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.
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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)
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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
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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)
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