Mixed miniature postsynaptic currents resulting from co-release of glycine and GABA recorded from glycinergic neurons in the neonatal respiratory network

Inhibitory neurons are involved in the generation and patterning of the respiratory rhythm in the adult animal. However, the role of glycinergic neurons in the respiratory rhythm in the developing network is still not understood. Although the complete loss of glycinergic transmission in vivo is lethal, the blockade of glycinergic transmission in slices of the medulla has little effect on pre-Botzinger complex network activity. As 50% of the respiratory rhythmic neurons in this slice preparation are glycinergic, they have to be considered as integrated parts of the network. We aimed to investigate whether glycinergic neurons receive mixed miniature inhibitory postsynaptic currents (mIPSCs) that result from co-release of GABA and glycine. Quantification of mixed mIPSCs by the use of different objective detection methods resulted in a wide range of results. Therefore, we generated traces of mIPSCs with a known distribution of mixed mIPSCs and mono-transmitter-induced mIPSCs, and tested the detection methods on the simulated data. We found that analysis paradigms, which are based on fitting the sum of two mIPSC templates, to be most acceptable. On the basis of these protocols, 2040% of all mIPSCs recorded from respiratory glycinergic neurons are mixed mIPSCs that result from co-release of GABA and glycine. Furthermore, single-cell reverse transcriptase polymerase chain reaction revealed that 46% of glycinergic neurons co-express mRNA of glycine transporter 2 together with at least one marker protein of GABAergic neurons. Our data suggest that significant co-transmission occurs in the pre-Botzinger complex that might be involved in the shaping of synaptic inhibition of respiratory glycinergic neurons.