PMC

A, Protein extracts of subdissected hippocampal area CA1 were generated, then HCN1 and HCN2 were coimmunoprecipitated with TRIP8b using α-TRIP8b antibody. Proteins were separated by SDS-PAGE and immunoblotted with gp α-HCN1 or gp α-HCN2 antibody. B, Interaction between TRIP8b and h channels was quantitated by densitometry. Interaction between h channels and TRIP8b was not significantly different 1 d after SE (percent coimmunoprecipitated, 1 d control vs. 1 d SE; HCN1: 12.6±0.8% vs. 12.0±0.9%; HCN2: 8.8±0.3% vs. 8.8±0.7%, n=4; p>0.7). At 28 d after SE, the interaction between TRIP8b and HCN1 was dramatically reduced, whereas interaction with HCN2 was unchanged (28 d control vs. 28 d SE; HCN1: 13.4±0.4% vs. 3.2±0.9 %, n=4, **p<0.05; HCN2: 9.9±0.5% vs. 9.1±1.0%, n=4, p>0.7).

A, Protein extracts from CA1 of age-matched control and SE animals were separated by SDS-PAGE, and blotted with α-HCN1, α-HCN2. Duplicate blots were labeled with α-tubulin and β-tubulin III (neuron-specific isoform) for loading controls. B, Intensity of HCN1 and HCN2 bands were quantified and normalized to the intensity of tubulin bands. No significant change in HCN1 protein expression was detected at 1 d after SE (SE, 103.5±5.5%). Slightly decreased HCN2 protein expression was observed at 1 d after SE (87.0±2.3%). C, Surface-expressed proteins in CA1 area hippocampus were biotinylated and precipitated by immobilized neutravidin. Proteins were separated by SDS-PAGE, and blotted with α-HCN1, α-HCN2, α-GluR1 and α-tubulin (as a non-surface control protein). Supernatant represents non-surface fraction of proteins. 30% input was loaded. D–E, Intensity of HCN1, HCN2 (D) and GluR1 (E) bands were quantified. A significant increase in surface expressed HCN1 proteins, but not HCN2 or GluR1 proteins, was detected at 1 d after SE. See section for detailed values. Error bars represent the SEM (n=6, **p<0.05).

A, Protein extracts from CA1 of age-matched control and SE animals were separated by SDS-PAGE, and blotted with α-HCN1, α-HCN2, and α-Kv4.2. Duplicate blots were labeled with α-tubulin and β-tubulin III (a neuron-specific isoform) for loading controls. B, Intensity of HCN1, HCN2 and Kv4.2 bands was quantified and normalized to the intensity of β-tubulin III bands. No significant change in HCN1 or HCN2 protein expression was detected 28 d after SE. Expression of Kv4.2 was decreased by 47.6±3.6% at 28 days after SE. Error bars represent the SEM (n=6, **p<0.05). C, Surface expressed proteins in CA1 area hippocampus were biotinylated and precipitated by immobilized neutravidin. Proteins were separated by SDS-PAGE, and blotted with α-HCN1, α-HCN2, α-GluR1 and α-tubulin (as an internalization control). Supernatant represents non-surface fraction of proteins. 30% input was loaded. D–E, Intensity of HCN1, HCN2 (D) and GluR1 (E) bands were quantified. A significant decrease in surface expressed HCN1 and HCN2 proteins, but not GluR1 proteins was detected at 28 d after SE. See section for detailed values. Error bars represent the SEM (n=3, **p<0.05).

A, representative traces showing the voltage responses measured at either the soma or distal dendrite from control (black) or 1–2 d after SE (red) CA1 pyramidal neurons. B, Summary graph showing that 1–2 d after SE input resistance measured at the soma was significantly reduced compared to control. Note that in control CA1 pyramidal neurons, input resistance was significantly lower in the distal dendrite compared to the soma. This difference is not present in CA1 pyramidal neurons 1–2 d after SE. **p< 0.01 compared to control; #p < 0.05 compared to soma. C, representative traces showing the voltage response to a hyperpolarizing current injection measured at either the soma or distal dendrite from CA1 pyramidal neurons in control (black) or 1–2 d after SE (red). D, Summary graph showing that 1–2 d after SE voltage sag measured at the soma was significantly increased compared to control. Note that in control CA1 pyramidal neurons, voltage sag was significantly higher in the distal dendrite compared to the soma. This difference is not present in CA1 pyramidal neurons 28–30 d after SE. *p< 0.05 compared to control; #p< 0.05 compared to soma. E, representative traces showing the voltage response to train of αEPSCs injected and recorded at either the soma or distal dendrite of control (black) or 1–2 d after SE (red). F, Summary graph showing that 1–2 d after SE temporal summation measured at the soma was significantly reduced compared to control. Note that in control CA1 pyramidal neurons, temporal summation was significantly lower in the distal dendrite compared to the soma. This difference is not present in CA1 pyramidal neurons 1–2 d after SE. *p< 0.05 compared to control; #p< 0.05 compared to soma. G, Representative somatic and dendritic impedance amplitude profile from a control (black) and 1–2 day post SE neuron (red). The dashed lines indicate the resonance frequency (f_R). Note the rightward shift in the SE neuron. H, Summary graph showing that 1–2 d after SE f_R measured at the soma was significantly increased compared to control. Note that in control CA1 pyramidal neurons, f_R was significantly higher in the distal dendrite compared to the soma. This difference is not present in CA1 pyramidal neurons 28–30 d after SE. *p< 0.05 compared to control; #p< 0.05 compared to soma.

A, representative traces showing the voltage responses measured at either the soma or distal dendrite from control (black) or 28–30 d after SE (red) CA1 pyramidal neurons. B, Summary graph showing that 28–30 d after SE input resistance measured at the distal dendrite was significantly reduced compared to control. Note that difference in input resistance between soma and dendrite is not present in CA1 pyramidal neurons 28–30 d after SE. *p< 0.05 compared to control; #p< 0.05 compared to soma. C, representative traces showing the voltage response to a hyperpolarizing current injection measured at either the soma or distal dendrite from CA1 pyramidal neurons in control (black) or 28–30 d after SE (red). D, Summary graph showing that 28–30 d after SE voltage sag measured at the distal dendrite was significantly reduced compared to control. Note that difference in voltage sag between soma and dendrites is not present in CA1 pyramidal neurons 28–30 d after SE. *p< 0.05 compared to control; ###, p< 0.001 compared to soma. E, representative traces showing the voltage response to 20 Hz train of 5 αEPSCs injected and recorded at either the soma or distal dendrite of control (black) or 28–30 d after SE (red). F, Summary graph showing that 28–30 d after SE temporal summation measured at the distal dendrite was significantly increased compared to control. Note that difference in temporal summation between soma and dendrites is not present in CA1 pyramidal neurons 28–30 d after SE. *p< 0.05 compared to control; #p< 0.05 compared to soma. G, Representative somatic and dendritic impedance amplitude profile from a control (black) and 28–30 day post SE neuron (red). The dashed lines indicate the resonance frequency (f_R). Note the rightward shift in the soma and leftward shift in the dendrite of the SE neuron. H, Summary graph showing that 28–30 d after SE f_R measured at the soma was significantly increased and f_R measured at the distal dendrite was significantly decreased compared to control. Note that difference in f_R between soma and dendrites is not present in CA1 pyramidal neurons 28–30 d after SE. *p< 0.05 compared to control; **p< 0.01 compared to control; ###p < 0.001 compared to soma.

A–B, Sagittal sections of control brain [A and B (a-b)] and brain fixed 28 d after SE (28 d SE) [A and B (c–d)] were immunolabeled with gp α-HCN1 or gp α-HCN2 and ms α-PSD95 and visualized with α-gp-alexa488 (left panels, green) and α-ms-cy3 (right panels, red), respectively, for fluorescence staining. HCN1 (A–a, arrow), and HCN2 (B–a, arrow) are both enriched in distal dendritic arborizations within the SLM. The distal dendritic enrichment of HCN1 is lost (A–c, asterisk), whereas high HCN1 immunoreactivity appears in the stratum pyramidale (SP) layer (A–c, arrowheads) of area CA1 of the 28 d SE brains. HCN2 remains enriched in SLM of area CA1 of 28 d SE brain (B–c, arrow), but novel somatic staining is observed (B–c, arrowheads). Note that the distribution pattern of PSD95 is unaltered (A–b, A–d, B–b, B–d), consistent with minimal neuronal damage. Insets (A–b, A–d) are 100x (with 2x digital zoom) images of SLM dendritic fields showing a punctate staining pattern of PSD95, that is similar in 28 d SE and control dendrites of hippocampal area CA1. C–E, The relative intensity of HCN1 (C), HCN2 (D), and PSD95 (E) immunoreactivity from stratum oriens (SO) to SLM layer was quantified and graphed. Note the increased HCN1 and HCN2 intensity in soma and decreased HCN1 but not HCN2 intensity in SLM layer 28 d after SE (**p< 0.05, ***p<0.001). Distribution of PSD95 immunoreactivity at 28 d after SE was not significantly different from control. Data points are mean ±SEM of 5 different animals (control) and 10 different animals (28 d SE). Scale bars: 100µm (5µm inset). See section for detailed description of the analysis method.

A–B, Rabbit α-TRIP8b antibody is specific in biochemical and immunohistochemical assays. A, Protein extracts from Cos-7 cells transfected with a TRIP8b-GFP-expressing plasmid or rat brain were separated by SDS-PAGE and blotted with α-TRIP8b antibody. Our custom antibody detected a ~110 kD band in transfected Cos-7 cells and band of ~78 kD in rat brain, consistent with the predicted size of the GFP-fusion and native protein, respectively. B, Parasagittal sections of rat brain were immunolabeled with α-TRIP8b antibody. Note the strong immunoreactivity in SLM of hippocampal area CA1 (arrow) and layer I-II of cortex (arrowheads). Scale bars: 200µm. (SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; SLM: stratum lacunosum moleculare). C, TRIP8b interacts with h channel subunits in the rat brain. Membrane fractions of rat brain extracts were generated and coimmunoprecipitation performed using antibodies against h channel subunits (Top, gp α-HCN1 antibody, gp α-HCN2 antibody, gp α-HCN4 antibody) or α-TRIP8b antibody (bottom). Immunoprecipitation using preimmune serum (PI) served as a negative control. D, Protein expression levels of TRIP8b were not altered in the CA1 hippocampus of SE animals. (Top) Hippocampal area CA1 was sub-dissected and membrane extract was generated. Proteins were separated by SDS-PAGE and immunoblotted using α-TRIP8b antibody or ms α-α-tubulin antibody. Protein expression levels of TRIP8b were quantitated by densitometry and normalized with the level of α-tubulin (as a loading control). (Bottom) Graphing the relative density of TRIP8b of SE and non-SE animals shows that TRIP8b protein expression levels were not significantly changed at either 1 d (1 d SE; 104.4±10.8% of 1 d control, n=4; p>0.7) or 28 d (28 d SE; 95.7±3.7% of 28 d control, n=4; p>0.7) after SE. E, Distribution of TRIP8b in CA1 hippocampus was unaltered after SE. 50µm parasagittal sections of control (left) and 28d SE (right) brains were immunolabeled with α-TRIP8b antibody and visualized with DAB staining. TRIP8b distribution was not changed in 28 d as compared to control CA1.