Proceedings of The Physiological Society

Physiology 2012 (Edinburgh) (2012) Proc Physiol Soc 27, C57

Oral Communications

On the role of reciprocal inhibition in locomotion rhythm generation - Brown's half centre hypothesis revisited a century later

P. Moult1, G. Cottrell1, W. Li1

1. University of St Andrews, St Andrews,, Fife, United Kingdom.

Background: Brown's half centre hypothesis proposed a century ago (Brown, 1911; Brown, 1914) emphasized the importance of reciprocal inhibition in the generation of locomotor rhythms. However, pharmacological blockade of reciprocal inhibition or lesion of commissural connections in all vertebrates has led to a widely accepted notion that reciprocal inhibition is not required in locomotor rhythm generation and neuronal pace-maker properties have been suggested to mediate the rhythms (Li, 2011). We use two new methods to suppress reciprocal inhibition on millisecond scales in the Xenopus tadpole swimming circuit to re-investigate the role of inhibition in locomotor rhythm generation. Methods: Procedures for producing tadpoles comply with UK Home Office Animals Act 1986 and have received local ethical approval. Xenopus tadpoles were anaesthetised with 0.1% MS-222 (3-aminobenzoic acid ester), immobilised with 10 μM α-bungarotoxin and dissected to expose the spinal cord. The two methods we used for rapidly suppressing reciprocal inhibitory interneuron (cIN) activity are 1) Optogenetics: we expressed the light-sensitive reverse proton pump archaerhodopsin-3 (ArCh) (Chow et al., 2010) on one side of the nervous system by injecting its cRNA into one blastomere at the two cell stage of embryo development. We then specifically inhibited that side of the nervous system by shining yellow light (585 nm) to activate ArCh. 2) Injecting large negative DC into single excitatory interneurons (dINs) which are electrically coupled and directly excite cINs during swimming. Results: We found that acutely inhibiting activity on one side could instantly stop swimming rhythms on both sides (median time for yellow light inhibition: 0.19 seconds or 2 swimming cycles, n=149 trials in 8 tadpoles; for -DC injections: 0.19 seconds or 2 cycles, n=107 trials in 13 dINs). Analysing changes in synaptic drives on the un-inhibited side showed that reciprocal inhibition was specifically weakened to 4.4 ± 2.6 % (IPSC size normalised to control, Wilcoxon Signed Rank test, n=11, p<0.001) in the last swimming cycle, leading to reduced dIN rebound firing reliability (from control of 99.6 ± 0.4 to 0.4 ± 0.4 %, Wilcoxon Signed Rank test, n=11, p<0.001). dIN activity drives tadpole swimming circuit and their rebound firing provides one mechanism to maintain normal tadpole swimming rhythms (Soffe et al., 2009; Li, 2011). Conclusion: Our data thus support Brown's original proposal that reciprocal inhibition is required in locomotor rhythm generation.

Where applicable, experiments conform with Society ethical requirements