A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter.

Abstract

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.

• Authors: Kathryn J Hilton, Alan N Bateson, Anne E King
• Journal: Brain research. Brain research reviews (Brain Res Brain Res Rev) Vol. 46 Issue 2 Pg. 191-203 (Oct 2004) Netherlands
• PMID: 15464207 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Biomarkers
• DNA, Recombinant
• Protein Precursors
• Receptors, Neurokinin-1
• Tachykinins
• preprotachykinin
• substance P, Sar(9)-Met(O2)(11)-
• Green Fluorescent Proteins
• Colforsin
• Substance P

Topics

• Animals
• Animals, Newborn
• Biolistics (instrumentation, methods)
• Biomarkers
• Cell Survival (genetics)
• Colforsin (analogs & derivatives)
• DNA, Recombinant (genetics)
• Female
• Green Fluorescent Proteins (genetics)
• Models, Biological
• Neuronal Plasticity (genetics)
• Organ Culture Techniques (instrumentation, methods)
• Posterior Horn Cells (cytology, drug effects, metabolism)
• Promoter Regions, Genetic (genetics)
• Protein Precursors (genetics)
• Rats
• Rats, Wistar
• Receptors, Neurokinin-1 (agonists, metabolism)
• Spinal Cord (metabolism)
• Substance P (analogs & derivatives, biosynthesis, pharmacology)
• Tachykinins (genetics)
• Transfection (instrumentation, methods)
• Up-Regulation (drug effects, physiology)