Immunohistochemical localization and biochemical characterization of hypocretin/orexin-related peptides in the central nervous system of the frog Rana ridibunda

In the present study, we have investigated the distribution and biochemical characteristics of hypocretin (hcrt)-like immunoreactivity in the central nervous system (CNS) of the frog Rana ridibunda; by using an antiserum dire cted against rat hcrt2. Immunoreactive cell bodies were only detected in fo ur diencephalic nuclei, including the anterior preoptic area and the suprac hiasmatic, magnocellular, and ventral hypothalamic nuclei. In contrast, hcr t2-immunoreactive fibers were widely distributed throughout the frog CNS. I n particular, a high density of hcrt-positive fibers was detected in severa l areas of the telencephalon, including the olfactory bulb, the nucleus of the diagonal band of Broca, and the amygdala. A dense network of hcrt-conta ining fibers was observed in all thalamic and hypothalamic nuclei. A low to moderate density of immunoreactive fibers was also found in the mesencepha lon, rhombencephalon, and spinal cord. Reversed-phase high performance liqu id chromatography analysis of frog brain extracts revealed that hcrt2-immun oreactive material eluted as two peaks, the major one exhibiting the same r etention time as synthetic rat hcrt2. The present data provide the first de tailed mapping of the hcrt neuronal System in the CNS of a nonmammalian ver tebrate. The occurrence of hcrt-containing cell bodies in the hypothalamus and the widespread distribution of hcrt-immunoreactive fibers throughout th e brain and spinal cord suggest that, in amphibians, hcrts may exert neuroe ndocrine, neurotransmitter, and/or neuromodulator activities. J. Comp. Neur al. 429:242-252, 2001. (C) 2001 Wiley-Liss, Inc.