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The midbrain periaqueductal grey (PAG) has been extensively studied with respect to descending control of nociception and is classically viewed as a source of descending inhibition, resulting in antinociception at the level of the spinal cord. However, recent studies suggest that facilitation can also be evoked from the PAG and descending facilitation from brainstem sites is hypothesised to contribute to the generation and maintenance of chronic pain. Recent work from this laboratory has demonstrated that descending control from the PAG discriminates between spinal processing of A- and C-nociceptive inputs. This has important consequences given the different roles of A- and C- nociceptors in physiological and pathophysiological pain.

Transient receptor potential vanilloid 1 (TRPVl) is an important molecular detector of painful stimuli and it is widely accepted that TRPVl is involved in the development of chronic pain conditions, which makes it an attractive target to control pain. The aim of this thesis is to study the molecular mechanisms that underlie the modulation of this receptor by vanilloids, cannabinoids and other lipid mediator using in vitro and in vivo rat models of pain. First, in vitro calcium imaging studies demonstrated that capsaicin and olvanil produced a robust calcium signal in cultured ORG cells, in a TRPVI-dependent manner. The capsaicin-evoked calcium response consisted of an initial peak followed by a decaying plateau phase; both phases of the response were abolished when cells were suprafused with calcium-free buffer. Switching to a calcium-free buffer immediately after the application of capsaicin did not, however, alter the plateau phase of the response. Furthermore, depleting the internal calcium stores using thapsigargin (1 μM) also did not alter the plateau phase. Olvanil evoked a smaller calcium signal, in comparison to capsaicin, but it was more effective at desensitising TRPV I. When comparing the kinetics of the calcium responses of these two agonists, / it was clear that there was no difference in the latency to the peak response to capsaicin or olvanil and, therefore, this is unlikely to. contribute to differences in desensitisation. The synthetic cannabinoid agonist HU210 (1J!M) significantly inhibited capsaicin- evoked calcium responses both in terms of the peak response and the decaying plateau response. The onset of action of HU210 was investigated, with cells suprafused with HU210 for varying intervals (1, 4 or 15 minutes). Maximal inhibitory effects of HU210 on capsaicin-evoked calcium responses were observed after 4 minutes of ii exposure. The inhibitory effects of HU21 0 were attenuated by pre-treatment with the selective CB 1 receptor antagonist SR 141716A (1 μM), and by pre-treatment with the G, protein inhibitor pertussis toxin (PTX, 1mg/ml). Pre-treatment with thapsigargin (1JlM) also decreased the inhibitory effects of HU210. Work in this thesis also demonstrated that forskolin pre-treatment (1JlM, 15 minutes) attenuated the ability of HU210 to inhibit the initial peak of the capsaicin-evoked calcium response, whereas the ability of HU21 0 to attenuate the whole response (peak + plateau) was unaltered by forskolin. This finding was further supported by the observation that there was a tendency for HU210 to inhibit forskolin-induced cAMP production in the DRG-like F 11 cell line. Finally, this thesis demonstrated that the essential fatty acid linoleic acid (1mM) and the linoleic acid metabolites 9- and 13-HODE (100μM) produced a calcium response in DRG cells in a TRPVI-dependent manner. Pre-exposing the cells to the selective 15-LOX inhibitor PD 146176 (10μM) significantly inhibited linoleic acid-evoked calcium responses, but did not alter the 9- or 13-HODE-evoked calcium responses. In the in vivo rat model of inflammatory pain, intraplantar injection of 2% carrageenan (100JlI) significantly increased the weight bearing difference between the two I hindpaws, and the paw oedema 4 h post-injection compared to saline-treated rats. Pre- administration of intraplantar PD146176 (50Jlg/50Jll) or anti-S-and 13-HODE (25Jlg each) antibodies significantly attenuated the carrageenan-induced hyperalgesia compared to vehicle. In conclusion, TRPV1 activity can be modulated by different lipid mediators and this modulation may have a therapeutic potential for the control of pain.

Unexplained medical symptoms are a major challenge for modern health care with high costs and patient/physician dissatisfaction. Pain, the archetypal symptom, can appear in excess or in absence of observed disease activity.

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