, 2004). Knockdown of TRIP8b led to a ∼70% increase in the t1/2 (time to decay by 50%) of PP EPSPs relative to control (TRIP8b siRNA: 28.36 ± 1.26 ms, n = 15; control siRNA: 47.79 ± 2.92 ms, n = 18; p < 0.01; Figure 1H). Addition of ZD7288 increased the t1/2 further, and to identical

values in both populations of neurons (TRIP8b siRNA: 62.91 ± 1.47 ms; n = 15; control: 63.45 ± 2.14 ms; n = 18). These results are consistent with the view that a reduction of TRIP8b protein in CA1 pyramidal neurons in vivo causes a substantial and specific decrease in somatodendritic Ih. To examine how reduction in TRIP8b causes loss of Ih, we used immunohistochemistry to examine the expression and localization of HCN1 protein (the predominant HCN isoform in pyramidal neurons) following siRNA-mediated knockdown of TRIP8b. Viral expression of TRIP8b siRNA, but not control siRNA, caused a marked Onalespib solubility dmso redistribution of HCN1 in CA1 neurons, with a significant TSA HDAC price increase in channel staining in the somatic layer and proximal dendrites in SR, compared with uninfected neurons in the same slice (Figure 2A). High magnification z-series sections revealed the appearance of HCN1 in discrete puncta in the cytoplasm surrounding the

nucleus of neurons expressing the TRIP8b siRNA (Figure 2B). Such puncta were not observed in neighboring uninfected cells or in neurons infected with control siRNA. These results were quantified by measuring the intensity of HCN1 staining in the pyramidal layer (SP), proximal dendrites (SR) and distal dendrites (SLM) of infected (EGFP +) and uninfected (EGFP -) CA1 neurons (Figure 2C). There was no significant difference in HCN1 staining between uninfected cells and cells infected with control siRNA. However, cells infected with TRIP8b siRNA exhibited a 43% increase in HCN1 staining intensity in the soma and a 22% increase in the SR, compared with uninfected neighboring cells in the same slice (N = 6

mice, 12 injections sites for TRIP8b siRNA, N = 4 mice, 8 injections sites for control. Each data point is the average of 17 regions). In contrast, there was no detectable difference in staining intensity in SLM between regions infected with TRIP8b siRNA versus uninfected regions. The punctate Phosphoprotein phosphatase pattern of HCN1 suggests that the increase in staining in the soma and proximal dendrites results from channel accumulation in an intracellular compartment, consistent with the observed decrease in Ih (see Figures 1D–1H). These results imply that a reduction in TRIP8b expression produces a defect in HCN1 membrane trafficking. The lack of change in HCN1 in SLM may reflect technical limitations of the immunohistochemical approach to detect a redistribution of HCN1 from the membrane to cytoplasmic compartments in the thin distal dendrites.