The influence of VEGFD expression on synaptic transmission in hippocampal neurons in culture was directly assessed by recording miniature excitatory postsynaptic currents (mEPSCs)

in the presence of TTX and the GABAA receptor blocker, gabazine. Neurons transfected with pAAV-shVEGFD or infected with rAAV-shVEGFD showed longer mEPSC interevent intervals (IEIs, 1/frequency) and smaller mEPSC amplitudes than their respective shSCR-expressing controls ( Figures 7E and 7F). The reduced mEPSC frequency in transfected hippocampal neurons suggests that the effect was not mediated by a reduced release probability presynaptically because the low transfection rate ensures that the majority of synaptic input to shRNA-expressing learn more cells comes from non-shRNA-expressing cells. The reduced mEPSC frequency is thus most likely indicative of fewer AMPA receptor-containing synapses per cell. The 21%–24% reduction in mEPSC amplitude also suggests a lower density of AMPA receptors at synapses in shVEGFD-expressing cells. mEPSCs of hippocampal neurons expressing shVEGFD also showed faster rise and decay time constants than their respective shSCR-expressing controls (

Figure 7C and Table S1), most likely due to reduced filtering of mEPSCs in their more compact dendritic trees. Alternatively, a synaptic NMDA receptor-mediated slow component of the mEPSC may have been reduced in shVEGFD-expressing neurons, although significant NMDA currents are unlikely in our Paclitaxel recording conditions (−71 mV holding potential, 1.3 mM Mg2+). Responses were also recorded to bath-applied AMPA, which produced a peak within 30 s whose amplitude was used as an indication of the total number of functional AMPA receptors per cell PDK4 ( Figure 7D). AMPA response amplitudes were smaller in hippocampal neurons expressing shVEGFD

( Figures 7D and 7G), indicative of a reduced total number of surface-expressed AMPA receptors per cell. Taken together, our patch-clamp analysis has identified a reduced plasma membrane surface area, as well as a reduced number of AMPA receptor-containing synapses, a reduced number of AMPA receptors per synapse, and a reduced total number of AMPA receptors in shVEGFD-expressing cells. These results are consistent with the reduced dendritic morphology identified by morphometric analyses. We next investigated the role of VEGFD in vivo. rAAV-shVEGFD or the appropriate control rAAVs were stereotaxically delivered to the dorsal hippocampus of 2-month-old C57BL/6 male mice. Infected neurons were readily identified by analysis of the mCherry fluorescence ( Figure S4). The morphology of neurons in the CA1 area of the hippocampus was assessed by manually tracing the basal dendrites of Golgi-stained brain slices obtained from animals 2.5 weeks after viral gene delivery.