The use of animal models in the understanding of the neurobiology of pain perception is essential for the development of new pharmacotherapies. Yet, in modeling human clinical experience, there is a lack of meaningful and appropriate dependent variables of pain-related behaviors in the mouse. In this thesis, we address the problem of finding behavioral measures of pain in mice that fully encompass the range of experience inherent to human pain conditions. Therefore, in this work complex behaviors were assessed as potential measures of persistent pain in mice

We investigated a wide range of quality of life behaviors in three classic pain models: spared nerve injury, chronic constriction injury and injection of complete Freund's adjuvant. Mechanical hypersensitivity is prominent in each of these conditions and persists for many weeks. To assess more complex behavioral outcomes, home cage behavior was continuously monitored after injury and a battery of motor disability and affective behavior tests were performed on these mice. No model of chronic pain produced long-lasting changes to behaviors of daily life, either in the home cage or in tests of affect and disability.

Next, we observed behaviors in three other models of persistent pain: osteoarthritis, disc-degeneration, and dental pulp exposure. In a pilot study of the former two models, mice with joint degeneration were tested for locomotor ability and motivation, but showed no signs of disability. Lastly, we measured behavior in the setting of dental pulp inflammation, for which there is no standard method of measuring pain levels. As with other models, pulpal injury also did not impact behavior in the home cage. Instead, we used an operant assay of sucrose consumption as a measure of dental pain in mice. Data from this task suggest that pain can, in fact, influence some elements of complex behavior. However, as alteration in daily life activities is the feature that is so disrupted in patients with chronic pain, our results suggest that many murine pain models do not fully reflect the human conditions and raise questions regarding the limitations of these models in pain research.

Noxious stimuli are detected by primary afferent neurons of the dorsal root ganglia (DRG). Such neurons, known as nociceptors, can be divided into several distinct populations based on the heterogeneous distribution of receptors, ion channels, and neurotransmitters, however, functional correlates of these anatomical differences are yet unidentified. The work in this thesis further examines the neurochemical and functional segregation of nociceptor subtypes through a genetic and pharmacological investigation of neurons that express the heat and capsaicin receptor, TRPV1.

TRPV1 is activated by noxious heat stimuli (>43 ^oC), and is robustly expressed by primary afferent nociceptors. In addition, some have argued that TRPV1 is widely distributed in cells outside of the DRG, although there is considerable disagreement as to the extent and localization of this expression. To address this question, we generated a line of mice in which TRPV1+ cells co-express two reporter genes: placental alkaline phosphatase and nuclear lacZ. These enzymes allow for the sensitive and accurate identification of TRPV1+ cells and processes. Using this approach, we observed that, in contrast to numerous previous reports, TRPV1 expression in the nervous system is largely limited to peptidergic, primary afferent neurons. We additionally found evidence for TRPV1 in arteriolar smooth muscle cells, highlighting an important substrate for the actions of heat, protons, and other TRPV1 agonists on vascular tone.

We then investigated the relative contribution of TRPV1+ and TRPV1- nociceptors to the behavioral responses to stimuli of different pain modalities. Surprisingly, despite the fact that most nociceptors show polymodal response properties in electrophysiological assays, we found that pharmacological ablation of the central branches of TRPV1+ nociceptors selectively abolished heat pain sensitivity without altering behavioral responses to mechanical or cold stimuli. Conversely, we found that genetic ablation of nociceptors expressing the G protein-coupled receptor, MrgprD, which marks a large subset of TRPV1- DRG neurons, selectively reduced behavioral sensitivity to noxious mechanical stimuli. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing as a result of distinct contributions from molecularly-defined nociceptor subtypes.

Venoms from spiders, snakes, cone snails and scorpions have evolved to produce a

vast pharmacopoeia of toxins that modulate receptor or channel function as a means of

producing shock, hemolysis, paralysis and pain. As such, venoms from these animals

represent a rich potential source of potent and selective toxins that can be exploited to

study and manipulate somatosensory and nociceptive signaling pathways. In this work, I

have identified and characterized two novel toxins from the Brazilian lancehead pit viper

(Bothrops moojeni) and Tanzania blue ringleg centipede (Scolopendra morsitans). The

Brazilian lancehead snake venom contains a novel secreted phospholipase A2

(sPLA2)-like protein that can promote ATP release from pannexin hemichannels.

Studies on the cellular and behavioral effects of this toxin reveal a role of regulated

endogenous nucleotide release in nociception. The discovered Tanzania blue ringleg

centipede toxin represents a new family of toxins that show evolutionary relationship

with α-scorpion toxins and could potentially function as potassium channel blockers.

The anti- migraine action of "triptan" drugs involves the activation of serotonin subtype 1D (5-HT1D) receptors expressed on "painresponsive" trigeminal primary afferents. In the central terminals of these nociceptors, the receptor is concentrated on peptidergic dense core vesicles (DCVs) and is notably absent from the plasma membrane. Based on this arrangement, we hypothesized that in the resting state the receptor is not available for binding by a triptan, but that noxious stimulation of these afferents could trigger vesicular release of DCVs, thus externalizing the receptor. Here we report that within 5 min of an acute mechanical stimulus to the hindpaw of the rat, there is a significant increase of 5-HT1D-immunoreactivity (IR) in the ipsilateral dorsal horn of the spinal cord. We suggest that these rapid immunohistochemical changes reflect redistribution of sequestered receptor to the plasma membrane, where it is more readily detected. We also observed divergent changes in 5-HT1D-IR in inflammatory and nerve-injury models of persistent pain, occurring at least in part through the regulation of 5-HT1D-receptor gene expression. Finally, we found that 5-HT1D-IR is unchanged in the spinal cord dorsal horn of mice with a deletion of the gene encoding the neuropeptide substance P. This result differs from that reported for the partial derivative-opioid receptor, which is also sorted to DCVs, but is greatly reduced in preprotachykinin mutant mice. We suggest that a "pain"-triggered regulation of 5-HT1D-receptor expression underlies the effectiveness of triptans for the treatment of migraine. Moreover, the widespread expression of 5-HT1D receptor in somatic nociceptive afferents suggests that triptans could, in certain circumstances, treat pain in nontrigeminal regions of the body.