The early development of serotonergic raphe-spinal interneurons in two related amphibians : 'Xenopus laevis' and 'Rana temporaria'
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1) A method has been devised to enable visualisation of 5HT immunoreactive neurons in the CNS of embryonic amphibians in wholemount. The technique relies upon a rather long incubation in primary antiserum to allow thorough access of antibodies to central neurons. 2) Using this technique, the results of van Mier et al (1986), using serially sectioned material, have largely been substantiated. 5HTi neurons in Xenopus embryos (developmental stage 37/38) comprise a cluster of some 60 cell bodies in the raphe region of the rostral, ventral hindbrain. A second, smaller cluster, noted by van Mier, in the caudal midbrain could not be detected in the present study until developmental stage 42. The descending axonal projections of raphe interneurons are located exclusively in the dorsolateral margins of the spinal cord of Xenopus embryos. At stage 42 about 70 somata are now present in the brainstem and their descending processes now also extend into the ventromedial regions of the cord. 3) Serotonergic neurons in Xenopus were succesfully ablated during embryogenesis, following exposure to the specific 5HT neurotoxin, 5,7 DHT, at ImM. This procedure enabled a preliminary physiological investigation of the effects of serotonergic input to the spinal cord on the development of spinal cord function. The absence of raphe interneurons appears to retard the normal development of neuronal circuitry underlying swimming movements. 4) A comparative study of serotonergic raphe-spinal interneurones in a related amphibian embryo, Rana temporaria was undertaken. In contrast to Xenopus embryos, the descending projections of serotonergic raphe neurons in Rana embryos at the time of hatching (stage 20; Gosner, 1960), are exclusively located in the ventral aspect of the spinal cord. 5) The results of raphe ablation studies in Xenopus larvae suggest that a causal link may exist between raphe projections and the developmental modulation of locomotor output in postembryonic larvae. The interspecific differences observed in the development of 5HTi projections in Rana, coupled with existing knowledge of the physiology of swimming in Rana compared with Xenopus, lend further support to this notion. The hypothesis is now amenable to rigorous experimental testing, using the techniques devised and presented in this thesis.
Thesis, MSc Master of Science
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