22 ± 0.03 pA/pF with control siRNA and 0.18 ± 0.02 pA/pF with TRIP8b siRNA; p > 0.5, t test). This provides strong evidence that the MDV3100 action of TRIP8b siRNA is specific for Ih with no obvious off-target effects. As HCN1 is not properly targeted to distal dendrites of hippocampal neurons in dissociated cell cultures, we examined the effect

of reducing TRIP8b levels on Ih expression in CA1 pyramidal neurons in vivo. Lentivirus encoding TRIP8b siRNA or control siRNA was injected under stereotactic control into the CA1 region of the hippocampus of 5-week-old mice. After 2 weeks, brains were dissected and sliced for immunohistochemical analysis. The EGFP-positive (infected) regions of CA1 from hippocampi expressing TRIP8b siRNA had reduced levels of TRIP8b staining compared with neighboring regions of CA1 that were not EGFP-positive (Figure S1). No change in TRIP8b staining was detected in slices infected with virus expressing control siRNA, confirming the in vivo efficacy and specificity of the siRNA. To examine the effect of in vivo knockdown of TRIP8b on HCN channel surface density, we obtained whole-cell recordings from EGFP-positive CA1 pyramidal neurons in acute slices from hippocampi injected with virus expressing either

TRIP8b siRNA or control siRNA. Because of limitations in achieving adequate voltage-clamp of CA1 dendrites in acute slices, we relied on current clamp measurements of electrophysiological parameters known to reflect Ih (Magee, 1998 and Magee, 1999) and HCN1 (Nolan et al., 2004). We found that CA1 neurons Selleck OSI-744 infected with

TRIP8b siRNA displayed a series of changes consistent with a marked reduction in Ih. First, there was a ∼3 mV negative shift in the resting Adenosine potential of neurons infected with TRIP8b siRNA (−72.1 ± 1.0 mV; n = 15) compared with control siRNA (−69.0 ± 0.9 mV; n = 18; p < 0.05, t test), consistent with a loss of the depolarizing influence of Ih. Moreover, this difference was eliminated in the presence of ZD7288 (TRIP8b siRNA: −78.7 ± 0.9 mV; n = 15; control siRNA: −78.4 ± 0.7 mV; n = 18), indicating a specific role of Ih (Figure 1B). Second, knockdown of TRIP8b caused a large increase in input resistance (TRIP8b siRNA: 140.5 ± 9.9 MΩ; n = 15; control siRNA: 89.67 ± 5.1 MΩ; n = 18; p < 0.01, t test), consistent with the loss of HCN channels (Figure 1F). This effect was also abolished by ZD7288 (TRIP8b siRNA: 189.9 ± 11.4 MΩ; n = 15; control: 180.1 ± 10.5 MΩ; n = 18). Third, the depolarizing sag in response to a hyperpolarizing current step, characteristic of Ih activation, was significantly decreased in CA1 neurons infected with TRIP8b siRNA (TRIP8b siRNA: sag ratio = 0.10 ± 0.02; n = 15; control: sag ratio = 0.24 ± 0.02; n = 18; p < 0.01, t test) (Figure 1G). In neurons expressing TRIP8b or control siRNA, the sag was eliminated by 10 μM ZD7288.