According to the NIH, 76.2 million Americans have suffered from pain lasting more than 24 hours, with millions more affected by chronic pain. With such a high prevalence and impact on health and quality of life, the understanding of pain is essential to effective treatment. Exploring the mechanism by which our neurons set the gain for what is perceived as painful stimuli is one way to further that knowledge. Different populations of neurons have different thresholds for activation, and oftentimes alterations to these thresholds can result in aberrant pain signaling. Phospholipase C (PLC) is a promising target to study, as it has been previously shown to potentiate the activation of pain neurons expressing Transient Receptor Potential cation channel subfamily A, member 1 (TRPA1). TRPA1 channels are responsive to noxious stimuli such as mustard oil (AITC). This study explores the effects of PLC using a PLC activator (m3m3FBS) and an inhibitor (U-71322) to examine its role in response to the noxious stimulus, AITC. We performed a locomotor assay of zebrafish larvae to explore behavioral effects, as well as neuronal imaging experiments using transgenic zebrafish with fluorescent calcium indicators in neural cells. We hypothesize that both behavioral responsiveness to pain and neuronal recruitment will likely increase with activation of PLC and decrease with deactivation of PLC because of PLC’s ability to modulate TRPA1 channel activity. With data collected thus far, the results look consistent with our hypothesis. We observed increased locomotion in response to activation of PLC with painful stimulus (AITC) compared to control and inhibitor groups. Likewise, zebrafish exposed to PLC activators exhibited greater numbers of AITC responsive neurons than zebrafish exposed to control or PLC inhibitors. Together, these results indicate that PLC is an important factor in modulating the sensitivity of TRPA1 expressing neurons.

Chronic pain is debilitating for millions of people worldwide and our understanding of how the central nervous system processes painful information is limited. If we are to create more effective therapeutics to treat chronic pain, it will be necessary to develop a better understanding of the circuitry and functionality of the somatosensory system. Our lab is interested in the calcitonin receptor-like (CALCRL) receptor, which is expressed throughout the central nervous system and is known to respond to pain-inducing neuropeptides. We aim to profile CALCRL receptor-expressing neurons within the dorsal horn to elucidate their functional roles in nociception and to determine how they process painful information. This was done by 1) utilizing an adeno-associated viral vector (AAV) to preferentially infect and fluorescently tag CALCRL-positive neurons in the dorsal horn, 2) utilizing a retrograde, monosynaptic rabies viral vector to infect neurons that are synaptically connected to CALCRL-positive neurons, and 3) immunohistochemical staining and analysis to detect a variety of neuronal markers of inhibitory and excitatory neurons, laminar markers, and several other known dorsal horn neuron subtypes. Thus far, CALCRL-positive neurons were successfully infected via direct injection using both AAV and rabies vectors into the dorsal horn. In addition, we have also been successful in costaining for multiple neuronal markers. We have begun to profile and classify these neurons according to known neuronal subtypes. Our next goal is to modulate the activity of this specific neuronal population followed by behavioral testing to better understand its functional role in the processing of painful stimulus. If we are able to create a clear profile of these neurons, then we may be able to identify future targets for specific therapeutics.

Both acute and chronic pain are universal, often debilitating sensations that lead to significant physiological, psychological and economic costs. Drug development and research have worked to counteract these adversities, but current therapies are often inadequate and have dangerous side effects. Targeted drug development, which relies on pre-selecting a target that is subjected to in-vitro testing, has been difficult, costly and ineffective in producing a drug that works to effectively relieve pain with minimal unfavorable consequences. An alternative approach would be to develop an untargeted screen in a system that employs complex pain behaviors. It would act as a means of modeling the nociceptive processes, which are the neural processes of encoding and processing noxious stimuli, in the organism. We utilize an unbiased, behavior-based, novel assay that uses zebrafish larvae to better understand pain sensation. We have screened thousands of small molecules on zebrafish larvae to identify ones that have analgesic properties. The potential analgesics should block sensitized temperature aversion which changes the larvae’s temperature zone inclination meaning we observe no preference between the two zones. Thus far, our untargeted screen identified three novel molecules with analgesic properties. We then performed a series of pharmacology and behavioral experiments to understand the impact of the compounds and to narrow down their effects in order to confirm if it is truly impacting nociception and/or temperature aversion with the intent to validate the compounds as prospective analgesics.