In agreement with previous studies (Suwa et al., 2001; Laube, 2002; Rigo et al., 2003; Hirzel et al., 2006), we provide evidence that GlyRs are not saturated in our preparation because the mIPSCs were potentiated by 1 m zinc, a high-affinity allosteric modulator that increases the receptor affinity for glycine (Bloomenthal et al., 1994; Laube et al., 1995, 2000; Suwa et al., 2001; Miller et al., 2005). were normalized by the first IPSC amplitude or by the average amplitude between APs 3 and 32. In semilogarithmic plots, IPSC amplitudes between the APs 4 and 1000 were averaged using a constant logarithmic bin of 0.1. Electrophysiological results are reported as mean SEM. All statistical assessments were nonparametric. The MannCWhitney test and the sign test EPZ-6438 (Tazemetostat) were used to assess differences between two impartial and two related samples, respectively. The KolmogorovCSmirnov test was used to assess the equality of the two distributions. For all those assessments, the number of asterisks in the figures corresponds to level of significance: * 0.05, ** 0.01, and *** 0.001. FM 4-64 imaging. GlyT2CEGFP spinal cord DHRS12 neurons were incubated for 3 min at 37C in a depolarizing extracellular answer containing the following: 102.4 mm NaCl, 40 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm glucose, 10 mm HEPES, pH 7.3, and 10 m FM 4-64 [are the average from 20C30 consecutive iontophoretic pulses. The coefficients of variance were 0.197, 0.074, and 0.026 at 10, 15, and 20 V activation, respectively. Open in a separate window Physique 6. Glycine release is not changed after GlyT2 uptake in control neurons. before (left) and after (right) glycine application ( 0.05) in control (solid collection) and in the presence of 70 m SR95531 (gray collection; SR; 0.05). 0.05), in control (open circle) and in the presence of 70 m SR95531 (gray circle; 0.05). 0.05) glycine application. Results We set out to study the role of GlyT2 for the refilling of synaptic vesicles with glycine, in pairs of cultured spinal neurons with recognized presynaptic GlyT2 neurons, using transgenic GlyT2CEGFP mice (Zeilhofer et al., 2005). GlyT2 transporter currents To confirm the functional expression of GlyT2 in green fluorescent neurons, we examined the glycine-evoked current recorded from GlyT2+ or GlyT2? neurons (Fig. 1= 127). In all neurons tested, fast application of glycine (200 m) generated large inward currents (Fig. 1= 4) (Fig. 1= 14) (Fig. 1= 5) (Fig. 1relationship EPZ-6438 (Tazemetostat) of the GlyT2-mediated current (values were normalized by their complete values at = 10). GlyT2 determines the neuronal glycinergic phenotype To examine synaptic transmission in GlyT2+ neurons, we recorded evoked postsynaptic currents (IPSCs) in pairs of connected neurons with an recognized GlyT2+ presynaptic element (Fig. 2= 16), although the low coefficient of variance (CV of 0.2 0.03), the short latency (1.6 0.1 ms), and the absence of failures all indicated that this connections were monosynaptic. SR95531, a specific GABAA receptor antagonist at EPZ-6438 (Tazemetostat) 5 m, blocked one-quarter of the IPSC amplitude (26.3 3.3%; = 30), whereas the remaining current was completely eliminated by strychnine (Fig. 2= 7) recorded in the presence of NBQX (2 m) and d-APV (50 m; left traces), SR95531 (5 m; middle traces; SR), or strychnine (3 m; right traces; stry) for control neurons (top, black) and EPZ-6438 (Tazemetostat) neurons preincubated for 15C24 h with 5 m “type”:”entrez-protein”,”attrs”:”text”:”ORG25543″,”term_id”:”1179172534″,”term_text”:”ORG25543″ORG25543 (bottom, gray). = 30) and neurons preincubated with 5 m “type”:”entrez-protein”,”attrs”:”text”:”ORG25543″,”term_id”:”1179172534″,”term_text”:”ORG25543″ORG25543 (gray; = 26). The values were normalized by the IPSC amplitude recorded in the presence of NBQX and d-APV. = 30; gray) and neurons preincubated with “type”:”entrez-protein”,”attrs”:”text”:”ORG25543″,”term_id”:”1179172534″,”term_text”:”ORG25543″ORG25543 (= 26; gray collection). = 30; gray) and neurons preincubated with “type”:”entrez-protein”,”attrs”:”text”:”ORG25543″,”term_id”:”1179172534″,”term_text”:”ORG25543″ORG25543 (= 26; white). 0.001), pure glycinergic ( 0.02), and pure GABAergic ( 0.03) IPSCs for control neurons (= 30; gray) and neurons preincubated with “type”:”entrez-protein”,”attrs”:”text”:”ORG25543″,”term_id”:”1179172534″,”term_text”:”ORG25543″ORG25543 (= 26; white). The majority of GlyT2+ neurons (27 of 30) displayed a dominant ( 50%) glycinergic phenotype (Fig. 2= 5 of 9), or glutamatergic, with evoked currents entirely blocked by a combination of NBQX and d-APV (= 4 of 9; EPZ-6438 (Tazemetostat) data not shown). Having characterized control inhibitory transmission, we first investigated the role of GlyT2 by preincubating the cultures with “type”:”entrez-protein”,”attrs”:”text”:”ORG25543″,”term_id”:”1179172534″,”term_text”:”ORG25543″ORG25543, a specific GlyT2 inhibitor (Caulfield et al.,.