1993); mouse anti-NR2B was diluted 1 : 500 (immunogen sequence a/a 892�C1051; BD Biosciences-Pharmingen, San Diego, CA, USA, Valle-Pinero et al. 2007); rabbit anti-NR2C (cat. no. PPS033, R&D Systems, Minneapolis, MN, USA) was diluted 1 : 1000; mouse anti-PSD-95 was diluted 1 : 2000 (immunogen sequence a/a 77�C299, cat. no. 73-028, NeuroMab, Davis, CA, USA). Sections were then washed 3�� 10 min in PBS with 0.1% Triton X-100 followed by incubation with secondary antibodies, either Alexa AZD9291 manufacturer
Fluor 488 or Alexa Fluor 546 (goat anti-rabbit 1 : 1000, goat anti-mouse 1 : 500; Molecular Probes) diluted in blocking buffer, for 2 h at room temperature. Negative controls were performed by incubating sections with secondary antibodies while omitting the primary antibody step. After a final washing step (4 �� 10 min), slides were mounted with Vectorshield HardSet Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained using a Leica fluorescence microscope (DM2500) fitted with a CCD camera (DFC350fx). For co-staining with mouse anti-NR2B and anti-PSD-95, after incubation with the pooled primary antibodies and subsequent washing, sections were first incubated for 1 h with the secondary antibody specific for isotype IgG2b, goat anti-mouse Alexa Fluor 488 (Molecular Probes) before addition of the non-IgG specific secondary antibody, goat anti-mouse Alexa Fluor 546 (Molecular Probes). Data are presented as means ��s.e.m. and significance verified using Student's two-tailed paired or unpaired t test or ANOVA as PR-171 solubility dmso
indicated, with P < 0.05 being considered significant. The aim of these experiments was to examine the development of synaptic NMDA receptors during maturation of the auditory system after the onset of hearing. We have used Lister Hooded rats and CBA/Ca mice to gain greater breadth of experimentation than was possible by using either species alone (see Methods). The evoked EPSC generated by the calyx of Held had a typical dual component response, with a fast AMPAR-mediated component and a slower-time-course voltage-dependent NMDAR-mediated current, as shown in Fig. 1A. To aid comparison with published data we first tested the temperature sensitivity of the NMDAR-mediated EPSC by comparing calyceal synaptic responses at 25 and 37��C in the same neurons (Fig. 1A; rat P10/11). This clearly shows that the YES1
slow EPSC increased in amplitude and had accelerated kinetics when the temperature was raised to 37��C. We confirmed that the slow EPSC is mediated by NMDAR by perfusion of the NMDAR antagonist d-AP5 (50 ��m) as shown in Fig. 1B. The temperature effect was similar across the whole voltage range as shown by the mean current�Cvoltage (I�CV) relationship in Fig. 1C. We have previously shown that raising temperature increases the amplitude of the fast EPSC by actions on postsynaptic AMPA receptors (Postlethwaite et al. 2007) while there is little net effect of temperature on transmitter release (Kushmerick et al. 2006).