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Due to the increasing resistance demonstrated by insects to conventional insecticides, the need for compounds with novel modes of action is becoming more urgent. Also, the discovery and production of new insecticides is vital as regulations and restrictions on conventional insecticides become increasingly stringent (Casida and Quistad 1998). Research in this area requires screening of many candidate compounds which is costly and time-consuming. The goal of this research was to produce in vitro insect neurons from Sf21 insect ovarian cell lines, which could lead to new high throughput screening methods and a way to mass produce insect material for basic research. This study used a culture of Sf21 cells and a mixture of differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, Sf21 cells began to express neuronal morphology, or the production of elongated, axon-like processes within 2-3 days. Maximal differentiation occurred when in the presence of 42 μM 20-HE or 10 μM insulin. Effects were maximal on day 2 for 20-E and day 3 for insulin. Insulin was more potent at day 2 for inducing differentiation (EC₅₀ = 247 nM) than 20-HE (EC₅₀ = 13 μM). In combination, 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system (CNS) stimulant, inhibited induction of elongated processes by 20-HE and/or insulin. Caffeine was a potent inhibitor of 42 μM 20-HE, with an IC50 of 9 nM, and the inhibition was incomplete, resulting in about one quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies studies with of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined. If they are present, high throughput screening for new insecticides will be accelerated and made more economical by the utility of this method. / Master of Science

The trigeminal ganglion (TG) is a complex sensory structure and multiple lines of evidence suggest that significant differences exist in anatomical, neurochemical and physiological properties between it and its equivalent structure in the somatosensory system, the dorsal root ganglion (DRG). This is likely to be a reflection, first on the unique areas of tissue innervation of the TG and second, on the unusual responses to injury which give rise to distinct pain symptoms such as toothache, migraine and temporomandibular joint disorders. In an attempt to address this disparity in knowledge, we have carried out an in-depth in vivo study investigating neurochemical populations and cell size distributions of sensory neurons within the rat TG. We have performed a detailed analysis of expression patterns for receptor components of important inflammatory mediators, NGF (TrkA), TNFα (p55) and IL-6 (gp130), along with the thermo-transducers TRPV1 and TRPM8. For each analysis we have compared our findings with those of the rat DRG. We have shown a significantly larger population of NF200+ neurons within the TG (51%) compared to the DRG (40%), and most interestingly, the majority of NF200+ neurons in the TG were within the small to medium cell size range, conferring a nociceptive phenotype. We have for the first time, determined expression of p55 and gp130 protein levels within neurochemically defined subpopulations of the TG. We show that a large proportion (33%) of TG neurons, in particular 27% of NF200+ neurons co-express p55, and thereby have the potential to respond directly to TNFα. Furthermore, we have observed gp130 protein expression to be ubiquitous within the TG, suggesting all neurons, including non-nociceptors, could respond to IL-6. In addition, we have utilised biochemical and electrophysiological techniques in vitro to measure the functional outcome of exposure of TG neurons to IL-6. We have demonstrated that IL-6 activates the JAK/STAT signalling pathway, preferentially within NF200+ neurons. Furthermore, we have shown that IL-6 sensitises the response of TG neurons to the TRPV1 agonist capsaicin, altering the gating properties and prolonging the opening time of the channel. Taken together, our findings support the emerging picture of a complex combinatorial pattern of co-expression of sensory neurochemicals, transducers and receptor components that help to define TG neuronal modality and function. We would advocate caution in making generalisations across sensory ganglia in particular in extrapolating data from the DRG to the trigeminal ganglion.