Cell-to-cell communication is one of the fundamental requisites of making multicellular organisms. G protein-coupled receptors (GPCRs) are one of the most abundant receptor-types within vertebrates. They canonically mediate their signal via hetrotrimeric G proteins, and G protein signaling is regulated by regulators of G protein-signaling (RGS). One of these RGS proteins, RGS4, is preferentially expressed in the central nervous system of humans and has been strongly connected to dopaminergic signaling, along with a number of severe neuronal diseases. rgs4 is not well studied in the model organism Danio rerio, the zebrafish, with only two publications. In this project, a newly constructed transgenic line, tg(rgs4:mCherry), with the fluorophore mCherry regulated by the promoter element of rgs4 was characterized in order to investigate fidelity to endogenous rgs4 expression and the utility of the transgenic line. The mCherry expression is apparent by 48 hours post fertilization, and expression is found mainly in neuronal tissue. Cell bodies are visible only in some labeled areas, while other areas show a more diffuse signal indicative of projections. There is only one transgenically labeled area that also unambiguously expresses rgs4; the pronephric tubule. This line is therefore not particularly well suited for rgs4-specifc studies, but this does not discredit the fidelity of the construct. A transgenic line made with a site-directed technique would most likely confer the fidelity of the promoter to the expression of the fluorophore. A way of increasing the labeling resolution includes exchanging the mCherry fluorophore for one with stronger signal and a lower tendency to aggregate, e.g. eGFP. Increasing the resolution of the characterization, e.g. to the level of sub-nuclei or neuronal types, would serve to enhance the utility of the line. As it is, the tg(rgs4:mCherry) zebrafish line has limited uses, and yet it is not without them.

We have investigated the role of the metabotropic glutamate receptor 7 (mGluR7) and the gastrin releasing peptide receptor (Grpr) population that are involved at different levels of itch transmission. We found that mGuR7 deficient mice displayed an anaphylaxis-like behavior when provoked with histamine. Analysis of blood revealed elevated plasma levels of histamine and mouse mast cell protease-1 (mMCP1), two indicators of anaphylaxis, in mGluR7 deficient mice compared with control mice. Inhibition of the neurokinin 1 receptor, by preventing binding of the corresponding ligand substance P (SP), prior to provocation with histamine prevented the development of anaphylaxis in mGluR7 deficient animals. However, blocking GRPR (gastrin releasing peptide receptor) only resulted in decreased itch levels in mGluR7 deficient mice but did not prevent the systemic anaphylaxis-like behavior. Our findings indicate that mGluR7 normally functions as a brake on histaminergic itch that is mediated through GRPR as well as anaphylaxis through Substance P. Grpr has previously been shown to mediate both histaminergic and non-histaminergic itch but little is known about the GRPR neuronal population. We used a BAC cloning strategy to construct a Grpr-Cre line, which we crossed with the reporter lines tdTomato and Viaat-egfp as well as with Vglut2-lox. We could conclude that Grpr-Cre neurons are mainly excitatory interneurons located in lamina II-IV, that convey itch using VGLUT2-mediated glutamatergic transmission to the next, currently unknown, step in the labeled line of chemical itch. To eventually deduce the function of the endogenous opioids dynorphin and enkephalin, which are hypothesized to be involved in gating pain and itch in the spinal cord, we constructed two Cre lines using BAC cloning that targeted the precursor proteins preprodynorphin and preproenkephalin, respectively. Preprodynorphin-Cre neurons were mainly located in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while the co-expression with the inhibitory markers Viaat-egfp and PAX2 was 13% and 28% respectively in the spinal cord. Preproenkephalin neurons were more localized to lamina III in the dorsal horn, furthermore single cell analysis showed that they overlapped to 94% with Vglut2 mRNA while 7% and 13% expressed Viaat-egfp and PAX2 respectively.