Distribution and Acute Stressor‐Induced Activation of Corticotrophin‐Releasing Hormone Neurones in the Central Nervous System of Xenopus laevis

Abstract

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.

Effect of starvation on Fos and neuropeptide immunoreactivities in the brain and pituitary gland of Xenopus laevis

“…It appeared that both peptides are widespread, indicating that they exert a large variety of actions, but they also show some overlap, mainly in the Mg (Calle et al, 2005a;Yao et al, 2004). Because of this coexistence of both feeding-related neuropeptides, we hypothesize that CRF and Ucn1 in the Mg are involved in the regulation of feeding.…”

“…The highly speciWc rabbit anti-Xenopus CRF (xCRF; dilution: 1:15) (Boorse and Denver, 2004;Calle et al, 2005a;Yao et al, 2004), rabbit anti-rat Ucn1 (1:30,000) (Bittencourt et al, 1999;Kozicz et al, 2002), rabbit antibovine ENK (1:1000) (Calle et al, 2005b) and rabbit anti-Xenopus NPY (xNPY; 1:8000) (Tuinhof et al, 1994;Ubink et al, 1997) sera had been used in our laboratories before. The C6-1 F4D4 CART-antiserum (1:100,000) had been raised in mice against the 109-126 fragment of rat CART peptide (Koylu et al, 1997;Lázár et al, 2004).…”

Stress hormones mediate predator-induced phenotypic plasticity in amphibian tadpoles

“…Once metamorphosis was complete (tail stub completely resorbed, 2 -3 days following removal from the tank), two metamorphic frogs originating from each tank were pooled and immediately flash frozen for baseline CORT rspb.royalsocietypublishing.org Proc R Soc B 280: 20123075 analysis (n ¼ 10 per treatment). To investigate whether the metamorphic frog stress response was influenced by prior exposure to predator chemical cue as a tadpole, an additional two metamorphic frogs from each tank were subjected to a confinement stressor ( placed into a 16.5 Â 15 cm plastic zip-top bag) for 90 min [38], after which they were pooled by tank and flash frozen for subsequent CORT analysis (n ¼ 10 per treatment).…”

The biological significance of acoustic stimuli determines ear preference in the music frog

“…Although the frog's telencephalon is not as well differentiated compared with that of mammals and birds (Butler and Hodos, 2005), limbic system structures that appear homologous to the amygdala and hippocampal formation of mammals have been described in frogs (Bruce and Neary, 1995), which possess ascending and descending connections comparable to the corresponding structures of mammals (Laberge et al, 2006). Yao et al (2004) studied the distribution of corticotrophin releasing hormone (CRH)-like peptides in the clawed frog (Xenopus laevis) brain, which conform to the distribution of CRH-positive neurons in mammals and are hypothesized to serve similar functions. It is notable that when clawed frogs are stressed by shaking, the concentration of CRH-like peptides in the medial amygdala significantly increases, similar to the effect of stressors in the mammalian amygdala.…”