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In mammals, corticotrophin-releasing hormone (CRH) and related peptides are known to play essential roles in the regulation of neuroendocrine, autonomic and behavioural responses to physical and emotional stress. In nonmammalian species, CRH-like peptides are hypothesized to play similar neuroendocrine and neurocrine roles. However, there is relatively little detailed information on the distribution of CRH neurones in the central nervous system (CNS) of nonmammalian vertebrates, and there are currently no comparative data on stress-induced changes in CRH neuronal physiology. We used a specific, affinity-purified antibody raised against synthetic Xenopus laevis CRH to map the distribution of CRH in the CNS of juvenile South African clawed frogs. We then analysed stress-induced changes in CRH immunoreactivity (CRH-ir) throughout the CNS. We found that CRH-positive cell bodies and fibres are widely distributed throughout the brain and rostral spinal cord of juvenile X. laevis. Strong CRH-immunoreactivity (ir) was found in cell bodies and fibres in the anterior preoptic area (POA, an area homologous to the mammalian paraventricular nucleus) and the external zone of the median eminence. Specific CRH-ir cell bodies and fibres were also identified in the septum, pallium and striatum in the telencephalon; the amygdala, bed nucleus of the stria terminalis and various hypothalamic and thalamic nuclei in the diencephalon; the tectum, torus semicircularis and tegmental nuclei of the mesencephalon; the cerebellum and locus coeruleus in the rhombencephalon; and the ventral horn of the rostral spinal cord. To determine if exposure to an acute physical stressor alters CRH neuronal physiology, we exposed juvenile frogs to shaking/handling and conducted morphometric analysis. Plasma corticosterone was significantly elevated by 30 min after exposure to the stressor and continued to increase up to 6 h. Morphometric analysis of CRH-ir after 4 h of stress showed a significant increase in CRH-ir in parvocellular neurones of the anterior preoptic area, the medial amygdala and the bed nucleus of the stria terminalis, but not in other brain regions. The stress-induced increase in CRH-ir in the POA was associated with increased Fos-like immunoreactivity (Fos-LI), and confocal microscopy showed that CRH-ir colocalized with Fos-LI in a subset of Fos-LI-positive neurones. Our results support the view that the basic pattern of CNS CRH expression arose early in vertebrate evolution and lend further support to earlier studies suggesting that amphibians may be a transitional species for descending CRH-ergic pathways. Furthermore, CRH neurones in the frog brain exhibit changes in response to a physical stressor that parallel those seen in mammals, and thus are likely to play an active role in mediating neuroendocrine, behavioural and autonomic stress responses.

In the amphibian Xenopus laevis, suprachiasmatic melanotrope-inhibiting neurons (SMINs) play an important role in the regulation of the background adaptation process. In this study, we investigated the innervation of the SMINs at the light- and electron- microscopical level. Immunocytochemistry in combination with confocal laser scanning microscopy revealed co-existence of neuropeptide Y (NPY) and synaptobrevin in spots in the direct vicinity of the SMINs, suggesting the existence of NPY-containing synapses on these cells. At the ultrastructural level, the SMINs showed a high degree of plasticity, containing more electron-dense vesicles and a larger extent of RER in white- than in black-adapted animals. In black-adapted animals, symmetric synapses (Gray type II) were observed on the soma of the SMINs, suggesting an inhibitory input to these cells. The synaptic profiles contained electron-lucent and electron-dense vesicles, indicating the involvement of both a classical neurotransmitter and a neuropeptide (possibly NPY) in this input. In white-adapted animals, synapses were only found at some distance from the SMIN somata. Our findings indicate a striking plasticity of the innervation of the SMINs in relation to background adaptation and support the hypothesis that the SMINs are innervated by NPY-containing interneurons that inhibit SMIN activity in black-adapted animals.