Acute and chronic pain conditions are often debilitating, inflicting severe physiological, emotional and economic costs and affect a large percentage of the global population. However, the development of therapeutic analgesic agents based primarily on targeted drug development has been largely ineffective. An alternative approach to analgesic development would be to develop low cost, high throughput, untargeted animal based behavioral screens that model complex nociceptive behaviors in which to screen for analgesic compounds. Here we describe the development of a behavioral based assay in zebrafish larvae that is effective in identifying small molecule compounds with analgesic properties. In a place aversion assay, which likely utilizes supraspinal neuronal circuitry, individually arrayed zebrafish larvae show temperature-dependent aversion to increasing and decreasing temperatures deviating from rearing temperature. Modeling thermal hyperalgesia, the addition of the noxious inflammatory compound and TRPA1 agonist allyl isothiocyanate sensitized heat aversion and reversed cool aversion leading larvae to avoid rearing temperature in favor of otherwise acutely aversive cooler temperatures. We show that small molecules with known analgesic properties are able to inhibit acute and/or sensitized temperature aversion.

Brain circuits comprise vast numbers of intricately interconnected neurons with diverse molecular, anatomical and physiological properties. To allow “user-defined” targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases (SSRs) based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo . They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally-precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.

Little is known about the capacity of lower vertebrates to experience itch. A screen of itch-inducing compounds (pruritogens) in zebrafish larvae yielded a single pruritogen, the TLR7 agonist imiquimod, that elicited a somatosensory neuron response. Imiquimod induced itch-like behaviors in zebrafish distinct from those induced by the noxious TRPA1 agonist, allyl isothiocyanate. In the zebrafish, imiquimod-evoked somatosensory neuronal responses and behaviors were entirely dependent upon TRPA1, while in the mouse TRPA1 was required for the direct activation of somatosensory neurons and partially responsible for behaviors elicited by this pruritogen. Imiquimod was found to be a direct but weak TRPA1 agonist that activated a subset of TRPA1 expressing neurons. Imiquimod-responsive TRPA1 expressing neurons were significantly more sensitive to noxious stimuli than other TRPA1 expressing neurons. Together, these results suggest a model for selective itch via activation of a specialized subpopulation of somatosensory neurons with a heightened sensitivity to noxious stimuli.