Excitatory synapses in the brain exhibit a remarkable degree of functional

Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). extra-synaptic AMPARs, but fails Daidzin price to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, we make compensatory mutations to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs. Excitatory synapses in the brain release the transmitter glutamate, which acts primarily on two subtypes of ionotropic receptors, the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and assessments. Fixation and Confocal Microscopy. Slice cultures were fixed for use in confocal microscopy with 4% paraformaldehyde/4% sucrose in PBS overnight at 4C. Images were obtained by compiling Z-stacks of images made at 100 with 0.5-m thick sections, using a Bio-Rad Confocal system attached to a Nikon microscope. Results Control of Synaptic AMPARs by PSD-95. We used organotypic slice cultures in conjunction with biolistic transfections to express GFP-tagged proteins in hippocampal pyramidal neurons. All experiments involved simultaneous patch-clamp recordings from a transfected cell and a neighboring untransfected cell. A stimulating electrode placed in stratum radiatum activated excitatory afferents. The relative Daidzin price response magnitudes evoked by activating the same presynaptic afferents were directly compared, allowing assessment of the effects of protein expression in the postsynaptic neuron on synaptic currents. A typical experiment, in this case with GFP-tagged PSD-95, is usually shown in Fig. ?Fig.1.1. Confocal microscopy of fixed slices (Fig. ?(Fig.22to dendritic spines, the contact site for excitatory synapses. Open in a separate window Physique 1 Slice culture recording configuration. Each experiment involved simultaneous whole-cell voltage-clamp recordings from both a transfected cell (PSD-95 GFP in this example, cell on right, gold particle in nucleus) and an immediately adjacent untransfected cell (left). ( 1 10?6, = 27 pairs), whereas NMDAR EPSCs are unchanged (= 0.81, = 23 pairs). (and = 26 pairs, = 0.48; NMDAR EPSCs, = 14 pairs, = 0.99). (= 3 pairs). (= 7 pairs, = 0.003). In every tests the amplitude was assessed by us from the AMPAR-mediated EPSC at ?70 mV and measured the amplitude from the NMDAR-mediated EPSC at +40 mV with a latency when the AMPAR EPSC got fully decayed (60 ms). Representative traces of averaged AMPAR and NMDAR EPSCs concurrently documented from a control and transfected cell (Fig. ?(Fig.22= 5). In several pairs we concurrently documented the response to bath-applied AMPA, which activated both synaptic and extra-synaptic AMPARs. No enhancement was detected in neurons expressing PSD-95 (Fig. ?(Fig.22and = 7, = 0.01). The effect of stargazin was specific to AMPARs, because responses to bath-applied NMDA (5 M) were unaffected (untransfected = 496 65 pA versus transfected = 419 147 pA; = 3). This result indicates that, in contrast to synaptic AMPARs, stargazin is usually limiting for expression of extra-synaptic surface AMPARs. AMPAR Recruitment to Synapses Involves Synaptic PDZ Interactions. To gain insights into the mechanisms of PSD-95 and stargazin function, we analyzed a series of mutant and deletion constructs. Removing the PDZ binding site of stargazin by deleting the last 4 aa at its C terminus (stargazinC) reduced both its clustering (Fig. ?(Fig.33and and and = 54 pairs, 1 10 ?9) while having no effect on NMDAR EPSCs (= 37 pairs, = 0.43). (Scale bars, traces: 10 pA, 20 ms.) (= 9 pairs, = 0.0006). We also analyzed PSD-95 deletion constructs to identify regions crucial to PSD-95 function. For each of the constructs illustrated in Fig. ?Fig.44we examined the degree of synaptic clustering and the degree of enhancement of AMPAR and NMDAR EPSCs. Our results with clustering (data not shown) generally agreed with previous findings (21) and, importantly, we found that the level of clustering correlated with the degree of enhancement of the AMPAR EPSC. The N-terminal palmitoylation of PSD-95 that is essential for its synaptic clustering was also necessary for enhancement of synaptic responses, as the PSD-95 C3,5S mutant did not enhance AMPAR EPSCs (Fig. ?(Fig.44 0.05; **, 0.01; ***, 0.001); the number of paired recordings is usually listed for each construct. Rabbit Polyclonal to RAB3IP (and and = 47 pairs, = 0.002) and significantly attenuated the enhancement caused by PSD-95 (GFP + PSD-95 = 202 13% increase, = 15 pairs; GFP-StarCterm + PSD-95 = 72 19% increase, = 14 pairs, 0.05). Although these results supported Daidzin price our model, expression of the C terminus of stargazin could have displaced other ligands that.