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The amphibian Xenopus laevis is able to adjust its skin color to the light intensity of the environment. Paling of the skin is achieved by inhibiting the release of alpha-melanophore-stimulating hormone (alpha-MSH) from the melanotrope cells in the pars intermedia of the pituitary gland. The release of alpha-MSH is inhibited by gamma-aminobutyric acid (GABA), neuropeptide Y (NPY), and dopamine (DA). To locate and identify neurons that might be responsible for the inhibitory input, double and triple immunocytochemistry, retrograde tracing from the pars intermedia with the carbocyanine membrane probe 1,1'dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzene-sulfonate (Fast DiI), and confocal laser-scanning microscopy were combined. Glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and NPY were found to coexist in an axonal network innervating the pars intermedia. The suprachiasmatic nucleus (SC) contained different populations of neurons that were single, double, or triple labelled for GAD, NPY, and TH. In the lateral SC, NPY+ neurons were observed. TH-immunoreactive (TH-IR) neurons occurred in the medial, dorsolateral, lateral, and ventrolateral SC. Neurons that were double labelled for NPY and TH and triple labelled for Fast DiI, NPY, and TH were present in the ventrolateral SC. This same area contained neurons that were triple labelled for GAD, NPY, and TH. It is concluded that the triple-labelled and probably the double-labelled ventrolateral SC neurons (suprachiasmatic melanotrope-inhibiting neurons) innervate the pituitary pars intermedia and are responsible for the NPY-, DA-, and GABA-mediated inhibition of melanotrope cell activity in Xenopus laevis.
Fig. 1. Transverse section of the pituitary gland of Xenopus laevis
triple-labelled with glutamic acid decarboxylase (GAD; A), neuropeptide
Y (NPY; B), and tyrosine hydroxylase (TH; C) antisera. Small
arrowheads mark varicosities where GAD, NPY, and TH coexist.
Large arrowheads indicate varicosities that were mainly stained for
NPY. Varicosities that stained predominantly for TH (large arrow) or
GAD (small arrow) are also indicated. PD, pars distalis; PI, pars
intermedia; PN, pars nervosa. Scale bar 5 25 μm.
Fig. 2. A,B: Immunofluorescence photomicrographs of a transverse
section of the Xenopus suprachiasmatic nucleus (SC) double
labelled with neuropeptide Y (NPY; A) and tyrosine hydroxylase
(TH; B) antisera. In the ventrolateral part of the SC, NPY and TH
coexist (arrows), whereas, in the medial part of the SC close to the
third ventricle (III), NPY and TH neurons occur clearly separated. OC,
optic chiasm. C: NPY-immunoreactive neurons in the ventromedial
thalamic nucleus (VM) and in the dorsomedial part of the suprachiasmatic
nucleus (SC). D: Diagram showing the locations of NPYimmunoreactive
(solid circles) and TH-immunoreactive (open circles)
neurons and neurons that are double labelled for both NPY and TH
(solid squares) or for GAD, NPY, and TH (open square). DL, dorsolateral
SC; M, medial SC; L, lateralSC; OC, optic chiasm; VL, ventrolateral
SC. Scale bars 5 25 μm.
Fig. 3. Immunofluorescence photomicrographs of the ventrolateral
part of the suprachiasmatic nucleus (SC) stained simultaneously with
glutamic acid decarboxylase (GAD; A), neuropeptide Y (NPY; B), and
tyrosine hydroxylase (TH; C) antisera. Thick arrows indicate neuron
that show coexistence of GAD, NPY, and TH; other neurons are double
stained for NPY and TH (arrowheads), and some stain for TH only
(thin arrow). Scale bar 5 25 μm.
Fig 4. Immunofluorescence photomicrographs of the ventrolateral
part of the suprachiasmatic nucleus triple labelled with Fast 1,18dioctadecyl-
3,3,38-tetramethylindocarbocyanine perchlorate (Fast DiI; A)
and by retrograde tracing from the pars intermedia with neuropeptide
Y (NPY; B) and tyrosine hydroxylase (TH; C) antisera. Most stained
neurons are triple-labelled for Fast DiI, NPY, and TH (arrowheads),
and some neurons lack Fast DiI but are double labelled with NPY and
TH antiserum (arrows). Scale bar 5 25 μm.
Fig. 5. Immunofluorescence photomicrographs of the projection of
13 1-μm optical sections of the ventrolateral part of the suprachiasmatic
nucleus stained with anti-neuropeptide Y (NPY). A: NPY
staining is found predominantly in discrete subcellular structures
(thick arrows). Thin arrow indicates an NPY1 varicosity in close
contact with an NPY1 neuron, as shown by vertical (B) and horizontal
(C) reslicing through the projected image. Scale bars 5 10 μm in A,
5 μm in B (also applies to C).
