Role of the broadly expressed olfactory receptor OlfCc1 in mediating amino acid detection in zebrafish.

摘要

A family of 63 class C GPCR olfactory receptors, termed the 'OlfC' family, is expressed in the microvillous cell population of the zebrafish olfactory epithelium and has been predicted to be involved in amino acid detection. While the majority of the receptors are expressed in a scattered, random fashion, the receptor OlfCc1 is present in most or all microvillous cells. The overlapping expression patterns of the broadly expressed OlfCc1 with the punctuate OlfCs suggests that there may be co-expression of these populations. I was interested in investigating the function of OlfCc1 as well as the prediction that the microvillous cells expressing it are amino acid responsive. My hypothesis is that OlfCc1 plays a critical role in mediating olfactory amino acid detection. In order to test this, I first generated a peptide antibody against OlfCc1 to better characterize its distribution in adult and larval zebrafish. I then employed an antisense morpholino mediated knockdown strategy to investigate the effects of loss of OlfCc1 function. An immunohistological assessment with a panel of markers revealed that the loss of OlfCc1 did not cause gross alterations to the cellular architecture of the developing olfactory system. To explore possible affects on odorant detection, I established an in vivo assay to examine odorant-evoked activity in the olfactory bulb of morphant zebrafish expressing the genetically encoded calcium sensor GCaMP1.6 under the neuronal promoter HuC. The results of these experiments indicated that the loss of OlfCc1 severely abrogates the olfactory bulb response to a pool of 9 amino acids containing members of each of the four amino acid classes: acidic, basic, and short chain and long chain neutral. The responses to pools of other odorants thought to act through different cellular pathways, the bile acids and the biogenic amines, as well as to a complex food extract, were not significantly affected. I showed in fish in which OlfcC1 expression had not been knocked down that sub-pools of amino acids, isoleucine and leucine vs. arginine and lysine, evoked differential patterns of activity. This suggests that these responses are mediated by different populations of receptors, and that the effects of OlfCc1 knockdown are consistent across these populations. The severe effect on amino acid evoked activity in the olfactory bulb, which receives innervation from all populations of olfactory neurons, also supports the hypothesis that OlfC expressing microvillous cells are primarily mediating the response to amino acids. Together, these results support the hypothesis that OlfCc1 is broadly critical for amino acid detection and raise the intriguing possibility that it may be acting as a heteromeric co-receptor with the other OlfC family members.