5 t test). Similarly, measurements of frequencies and amplitudes of mIPSCs in cells expressing GPHN.FingR-GFP (Figures 7H and 7I; f = 4.2 ± 0.5 s−1, A = 14.1 ± 1.0 pA, n = 8 cells) were not significantly different from comparable measurements in control cells (f = 4.3 ± 0.5 s−1, A = 13.5 ± 1.1 pA, n = 9 cells; p > 0.1 t test). Thus, our results Selleckchem Screening Library indicate that expressing PSD95.FingR-GFP and GPHN.FingR-GFP does not cause changes in synaptic physiology and has no effect on the number or neurotransmitter receptor content of individual synapses. To determine

whether GPHN.FingR-GFP signals represent functional inhibitory synapses, we measured IPSCs evoked by GABA photolysis at individual green punta. Hippocampal CA1 pyramidal neurons were transfected with GPHN.FingR-GFP and TdTomato to visualize inhibitory synapses and neuronal structure (Figure 8A). Two-photon GABA uncaging 0.5 μm away from puncta of GPHN.FingR-GFP triggered IPSCs. IPSC amplitude diminished when uncaging

occurred further away from the dendrite, demonstrating that IPSCs originated from the activation of receptors localized in dendrites of the recorded neuron (Figures 8B and 8C). When GABA was photoreleased on the dendritic shaft at locations where GPHN.FingR-GFP was present, robust IPSCs were evoked. However, GABA photorelease at two locations, one in a dendritic spine and a second on a dendritic shaft, where there was no GPHN.FingR-GFP signal elicited small or negligible IPSCs (Figures 8D and 8E). These data confirm that GPHN.FingR-GFP does indeed label functional Rutecarpine inhibitory synapses. PSD95.FingR and GPHN.FingR label their endogenous target proteins in dissociated check details neurons, as well as in neurons in slices. To determine whether FingRs can be used to label endogenous proteins in vivo, we transfected PSD95.FingR-GFP into neurons in mouse embryos in utero using electroporation and then assessed expression at approximately 7 weeks of age. Images of dendrites of layer V cortical pyramidal neurons coexpressing HA-mCherry and taken from unstained sections cut from perfused, fixed brains clearly show punctate patterns of GFP expression consistent

with labeling of PSD-95 (Figures 9A and 9B). In addition, lower-magnification images show labeling of layer V and layer II/III pyramidal neurons that is also consistent PSD-95 labeling (Figures 9C and 9D). Finally, an image obtained from a living animal of PSD95.FingR-GFP expressed in an apical tuft from a cortical pyramidal neuron (Figure 9E) demonstrates that PSD95.FingR-GFP can be imaged in vivo. In this paper we demonstrate that Fibronectin intrabodies generated with mRNA display (FingRs) can be used to visualize the localization of the endogenous postsynaptic proteins Gephyrin and PSD-95 in living neurons without affecting neuronal structure and function. FingRs represent a substantial improvement over traditional antibody approaches that, in general, require that cells be fixed and permeabilized prior to staining.