neuronal Plasticity / Q000235:genetics; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods; D020671:Posterior Horn Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism*; D013373:Substance P / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000494:pharmacology; D015320:Tachykinins / Q000235:genetics*; D014162:Transfection / Q000295:instrumentation / Q000379:methods*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D015415:Biomarkers; D004274:DNA, Recombinant; D011498:Protein Precursors; D018040:Receptors, Neurokinin-1; D015320:Tachykinins; C044206:preprotachykinin; C062618:substance P, Sar(9)-Met(O2)(11)-; D049452:Green Fluorescent Proteins; D005576:Colforsin; D013373:Substance P