From behavior to circuitry: The role of glycine regulation in CNS motor output

One central goal in neurobiology is to establish the link between genes and behavior. The gap between the motor behaviors of whole organisms and our growing knowledge of the molecules and cells in the nervous system remains substantial. We have used zebrafish in a forward genetic strategy to discover the genetic pathways and neuronal circuits important for locomotion. Our analysis of the shocked (sho) mutation demonstrated the importance of glycine regulation for proper signaling within the central nervous system.; sho embryos have deficits in early motor behaviors that include reduced spontaneous coiling of the trunk, diminished escape responses when touched, and an absence of swimming before 2 days. Positional cloning found a missense mutation in the slc6a9 gene that encodes a glycine transporter (GlyT1) as the cause of the sho phenotype. Antisense knockdown of GlyT1 in wild-type embryos phenocopies sho , and injection of wild-type GlyT1 mRNA into mutants rescues them. The mutation renders the transporter non-functional when the mutant protein is expressed in Xenopus oocytes. Glyt1 is expressed in non-neuronal cells in the hindbrain and spinal cord. Lesion experiments showed that the caudal hindbrain normally provides the excitatory descending drive to the spinal cord during swimming, and that this site is severely affected by the loss of GlyT1 in sho. This suggests that GlyT1 function is critical for proper signaling in motor circuits.; The analysis of CNS physiology in sho demonstrated perturbations to both glycinergic and glutamatergic synaptic transmission. Glial glycine transporters are thought to clear synapses of synaptically released glycine. Since glycine serves as an inhibitory neurotransmitter, sho mutants should exhibit high levels of extracellular glycine and enhanced glycinergic inhibition. Indeed, sho motoneurons failed to receive the NMDA-dependent excitatory synaptic drive that normally gives rise to swimming. Furthermore blocking glycine-gated Cl- channels with strychnine partially restored motor behaviors in sho embryos. Likewise, washing out the excess glycine in the sho CNS restored normal swimming while the introduction of exogenous glycine re-introduced the abnormal phenotype. These findings suggest that the regulation of glycine levels by GlyT1 is critical in glycinergic transmission and in the generation of locomotor pattern.