Characterization of CRF immunoreactive neurons in the anuran optic tectum
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In amphibians, it has been determined that CRF has an affect on subcortical visual pathways that regulate prey capturing ability. The pathways targeted by CRF effects on visually guided feeding are unknown, but CRF-ir fibers have been reported in the superficial layers of the amphibian optic tectum that collect retinal innervation. These findings have led to the hypothesis that CRF may originate from retinal afferents that innervate the superficial layers of the tectum. A series of experiments were carried out in the cane toad (Bufo marinus) and the African clawed frog (Xenopus laevis) to examine an alternate hypothesis that CRF neurons intrinsic to the optic tectum may contribute to the CRF content innervation of this visual center in the amphibian brain. In both species, CRF-immunoreactive neurons were observed in tectal layers 6 and 7 with axons directed toward the more superficial layers of the tectum. To confirm the identity of CRF neurons in the tectum, I examined the expression of CRF and CRF related genes in the tectum by reverse transcriptase PCR using primers generated against sequences in the Xenopus genes for CRF, urocortin-1 (UCN-1), the CRF receptors CRF R1, CRF R2, CRF binding protein and the ribosomal protein, rpL8, as a housekeeping gene. RT-PCR showed the relative expression of CRF, CRF R1, CRF-R2, UCN-1, CRF-BP and rpL8 mRNA in four different brain regions of X. laevis. Results showed the forebrain to contain CRF, CRF R1 and R2, and rpL8. The hypothalamus and brain stem contained all 6 mRNA’s, and the absence of CRF R2 and CRF BP was shown in the optic tectum. Finally, we used a radioimmunoassay to investigate CRF peptide content in differential brain regions of the toad and the effects of unilateral eye ablation on CRF content in the tectum to discern the relative contribution of retinal CRF to tectal CRF content. There were no significant differences in CRF content between right and left optic tectum after unilateral eye ablation, suggesting that the majority of CRF in the tectum arises from intrinsic tectal CRF neurons. Therefore, we conclude that CRF in the optic tectum does not arise from retinal afferents that innervate the superficial layers of the tectum.