Ramanathan S et al. (2006), Developmental and regional expression of NADPH-...

XB-ART-34721

Eur J Neurosci 2006 Oct 01;247:1907-22. doi: 10.1111/j.1460-9568.2006.05057.x.

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Developmental and regional expression of NADPH-diaphorase/nitric oxide synthase in spinal cord neurons correlates with the emergence of limb motor networks in metamorphosing Xenopus laevis.

Ramanathan S , Combes D , Molinari M , Simmers J , Sillar KT .

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Metamorphosis in anuran amphibians requires a complete transformation in locomotor strategy from undulatory tadpole swimming to adult quadrupedal propulsion. The underlying reconfiguration of spinal networks may be influenced by various neuromodulators including nitric oxide, which is known to play an important role in CNS development and plasticity in diverse species, including metamorphosis of amphibians. Using NADPH-diaphorase (NADPH-d) staining and neuronal nitric oxide synthase (nNOS) immunofluorescence labelling, the expression and developmental distribution of NOS-containing neurons in the spinal cord and brainstem were analysed in all metamorphic stages of Xenopus laevis. Wholemount preparations of the spinal cord from early stages of metamorphosis (coincident with emergence of the fore- and hindlimb buds) revealed two clusters of NOS-positive neurons interspersed with areas devoid of stained somata. These cells were distributed in three topographic subgroups, the most ventral of which had axonal projections that crossed the ventral commissure. Motoneurons innervating the fore- and hindlimb buds were retrogradely labelled with horseradish peroxidase (HRP) to determine their position in relation to the two NOS-expressing cord regions. Limb motoneurons and NOS-positive cells did not overlap, indicating that during early stages of metamorphosis nitrergic neurons are excluded from regions where spinal limb circuits are forming. As metamorphosis progresses, NOS expression became distributed along the length of the spinal cord together with an increase in the number and intensity of labelled cells and fibers. NOS expression reached a peak as the forelimbs emerge then declined. These findings are consistent with a role for nitric oxide (NO) in the developmental transition from undulatory swimming to quadrupedal locomotion.

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Species referenced: Xenopus laevis
Genes referenced: mhc2-dab myh3 nos1 nos3

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	Fig. 6. NADPH-d reactivity in wholemount spinal cord of a late pro-metamorphic stage 57 tadpole. Schematic of wholemount CNS at left indicates the positions and relative densities of NOS-expression, and the levels (A and B) at which transverse sections were made. NOS-positive staining is now observed along the entire length of the spinal cord. Note in A the three groups of neurons as labelled at earlier stages. (B) Similar staining at the level of the lumbar enlargement but, in comparison to early pro-metamorphic stages, Group 2 neurons now divide into distinct subgroups (seen at higher magnification in Bi and ii). Scale bars, 500 lm (A and B) and 100 lm (Bi and ii).
	Fig. 7. NADPH-d reactivity in the spinal cord of a stage 64 froglet after metamorphic climax. Schematic of the CNS wholemount at left indicates levels at which transverse sections were obtained. Hindbrain spinal cord boundary is marked by the dotted line. (A) Cross section at the thoracic level showing three groups of NADPH-d reactive spinal neurons: a dorsal group of cells (equivalent to Grp 1) intermingled with nitrergic fibers (*; see also dorsal view in Ai); a second medial group (Grp 2) of multipolar neurons with axonal processes that project both dorsally and ventrally; a third ventral group (Grp 3; magnified in Aii) consisting of spindle-shaped neurons (arrowhead) that send axonal processes bilaterally, many of which cross the midline. (B) In the lumbar region, labelling continues to occur in the dorsal group 1 area (starred) and in a more ventro-lateral area (expanded in Bi), but no neurons stain in the region immediately ventral to the central canal (circled, see text). Scale bars, 200 lm (A and B); 100 lm (Ai, ii and Bi) and 5 mm (froglet schematic).
	Fig. 8. HRP and NADPH-d double-labelling in the spinal cord of a stage 51 tadpole. (A) Wholemount CNS after HRP injections into the right and left forelimbs and the left hindlimb. The CNS was reacted with DAB to reveal retrogradely labelled motoneurons innervating the limb buds (brown reaction product), then NOSpositive structures in the brainstem and spinal cord were labelled with NADPH-d (blue reaction product). Due to the tissue thickness and high level of background staining, a camera lucida drawing of the CNS is also presented in (B) showing the position of NOS-positive neurons (in blue) and HRP backfilled motoneurons (in red). NADPH-d reactive neurons in the spinal cord did not overlap with regions occupied by HRP-backfilled motoneurons. (C) Cross section through the NOSpositive spinal cluster showing three groups of NADPH-d reactive neurons; labelled motoneurons were never observed in sections where NOS staining was present. (D) Cross sections from spinal cord where labelled motoneurons were located in wholemount preparations. (Di) Higher magnification shows labelled motoneurons are bipolar and have large spindle-shaped cell bodies (arrows). Scale bars, 500 lm (A and B) and 100 lm (C and D).
	Fig. 9. Pattern and distribution of NADPH-d reactivity in the CNS of Xenopus laevis during metamorphosis. The schematic summarizes the approximate position, sequence of appearance and intensity (as a function of shading) of NADPH-d neuronal staining in the brainstem and spinal cord in representative metamorphic stages. In free-swimming larval stages (stages 43â47), the most prominent group of NOS-positive neurons are found in the brainstem and only a few isolated neurons in the spinal cord are NOS-positive (McLean & Sillar, 2001). NOS-positive spinal neurons are first observed at pre-metamorphic stage 48, coinciding with limb bud emergence and by stage 51 two longitudinal clusters are clearly evident. Through later pre-metamorphic stages, staining is restricted to the two spinal clusters, but with increasing NADPH-d reactivity. These clusters (shaded) are in spinal areas flanking the emerging limb motor pools (hatched). During pro-metamorphosis, when both axial- and limb-based swimming begins to occur, neurons in previously unstained spinal areas begin to show weak NADPH-d reactivity, which then also increases as metamorphosis progresses. By pro-metamorphic stage 57, intense neuronal staining is seen throughout the length of the spinal cord, and this persists until the metamorphic climax when a decline in staining in the lumbar region is observed.