Análisis de los antecedentes investigativos

In all species where it has been studied, electrical stimulation of the brainstem or other supra-spinal centres is able to generate properly coordinated fictive locomotion (Shik et. al, 1966; Skinner & Garcia-Rill, 1984; Fetcho & Svoboda, 1993; Sirota et. al, 2000; Kyriakatos et al. 2011). In Xenopus tadpoles this is also the case (Fig. 1.13). However, unlike many other species, a transient stimulation results in sustained locomotion. To generate normal locomotor-like activity via the MLR a tonic electrical stimulation is required. In the case of the mammalian MLR, increasing the intensity of stimulation sequentially engages the postural system before eliciting coordinated fictive locomotion (Shik et. al, 1966). As the frequency of the tonic stimulation is increased the speed of fictive locomotion also increases and this is also seen in the fictive swimming of lampreys (Sirota et al, 2000) and goldfish (Fetcho & Svoboda, 1993). In pro-

metamorphic Xenopus tadpoles, brief (1-20ms) stimulation of the optic tectum produces reliable self-sustaining swimming. Although it has not been tested systematically, the most reliable sites for stimulation appear to be dorsally on the caudal portion of the optic tectum. In the lamprey, which shares broad anatomical similarities with the

tadpole brain, the MLR has been localized to a similar area of the midbrain (Sirota et al, 2000). However, the MLR is located both more ventral and more medial than the site I stimulated. As such, it is not clear whether the stimulus used here would be appropriate to activate the equivalent area of the tadpole brain. Moreover, activation of the MLR in the lamprey requires a relatively specific stimulation with activation of regions just 25m away producing asymmetrical or disrupted swimming patterns (Sirota et al, 2000). One possibility is that activation of a pathway within the optic tectum projects to the MLR or bypasses it completely and directly activates reticulospinal centres within the hindbrain.

In fish, an overlapping and perhaps analogous region, the MLF, is also implicated in locomotion (Sankrithi & O’Malley, 2010). One major role for the MLF is likely in relaying visual stimuli to the hindbrain and spinal cord and thus mediating optomotor responses (Uematsu & Todo, 1997; Sankrithi & O’Malley, 2010). The fact that

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stimulation of multiple sites on the optic tectum elicits episodes of fictive locomotion is further evidence for the importance of this structure in visually guided motor behaviour (Dong et al. 2009). In addition to the optic tectum, stimulation of vestibulocochlear nerve as well as sites in the hindbrain were all capable of eliciting fictive locomotion in the tadpole, highlighting the convergent nature of the supra-spinal control of

locomotion.

The ability to produced sustained swimming from a transient stimulus is a useful tool to study the intrinsic capability of the spinal CPG to produce locomotion and to generate rhythmic activity. This has been one of the major benefits of the embryonic and early larval models of locomotion in Xenopus. Moreover, it avoids the need to use

pharmacological means to activate the spinal network as is the case with the in vitro

preparations in the lamprey (Cohen & Wallen, 1980) and the neonatal mammal (Kudo & Yamada, 1987). This is important since the application of excitatory receptor agonists such as NMDA, 5-HT and DA will alter the basal state of the nervous system and could potentially mask the effects of subsequent drug applications during experimental

manipulations. Furthermore, pharmacologically activated preparations tend to be phase locked into a set rhythm, and as such studying the network activity over its full dynamic range is no longer possible. A similar approach for evoking episodes of swimming has been developed recently in the adult zebrafish (Kyriakatos et al, 2011). This work highlighted the potential pitfalls of pharmacologically activated preparations as prolonged exposure to NMDA changed the amplitude of synaptic drive and action potentials in MNs, and led to MNs firing during the rising phase of the synaptic drive rather than the during the plateau depolarisation, as seen following evoked episodes.

When the optic tectum is stimulated at regular intervals, episodes of evoked swimming are relatively consistent in duration (Fig. 1.13C). When the interval between episodes is varied, however, the episode duration displays a linear relationship: the longer the interval the longer the subsequent episode (Fig. 1.13C). It should be noted that this relationship would not be truly linear and would level off at intervals greater than 2 minutes. Eventually activity occurs spontaneously and the preparations effectively set their own interval that stays relatively stable (see Fig. 1.11Bi for example). At early stages of Xenopus development this relationship is also present and has been linked to an innate mechanism akin to short-term memory within the spinal network (Zhang & Sillar, 2012) Spinal neurons at these stages display an approximately minute-long, ultra-

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slow afterhyperpolarization (usAHP), which is generated by up regulation of Na+/K+ pump function during activity. CPG neurons are essentially able to integrate spike frequency over time and use previous activity to control the excitability of the network. Similar cellular phenomena are also found at pro-metamorphic stages and these will be discussed later in this thesis (Chapter 3). At the level of motor behaviour this means that after a long episode of swimming, the tadpole is subsequently only capable of

generating a shorter episode.