DISCUSION Y COMENTARIOS

Fictive locomotion recorded from the isolated nervous system of pro-metamorphic

Xenopus tadpoles correlates well with their motor behaviour at these stages of development. Episodes of sustained, rhythmic locomotion occur spontaneously (Fig. 1.11; Combes et al, 2004) and the pattern of recruitment, with caudal sections active first and more rostral sections incorporated as the episode progresses, is retained in vitro

(Fig. 1.12A; Rauscent, 2008).

At the level of the neural control of locomotion, the switch from sessile embryos to free-swimming larvae is presumed to be linked to the degradation of the GABAergic pathway that controls the stopping response in young tadpoles (Boothby & Roberts, 1992a,b; Perrins et al, 2002). At larval stage 42, the GABAergic mhr neurons are still present (Reith, 1996) and, although the pathway connecting them to the cement gland is degrading, they are thought to be incorporated into a central circuit where they may continue to contribute to the cessation of swimming episodes. Evidence for this comes from the fact that episodes of swimming often terminate coincident with a barrage of GABAergic IPSPs (Reith & Sillar 1999). In general, sporadic GABA IPSPs are thought to contribute to an inhibitory tone that counteracts the tonic depolarisation of spinal neurons during swimming. The short, intense episodes of swimming seen in the

presence of bicuculline at stage 42 support this idea (Reith & Sillar, 1999). Input to mhr neurons from the cement gland is not a factor at pro-metamorphic stages, due to the afferent limb of the pathway having fully degraded. Despite this, there is also evidence of a continued role for descending GABAergic inhibition of the spinal CPG. The blockade of GABAA-receptors highlights this, as disinhibited preparations show

increased spontaneous activity well above control levels (see Fig. 1.18A). However, these experiments have not addressed the site of action of GABAA blockade and

therefore could also involve disinhibtion of supra-spinal centres. This would mirror the situation in many other vertebrates, where local application of bicuculline to the MLR activates locomotor activity (Garcia-Rill et al, 1990; Menard et al, 2007).

It is worthy of note, however, that even at much later stages of development (stage 53- 62) the tadpoles show signs of residual activity of a descending inhibitory pathway. Sensory stimulation of most parts of the tadpoles head will reliably elicit an aversive

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response and cause the animal to swim away but touching the tadpole directly between the nasal pits, at the rostral extent of the head, has no such effect. In fact, this form of stimulation seems to actively inhibit motor activity (personal observation). Given the dramatic and reliable effects of disinhibition in these preparations it is certainly worthy of further experiments to investigate both the extrinsic and intrinsic modulation of the tadpole spinal CPG by GABA.

As well as simply being more active during free-swimming larval life, Xenopus tadpoles spend most of their time feeding, with their bodies tilted in a head down hover (Hoff & Wassersug, 1986). They maintain this posture by sculling with the caudal most parts of the tail and swim faster by recruiting more of the tail. This recruitment pattern is also seen in fictive locomotion (Fig. 1.12A). The recruitment and de-recruitment of the tail musculature can be seen as the sequential recruitment of more rostral ventral roots. To achieve smooth, uninterrupted swimming while incorporating more of the tail into the movement is quite a feat. The prevailing theory is that a gradient of excitability must exist within the spinal circuitry along the length of the cord (Rauscent, 2008). For instance, if the caudal most sections of the cord have the lowest threshold for activation then low levels of excitation will be enough to cause swimming with the tip of the tail. Subsequently increasing the descending excitation would then lead to the sequential recruitment of more rostral areas.

A similar pattern of recruitment to increase locomotor speed has been described during swimming in zebrafish (McLean et al, 2007; McLean & Fetch, 2009). The recruitment does not occur along the length of the cord, since fish tend to swim with the whole tail at all speeds, but instead, spinal neurons are recruited based on their relative dorso- ventral position. There is evidence that this recruitment is based on topographic

gradients of excitability such that cells with the high IR are recruited first (McLean et al, 2007). For the Xenopus swim network to function like this, it is apparent that the caudal segments of the cord must be able to produce properly patterned output in partial

isolation from the rest of the cord. In the lamprey, it has been known for a long time that the spinal CPG is compartmentalised at multiple levels along the cord. In this

preparation, isolated sections of spinal cord can still produce normal locomotor activity when activated pharmacologically (Cohen & Wallen, 1980; Wallen & Williams, 1984). This has led to the proposal that the spinal CPG is organised as a series of coupled oscillators (Cohen & Wallen, 1980; Buchanan, 1992). In the case of the lamprey, where

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the whole body is involved in swimming at all speeds, the speed of swimming is a function of the phase relationship between the oscillator units. In Xenopus the speed would have to be primarily based on a gradient of excitability between oscillator units.

The tonic activity, which can be seen before and after rhythmic ventral root activity (Fig. 1.12B), can perhaps provide some clues as to how the caudo-rostral system of recruitment works and what its function might be. Firstly, it is evident that MNs are active at times other than when directly involved in propulsive locomotion. One theory to explain this would be that, at sub-threshold levels for discrete bursting, the MNs are involved in postural control. In mammalian studies where locomotion was induced via stimulation of the MLR, the lowest levels of stimulation engaged the postural system causing the animals to stand and bear their own weight (Shik et al, 1966). Perhaps a similar initial state is activated in Xenopus as excitation builds up within the more rostral segments and helps stabilise the regions immediately adjacent to those actively involved in propulsion. Biomechanically, actively stiffening the body can be very important for swimming in fish as it helps counteract self-generated rotational forces (Webb 2002). This is known to be especially important during slow swimming, like the characteristic hover of the Xenopus tadpole, since the hydrodynamic trimming forces associated with forward swimming are reduced.

Even if recruitment within the cord is based on a gradient of excitability, you would expect the neurons at a given level to be a heterogeneous group, with some cells more excitable than others. Given that situation, the tonic activity could be the sporadic output of low threshold MNs. Only when excitation reaches a level high enough to activate a critical proportion of the network would the coordinated bursting emerge from this background excitation. Interestingly, I have recorded from neurons in the spinal cord, including MNs, that show a good correlate for this type of activity. First, they depolarise and fire tonically, coincident with the low amplitude tonic activity in the ventral root, before switching into a pattern of rhythmic firing that matches the rhythmic output of the network. (see Chapter 3 for further discussion). In the context of Brown’s half centre model (Brown, 1914), this would be equivalent to the tonic descending excitation needed to bring the pattern generating component of the network to threshold. Effectively the tadpole may have ‘units’ of the CPG located at each segment of the spinal cord that have their own threshold for activation. Once a critical number of neurons within a given segment are depolarised, the intrinsic organisation within the

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network should provide the appropriate synaptic excitation and inhibition required to produce rhythmic firing.Of course, the trigger for rhythmic activity may not be

dependent on a critical number of neurons becoming active but could rely on a specific type of neuron becoming active. For instance, the descending interneurons (dINs) are known to be the primary source of excitatory drive in the embryonic tadpole spinal cord (Li et al, 2006; see Chapter 3). If these neurons persist during development, as my preliminary data suggests (Fig.3.14), then perhaps their recruitment is the primary factor determining the activation of more rostral segments of the cord during swimming.

The emergence of tightly coordinated bursting from an initial tonic depolarisation of spinal neurons may also be related to coupling between adjacent areas of the cord. The first and last few bursts in an episode of spontaneous activity are often less sharply tuned than those in the middle of the episode (see Fig. 1.12Bii &iii). In simulations of axial swimming in the lamprey, the coupling between adjacent segments is important to phase-lock their activity (Buchanan, 1992). In Xenopus swimming, which involves recruitment and de-recruitment of spinal segments, it seems likely that the activity of a given segment will be better coordinated when those either side of it are active.

Therefore, the first and last few bursts in an episode, which will have less rhythmic input from more rostral segments, should be expected to be less sharply tuned than those in the middle of the episode.

The in vitro preparations are capable of swimming continually, just as in the intact animal (the tail in semi-intact preparations sometimes swims for several hours without stopping). However, the full extent of the locomotor-like activity is not recorded since the most caudal sections are too small to make reliable extracellular recordings.

Nevertheless, what is recorded is at approximately the same frequency as during natural swimming in the intact animal. The natural tail beat frequency ranges between 8-12Hz for all but the fastest swimming speeds where it can occasionally reach 20Hz (Hoff & Wassersug, 1986). This correlates well with the tail beat frequency in semi-intact (tail- attached) preparations where the tail beat is at around 8Hz (Rauscent, 2008). One confound between these two data sets, however, is that in vitro the recruitment of more rostral segments associated with increased swimming speeds is characterised by a reduction in ventral root burst frequency (Rauscent, 2008). This difference may be due to the absence of sensory feedback in the in vitro preparation. In the lamprey, the bending of the body during swimming activates contralateral stretch receptors called

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edge cells, which reinforce the left-right alternation coordinated by the central circuit (Grillner et al, 1984; Di Prisco et al, 1990). If similar cells are involved in swimming in

Xenopus, the lack of movement in the in vitro preparation may result in an inability to maintain high frequency swimming during recruitment of more rostral spinal segments.