Limits...
Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish.

Menelaou E, Husbands EE, Pollet RG, Coutts CA, Ali DW, Svoboda KR - Eur. J. Neurosci. (2008)

Bottom Line: Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development.When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos.These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

ABSTRACT
Zebrafish embryos exhibit spontaneous contractions of the musculature as early as 18-19 h post fertilization (hpf) when removed from their protective chorion. These movements are likely initiated by early embryonic central nervous system activity. We have made the observation that narrowminded mutant embryos (hereafter, nrd(-/-)) lack normal embryonic motor output upon dechorionation. However, these mutants can swim and respond to tactile stimulation by larval stages of development. nrd(-/-) embryos exhibit defects in neural crest development, slow muscle development and also lack spinal mechanosensory neurons known as Rohon-Beard (RB) neurons. At early developmental stages (i.e. 21-22 hpf) and while still in their chorions, nrd siblings (nrd(+/?)) exhibited contractions of the musculature at a rate similar to wild-type embryos. Anatomical analysis indicated that RB neurons were present in the motile embryos, but absent in the non-motile embryos, indicating that the non-motile embryos were nrd(-/-) embryos. Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development. These errors were reversed when nrd(-/-) embryos were raised in high [K(+)] beginning at 21 hpf, indicating that the abnormal axonal phenotypes may be related to a lack of depolarizing activity early in development. When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos. These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

Show MeSH

Related in: MedlinePlus

Muscle physiology in nrd+/? and nrd−/− larval zebrafish. (A) Averaged traces of 26–48 mEPCs shown with single (orange) and double (magenta) exponential fits for the decay component obtained from 72-hpf larval zebrafish. (B) Bar graphs quantifying the SSE for the single and double exponential fits of the decay component for the traces shown in A, wild-type (n= 6), nrd+/? (n= 8) and nrd−/− (n= 17). The SSE for a double exponential fit is significantly smaller for the nrd−/− mutants (*P< 0.05, n= 17). (C) Bar graphs of single exponential fits for the decay component showing a significantly longer decay for the mEPCs acquired from nrd−/− larval zebrafish.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2741004&req=5

fig06: Muscle physiology in nrd+/? and nrd−/− larval zebrafish. (A) Averaged traces of 26–48 mEPCs shown with single (orange) and double (magenta) exponential fits for the decay component obtained from 72-hpf larval zebrafish. (B) Bar graphs quantifying the SSE for the single and double exponential fits of the decay component for the traces shown in A, wild-type (n= 6), nrd+/? (n= 8) and nrd−/− (n= 17). The SSE for a double exponential fit is significantly smaller for the nrd−/− mutants (*P< 0.05, n= 17). (C) Bar graphs of single exponential fits for the decay component showing a significantly longer decay for the mEPCs acquired from nrd−/− larval zebrafish.

Mentions: We sought to determine if there were any changes in the properties of mEPCs (muscle synaptic physiology) arising from the activation of nAChRs associated with axial muscles in nrd−/− larvae because there was a lack of activity and an increase in the branching pattern of primary motoneurons in these fish. Primary motoneurons are not thought to innervate slow fibers (van Raamsdonk et al., 1983; de Graaf et al., 1990; van Asselt et al., 1993) and the slow fibers were never present in the nrd−/− larvae; therefore, we only recorded mEPCs from fast fibers. We found that there was a small but significant change in the decay kinetics of the mEPCs, while the majority of cellular and other mEPC properties were unchanged between wild-type and mutant larvae (Table 2). Specifically, we found that the mEPCs from nrd−/− larvae were significantly better fit with a double exponential decay rather than a single exponential decay as in the wild-type and heterozygous siblings (Fig. 6A). A comparison of the SSEs (Fig. 6B) shows that a double exponential fit is significantly better than a single fit for the mEPCs from nrd−/− larvae (P< 0.05), suggesting that there is a change in the off-kinetics of mEPCs in nrd−/− larvae. Furthermore, we found that mEPCs recorded from fast fibers of nrd−/− larvae took significantly longer to decay to baseline (τ = 0.37 ± 0.01 ms, n= 17) than heterozygous siblings (τ = 0.29 ± 0.01 ms, n= 8) and wild-type fish (τ = 0.32 ± 0.02 ms, n= 6) when the decay was forced-fit with a single exponential component (Fig. 6C, P< 0.05). Even though we detected a change in muscle physiology, where the decay times of mEPCs in the mutants differed from siblings, this difference did not compare with the differences in decay times documented for twister mutants and stage-matched twister siblings (see Lefebvre et al., 2004; Figure 6). In that study, decay time differences were of the order of 100 ms, whereas in nrd−/− larvae, the differences in mEPC decay times was about 0.08 ms (see Table 2 for summary). Thus, we conclude that the muscle-specific nAChR is not overactive in nrd−/− larvae and does not likely contribute to the motoneuron pathfinding errors.


Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish.

Menelaou E, Husbands EE, Pollet RG, Coutts CA, Ali DW, Svoboda KR - Eur. J. Neurosci. (2008)

Muscle physiology in nrd+/? and nrd−/− larval zebrafish. (A) Averaged traces of 26–48 mEPCs shown with single (orange) and double (magenta) exponential fits for the decay component obtained from 72-hpf larval zebrafish. (B) Bar graphs quantifying the SSE for the single and double exponential fits of the decay component for the traces shown in A, wild-type (n= 6), nrd+/? (n= 8) and nrd−/− (n= 17). The SSE for a double exponential fit is significantly smaller for the nrd−/− mutants (*P< 0.05, n= 17). (C) Bar graphs of single exponential fits for the decay component showing a significantly longer decay for the mEPCs acquired from nrd−/− larval zebrafish.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2741004&req=5

fig06: Muscle physiology in nrd+/? and nrd−/− larval zebrafish. (A) Averaged traces of 26–48 mEPCs shown with single (orange) and double (magenta) exponential fits for the decay component obtained from 72-hpf larval zebrafish. (B) Bar graphs quantifying the SSE for the single and double exponential fits of the decay component for the traces shown in A, wild-type (n= 6), nrd+/? (n= 8) and nrd−/− (n= 17). The SSE for a double exponential fit is significantly smaller for the nrd−/− mutants (*P< 0.05, n= 17). (C) Bar graphs of single exponential fits for the decay component showing a significantly longer decay for the mEPCs acquired from nrd−/− larval zebrafish.
Mentions: We sought to determine if there were any changes in the properties of mEPCs (muscle synaptic physiology) arising from the activation of nAChRs associated with axial muscles in nrd−/− larvae because there was a lack of activity and an increase in the branching pattern of primary motoneurons in these fish. Primary motoneurons are not thought to innervate slow fibers (van Raamsdonk et al., 1983; de Graaf et al., 1990; van Asselt et al., 1993) and the slow fibers were never present in the nrd−/− larvae; therefore, we only recorded mEPCs from fast fibers. We found that there was a small but significant change in the decay kinetics of the mEPCs, while the majority of cellular and other mEPC properties were unchanged between wild-type and mutant larvae (Table 2). Specifically, we found that the mEPCs from nrd−/− larvae were significantly better fit with a double exponential decay rather than a single exponential decay as in the wild-type and heterozygous siblings (Fig. 6A). A comparison of the SSEs (Fig. 6B) shows that a double exponential fit is significantly better than a single fit for the mEPCs from nrd−/− larvae (P< 0.05), suggesting that there is a change in the off-kinetics of mEPCs in nrd−/− larvae. Furthermore, we found that mEPCs recorded from fast fibers of nrd−/− larvae took significantly longer to decay to baseline (τ = 0.37 ± 0.01 ms, n= 17) than heterozygous siblings (τ = 0.29 ± 0.01 ms, n= 8) and wild-type fish (τ = 0.32 ± 0.02 ms, n= 6) when the decay was forced-fit with a single exponential component (Fig. 6C, P< 0.05). Even though we detected a change in muscle physiology, where the decay times of mEPCs in the mutants differed from siblings, this difference did not compare with the differences in decay times documented for twister mutants and stage-matched twister siblings (see Lefebvre et al., 2004; Figure 6). In that study, decay time differences were of the order of 100 ms, whereas in nrd−/− larvae, the differences in mEPC decay times was about 0.08 ms (see Table 2 for summary). Thus, we conclude that the muscle-specific nAChR is not overactive in nrd−/− larvae and does not likely contribute to the motoneuron pathfinding errors.

Bottom Line: Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development.When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos.These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

ABSTRACT
Zebrafish embryos exhibit spontaneous contractions of the musculature as early as 18-19 h post fertilization (hpf) when removed from their protective chorion. These movements are likely initiated by early embryonic central nervous system activity. We have made the observation that narrowminded mutant embryos (hereafter, nrd(-/-)) lack normal embryonic motor output upon dechorionation. However, these mutants can swim and respond to tactile stimulation by larval stages of development. nrd(-/-) embryos exhibit defects in neural crest development, slow muscle development and also lack spinal mechanosensory neurons known as Rohon-Beard (RB) neurons. At early developmental stages (i.e. 21-22 hpf) and while still in their chorions, nrd siblings (nrd(+/?)) exhibited contractions of the musculature at a rate similar to wild-type embryos. Anatomical analysis indicated that RB neurons were present in the motile embryos, but absent in the non-motile embryos, indicating that the non-motile embryos were nrd(-/-) embryos. Further anatomical analysis of nrd(-/-) embryos revealed errors in motoneuron axonal pathfinding that persisted into the larval stage of development. These errors were reversed when nrd(-/-) embryos were raised in high [K(+)] beginning at 21 hpf, indicating that the abnormal axonal phenotypes may be related to a lack of depolarizing activity early in development. When activity was blocked with tricaine in wild-type embryos, motoneuron phenotypes were similar to the motoneuron phenotypes in nrd(-/-) embryos. These results implicate early embryonic activity in conjunction with other factors as necessary for normal motoneuron development.

Show MeSH
Related in: MedlinePlus