We have previously carried out detailed characterization and identification of Fos expression within the trigeminal nucleus after tooth pulp stimulation in ferrets. The aim of this study was to determine the effect of pulpal inflammation on the excitability of central trigeminal neurons following tooth pulp stimulation. Adult ferrets were prepared under anesthesia to allow tooth pulp stimulation, recording from the digastric muscle, and intravenous injections at a subsequent experiment. In some animals, pulpal inflammation was induced by introducing human caries into a deep buccal cavity. After 5 d, animals were re-anaethetized, and the teeth were stimulated at 10 times the threshold of the jaw-opening reflex. Stimulation of all tooth pulps induced ipsilateral Fos in the trigeminal subnuclei caudalis and oralis. All non-stimulated animals showed negligible Fos labeling, with no differences recorded between inflamed and non-inflamed groups. Following tooth pulp stimulation, Fos expression was greater in animals with inflamed teeth than in animals with non-inflamed teeth, with the greatest effect seen in the subnucleus caudalis. These results suggest that inflammation increases the number of trigeminal brainstem neurons activated by tooth pulp stimulation; this may be mediated by peripheral or central mechanisms.

HighlightsChronic inflammation of tooth pulp activates pERK and pp38 in the trigeminal nucleusActivation is persistent and bilateral, and further increased by acute stimulationThis altered signaling may be relevant in the development of chronic pulpitic painpERK and pp38 are more sensitive markers of central change than Fos expressionSequential activation in different cell types may be linked to pain progression

Expression of the inducible transcription factor Fos in the spinal dorsal horn in vivo is associated with nociceptive afferent activation, but the underlying stimulation-transcription pathway is less clear. This in vitro spinal cord study concerns the role of protein kinase A and C second messengers in substance P receptor (NK1R)-mediated or nociceptive afferent-evoked neuronal excitation and Fos expression. Nociceptive afferent (dorsal root) stimulation of isolated spinal cords (10-14 day old rats) evoked a 'prolonged' excitatory polysynaptic potential (DR-EPSP) that was attenuated (P < 0.05) by: the protein kinase A inhibitor, Rp-cAMP; the protein kinase C inhibitor, bisindolymaleimide I; and the selective NK1R antagonist, GR82334. Neuronal excitations induced by the NK1R agonist [Sar9,Met(O2)11]-SP were attenuated by Rp-cAMP, bisindolymaleimide I and GR82334. Effects of the protein kinase A and C inhibitors on the DR-EPSP or the [Sar9,Met(O2)11]-SP-induced depolarization were nonadditive, suggesting convergence of these intracellular signalling pathways onto a common final target. Nociceptor afferent-induced Fos, detected by immunohistochemistry in superficial and deep dorsal horn laminae, was attenuated by Rp-cAMP, bisindolymaleimide I and GR82334. In spinal cords pretreated with TTX to eliminate indirect neuronal activation, [Sar9,Met(O2)11]-SP (1-20 microM) elicited a dose-related expression of Fos that was reduced by Rp-cAMP, bisindolymaleimide I and GR82334. The effects of these inhibitors were most pronounced in the deep laminae. These data support a causal relationship between protein kinase A- or C-dependent signal transduction, nociceptive afferent- or NK1R-induced neuronal excitation and Fos expression in dorsal horn. Implications for short- versus long-term modulation of nociceptive circuitry are discussed.

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Dorsal horn neurons that express the neurokinin 1 receptor (NK-1R) play an important role in nociceptive processing. The targetting of NK-1R neurons by serotoninergic (5-hydroxytryptamine, 5-HT) axons would provide a straightforward means to exert an inhibitory analgesic effect at spinal level. This study used single cell electrophysiology to analyse and correlate the responses of rat deep DH neurons in vitro to both 5-HT and the NK-1R agonist [Sar9,Met(O2)11]-substance P (SP). Subsequently a combination of immunocytochemistry and confocal imaging was applied to biocytin-filled laminae III-VI neurons to reveal putative 5-HT innervation in this neuronal sample. A population of neurons was identified in which 5-HT (50 microM) significantly attenuated the dorsal root-evoked excitatory postsynaptic potential and [Sar9,Met(O2)11]-SP (2 microM) induced a direct tetrodotoxin-resistant depolarisation. Immunolabelling revealed that all of these neurons were inhibited by 5-HT, including those that were excited by [Sar9,Met(O2)11]-SP, were overlaid by a plexus of 5-HT immunoreactive fibres and in some instances, closely apposed putative contacts with somata and proximal dendrites identified although their incidence was low. Inhibition by 5-HT of deep DH neurons directly responsive to SP may account at least in part for monoamine-induced modulation of nociceptive processing in the spinal cord.