Lentivirus

Lentivirus PF-01367338 solubility dmso expressing shRNA-HCN1 was infused in the CA1 region of the dorsal hippocampus, which expressed on 7 days postinfusion (DPI) and up to at least six months (Figure 1B) and spread mediolaterally (about 0.7–1.0 mm) and anteroposteriorly (about 1.2–1.6 mm)

(Figure 1C). We quantified the local silencing efficiency of HCN1 protein by immunohistochemistry and western blotting. The HCN1 protein expression was significantly decreased without alteration in HCN2 and MAP2 protein expression in the shRNA-HCN1-infected region as compared to non-infected or shRNA-control-infected CA1 regions (Figures 2A–2D). Quantification of protein expression from isolated lentiviral shRNA-HCN1-infected dorsal CA1

region showed a 58% reduction in HCN1 protein selleck chemical expression without change in HCN2 and β-tubulin protein expression as compared to shRNA-control-infected region (Figure 2E), suggesting specificity for knockdown of HCN1 channels. To determine whether silencing of HCN1 gene had an effect on the physiology of the dorsal CA1 pyramidal neurons, Ih-sensitive electrophysiological parameters were measured using the whole-cell current-clamp method ( Narayanan and Johnston, 2007; Figures 3 and S2). ShRNA-HCN1-infected CA1 pyramidal neurons had hyperpolarized resting membrane potentials ( Figures 3C), higher steady-state input resistance ( Figure 3D), and slower membrane time constant ( Figure 3E) than noninfected or shRNA-control-infected CA1 pyramidal

neurons. For proper comparison between groups, we held membrane potentials at −65 mV with current injection and compared electrophysiological properties ( Figures 4 and S3). ShRNA-HCN1-infected CA1 pyramidal neurons had less voltage sag ( these Figure 4A) and lower resonance frequency ( Figure 4B) compared to noninfected or shRNA-control-infected CA1 pyramidal neurons. In addition, shRNA-HCN1-infected CA1 pyramidal neurons generated more action potentials in response to depolarizing current steps (30–300 pA in 30 pA increments for 750 ms) ( Figure 4C), suggesting increased cellular excitability ( Shah et al., 2004). Similar results, however, were also obtained with neurons at their normal resting potentials ( Figure S2). To examine subthreshold synaptic integration (αEPSP), the response to repetitive current injections similar to multiple excitatory postsynaptic currents were measured using a train of 5 alpha current injections (α = 0.1, 20 Hz) ( Brager and Johnston, 2007; Dembrow et al., 2010; Poolos et al., 2002). ShRNA-HCN1-infected CA1 pyramidal neurons had larger αEPSP summation than noninfected or shRNA-control-infected CA1 pyramidal neurons ( Figure 4D). In agreement with our biochemical results, these data indicate that silencing of the HCN1 gene by shRNA-HCN1 produced electrophysiological changes consistent with a reduction in Ih.

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