Inhibitory synapses are established during development but continue to be generated and modulated in strength in the mature nervous system. In the spinal cord and brainstem, presynaptically released inhibitory neurotransmitter dominantly switches from GABA to glycine during normal development in vivo. While presynaptic mechanisms of the shift of inhibitory neurotransmission are well-investigated, the contribution of postsynaptic neurotransmitter receptors to this shift is not fully elucidated. Synaptic clustering of glycine receptors (GlyRs) is regulated by activation-dependent depolarization in early development. However, GlyR activation induces hyperpolarization after the 1st postnatal week, and little is known whether and how presynaptically released glycine regulates postsynaptic receptors in depolarization-independent manner in mature developmental stage. Here we developed spinal cord neuronal culture of rodents using chronic strychnine application to investigate whether initial activation of glycine receptors (GlyRs) in mature stage could change postsynaptic localization of GlyRs. Immunocytochemical analyses demonstrate that chronic blockade of GlyR activation until mature developmental stage resulted in smaller clusters of postsynaptic GlyRs that could be enlarged upon receptor activation for one hour in mature stage. Furthermore, live cell imaging techniques show that GlyR activation decreases its lateral diffusion at synapses, and this phenomenon is dependent on PKC, but neither Ca2+ nor CaMKII activity. These results suggest that the GlyR activation can regulate receptor diffusion and cluster size at inhibitory synapses in mature stage, providing not only new insights into the postsynaptic mechanism of shifting inhibitory neurotransmission, but also the inhibitory synaptic plasticity in mature nervous system.
Significant Statement Clustering of postsynaptic glycine receptors (GlyRs) is critical for developmental shift from GABAergic to glycinergic inhibitory neurotransmission in the spinal cord and brainstem. Synaptic GlyR localization is mediated by the receptor activation and followed by depolarization-dependent Ca2+ influx in immature stage. However, little is known whether and how presynaptically released glycine enhances postsynaptic GlyR clustering in mature stage when glycinergic transmission is upregulated without depolarization. Here we report postsynaptic GlyR clustering induced by the receptor activation in mature neurons. Furthermore, postsynaptic stabilization of laterally diffusive GlyRs is mediated in Ca2+-independent PKC activity at sites expressing gephyrin, a postsynaptic scaffolding protein. Our results provide new insights into homeostatic receptor dynamics underlying activation-dependent modulation of inhibitory synaptic strength in mature nervous system.
The authors declare no competing financial interests.
3 This work was supported by Grants-in-Aid for Scientific Research [KAKENHI] from JSPS to Y.N. (11J02839, 14J30003, 26870826) and to J.N. (25253017).