IV. RESULTADOS Y DISCUSIÓN
4.2. Análisis inferencial y contrastación de hipótesis
4.2.2. Análisis inferencial del modelo 2 obtenido
Paradoxically, for an animal used to study motor behaviour, the tadpole embryo is primarily a sessile creature, swimming only in response to sensory stimulation. Instead of swimming around, the embryos spend the vast majority of their first day post- hatching hanging from a mucous-secreting cement gland located ventro-medially on their head. The cement gland and head skin contain mechanosensory nerve endings whose cell bodies are located in the trigeminal ganglia (Roberts & Blight, 1975;
Roberts, 1980). When activated, for example after the tadpole swims into a solid object or the water surface, the action potentials of trigeminal neurons project to the hindbrain, activating GABAergic midhindbrain reticulospinal (mhr) neurons, which in turn project
32 Stage 37/38 Stage 45
A
B
1cmCi
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0 10 20 30 40 50 60 70 80 90 100 % S wi m m in g A c ti v it y 37/38 43 45 47 Developmental Stage*
* **33
Figure 1.6 The switch to free swimming in Xenopus laevis larvae.
During embryonic and early larval development, Xenopus laevis tadpoles are predominantly sessile creatures. At stage 45 there is a dramatic switch in their behaviour, where they take to
the water column and swim almost continually. A, Spontaneous swimming occurs very
occasionally in stage 37/38 embryos but by stage 45 these episodes are far more frequent and
the total time spent swimming has increased significantly. B, A traced trajectory for a single
example of a spontaneous swimming episode from a stage 37/38 and stage 45 tadpole. Not only are these events very rare in the embryo, they are significantly shorter than those occuring at
stage 45. Ci, This picture of a stage 50 Xenopus larvae illustrates the typical head down hover
maintained while the animal feeds. This slow swimming involves just the caudal portion of the tail as can be seen by the travelling wave that begins more than half way along the animal in this
image. Cii, At these stages Xenopus swimming differs from almost all known aquatic animals
in that it does not display a linear relationship between tail beat frequency and swimming speed (see open white circles). The relatively high tail beat frequencies at slow swimming speeds in Xenopus are due to it initially controlling changes in swim speed via recruitment and de- recruitment of the axial muscles along the length of its tail. For comparison the same
measurements are displayed for swimming in a Rana tadpole, which swims with its whole tail at
all swimming speeds (filled black circles and linear plot). Figures adapted from Scott (2012; A&B) and Hoff & Wassersug (1986; C).
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to the spinal cord and whose activity inhibits spinal neurons and prematurely terminates on-going swimming (Boothby & Roberts, 1992; Perrins et al, 2002).
As well as the additional influence of biogenic amines and other neuromodulators, the development of swimming behaviour in Xenopus is altered by the degradation of this GABAergic pathway (Boothby & Roberts, 1992). The cement gland is gradually lost during development and there is a coincident reduction in the fidelity of the stopping response. Depspite this, there is evidence that the GABAergic mhr neurons persist until at least stage 42 where despite being detached from the cement gland they may still contribute to the termination of swimming, which at these stages is characterised by a barrage of GABA IPSPs (Reith, 1999).
By stage 45/46 (4 days post fertilisation; dpf) the animal is free-swimming and the cement gland has completely degraded. At these stages, fictive locomotion also displays an increase in episodes of an apparently spontaneous nature (Fig. 1.6; Scott, 2012). At the earliest larval stages (stages 42-43) there are only sporadic episodes of spontaneous swimming but by stage 45 the animal is active about 20% of the time. Moreover the spontaneous activity persists after removal of both the forebrain and mid-brain but is absent completely in spinalised preparations (Scott, 2012).
A series of in vitro brainstem-spinal cord preparations from specific time points during
Xenopus metamorphosis produce stage-specific locomotor output in isolation from the rest of the animal (Combes et al. 2004; Fig. 1.7). At pre-metamorphic stages (stages 50- 54) the fictive motor output recorded from exposed ventral roots is very similar to that during embryonic and early larval development: it displays left-right alternation across the spinal cord and a rostro-caudal delay. Moreover, the frequency of the motor bursts is appropriate for the corresponding stages in vivo. In post-metamorphic juvenile froglets (stage 64) fictive motor output can now be recorded from lumbar nerve branches that would normally innervate extensor or flexor muscles in the hind limbs. Again, the in vitro activity was found to correlate well with the real behaviour, with synchronous bi- lateral activity between extensor or flexor pairs across the body and ipsilateral flexor- extensor alternation.
During metamorphosis the larval tadpoles transition between purely tail-based axial undulations and purely limb-based appendicular kicking. The intervening stages are characterised by two distinct periods. The first occurs when the hind limbs first become
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Ai
ii
iii
Bi
ii
iii
Ci
ii
iii
Di
ii
iii
E
F
50ms 1s expanded in E36
Figure 1.7 The transition from axial to appendicular locomotor strategies studied
in vitro during amphibian metamorphosis.
The isolated brainstem-spinal cord preparations developed by Combes et al (2004) illustrate the development of the neural circuitry underlying the dramatic switch in locomotor strategy during
metamorphosis in Xenopus laevis. A, In pre-metamoprhic larvae (Ai; stage 50) extracellular
recordings from exposed spinal ventral roots (Aii) ehibit the left-right alternation and rostro-
caudal phase lag characteristic of fictive locomotion in earlier stages of Xenopus development
(Aiii). B, During early pro-metamorphic stages (Bi; stage 58) additional extracellular recordings
can be made from exposed nerve roots that ordinarily innervate flexor and extensor muscles
(Bii). The fictive motor output recorded at these stages is unusual in that ipsilateral flexors and
extensors are active in synchrony while contralateral limb muscles are active in alternation (Biii
and on an expanded time base in E). Moreover, the limb activity is functionally coupled to the
axial rhythm alternating across the cord in phase with spinal ventral root activity. C, During
metamorphic climax (Ci; stage 61) the fictive motor output (Ciii) highlights the emergence of
two distinct rhythms, which can be recorded simultaneously from the isolated brainstem-spinal
cord preparation (Cii). D, In post-metamorphic froglets (Di; stage 64) the tail has been resorbed
and fictive locomotion recorded from the exposed nerve roots of the hindlimbs (Dii) reveals the
functional appendicular rhythm with bilateral synchrony between homologous muscles and
ipsilateral alternation between flexors and extensors (Diii). F, A summary of the emergence of
the functional appendicular rhythm (red) from the distinct pre-existing axial rhythm (blue). Figures adapted from Combes et al (2004; A-E) & Sillar et al (2008; F).
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motile and are swept back against the tail during undulatory swimming. The in vitro
preparations from this period (stage 58) illustrates the neuromuscular basis of this behaviour with ipsilateral flexor-extensor activity synchronised and alternating with the corresponding nerve roots from the opposite side of the spinal cord. This activity is not only unlike rhythmic activity in the limbs later in development, it is coordinated with axial ventral root activity recorded further down the tail. The final preparation
developed by Combes et al (2004) was representative of metamorphic climax when the tadpoles swim using a combination of axial and appendicular propulsive modes. The in vitro preparations from this period (stage 61) display both axial and appendicular rhythms, that may occur either simultaneously or independently (Fig. 1.7).
Together these data suggest that the emergence of the circuitry for limb-based locomotion is characterised by a period when the functional output of the neural
network is coupled to the pre-existing axial network. The anatomical and physiological basis of this coupling remains to be determined but preliminary data suggests that electrical coupling between limb and axial MNs that may contribute to the initial functional development of the appendicular circuitry (Wagner 2013, unpublished observations).