Glycine has two main roles in the vertebrate nervous system: (i) as an inhibitory amino acid transmitter released from spinal interneurones which binds to postsynaptic, strychnine-sensitive receptors in the brainstem and spinal cord; and (ii) as an obligatory agonist at the glycine binding site on the NMDA-type glutamate receptor. The levels of glycine in the extracellular fluid are normally saturating, questioning the availability of this site for physiological regulation. However, at least two forms of glycine transport mechanism can regulate extracellular glycine levels: GLYT2 transport proteins serve to sequester glycine released at inhibitory synapses; GLYT1 proteins have been associated with glia and may control glycine levels around glutamate receptors.
We are exploring glycine transporters in a simple model system, the neural network controlling swimming in pre-feeding Xenopus laevis tadpoles (stages 37/8-42; Nieuwkoop & Faber, 1956). Sarcosine (n-methyl glycine) is a naturally occurring competitive inhibitor of GLYT 1b. When bath-applied to pre-feeding α-bungarotoxin-immobilized Xenopus tadpoles, sarcosine (250 µM-5 mM; n = 36) produced a dose-dependent, reversible inhibition of ‘fictive’ swimming: sarcosine decreased swim episode and ventral root bursts durations, burst amplitudes and cycle frequencies. At the highest concentrations tested sarcosine abolished all activity. These effects are due to the activation of inhibitory glycine receptors since they were reversed by strychnine (1 µM; n = 5), but not by the GABAA receptor antagonist, bicuculline (20-50 µM; n = 3). In intracellular recordings from swim motorneurones (n = 8), sarcosine produced a large strychnine-sensitive increase in membrane conductance, an effect which was also resistant to application of tetrodotoxin (1 µM; n = 3).
Finally, the effects of sarcosine were time dependent. The inhibition was often preceded by prolongation of swimming episodes and the initiation of spontaneous bouts of swimming. This suggests that sarcosine first produces facilitation and activation of swimming activity followed by activation of inhibitory glycine receptors. To test this possible action of glycine at the NMDA receptor binding site we looked for effects of sarcosine following block of glycine receptors with strychnine (n = 7). We find that the duration of motor bursts increases and the cycle periods decrease following sarcosine applications in the presence of strychnine. We conclude that the excitatory drive for swimming increases in the presence of sarcosine and that the glycine-binding site on the NMDA receptors must normally be held below saturation by the actions of GLYT1 receptors. This suggests that the site is available for physiological regulation and could thus be used to control levels of excitability in spinal motor networks.
This work was supported by The Wellcome Trust.