Downregulation of dendritic HCN channel gating in epilepsy is mediated by altered phosphorylation signaling

Abstract

The onset of spontaneous seizures in the pilocarpine model of epilepsy causes a hyperpolarized shift in the voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated current (Ih) in CA1 hippocampal pyramidal neuron dendrites, contributing to neuronal hyperexcitability and possibly to epileptogenesis. However, the specific mechanisms by which spontaneous seizures cause downregulation of HCN channel gating are yet unknown. We asked whether the seizure-dependent downregulation of HCN channel gating was due to altered phosphorylation signaling mediated by the phosphatase calcineurin (CaN) or the kinase p38 mitogen-activated protein kinase (p38 MAPK). We first found that CaN inhibition upregulated HCN channel gating and reduced neuronal excitability under normal conditions, showing that CaN is a strong modulator of HCN channels. We then found that an in vitro model of seizures (1 h in 0 Mg2+ and 50 microM bicuculline at 35-37 degrees C) reproduced the HCN channel gating change seen in vivo. Pharmacological inhibition of CaN or activation of p38 MAPK partially reversed the in vitro seizure-induced hyperpolarized shift in HCN channel gating, and the shift was fully reversed by the combination of CaN inhibition and p38 MAPK activation. We then demonstrated enhanced CaN activity as well as reduced p38 MAPK activity in vivo in the CA1 hippocampal area of chronically epileptic animals. Pharmacological reversal of these phosphorylation changes restored HCN channel gating downregulation and neuronal hyperexcitability in epileptic tissue to control levels. Together, these results suggest that alteration of two different phosphorylation pathways in epilepsy contributes to the downregulation of HCN channel gating, which consequently produces neuronal hyperexcitability and thus may be a target for novel antiepileptic therapies.

Figure 1.

CaN inhibition upregulates I_h activation in dendrites of CA1 hippocampal pyramidal neurons from naive animals. A, I_h voltage-dependent activation after treatment with CaN inhibitor FK506 (open circles) was depolarized compared with control (solid circles), while treatment with a nonspecific CaN activator, A23187, produced a hyperpolarized shift (open triangles). Representative current traces shown are in response to voltage commands of −88, −90, and −92 mV in pyramidal neuron dendrites from control, FK506-treated, and A23187-treated slices, respectively. The dendritic recording distances were 220, 200, and 180 μm, respectively. The approximate dendritic recording location is shown in the representation of a CA1 hippocampal pyramidal neuron. B, I_h amplitudes at maximal activation, obtained after CaN inhibition by FK506 or activation by A23187, were not significantly different from control. Representative current traces shown are in response to voltage commands of −155, −150, and −154 mV in pyramidal neuron dendrites from control, FK506-treated, and A23187-treated slices, respectively. The dendritic recording distances were 180, 200, and 180 μm from the soma, respectively.

Figure 2.

CaN inhibition decreases neuronal excitability in CA1 hippocampal pyramidal neurons from naive animals. All measurements were performed with resting potential held at −65 mV. A, Current-clamp recording from pyramidal neuron dendrites showed that the mean IR from FK506-treated neurons was significantly reduced compared with control (**p < 0.01). Representative traces in response to 100 pA hyperpolarizing current injection are shown. The dendritic recording distances in control and FK506-treated neurons were 180 and 170 μm, respectively. IR from control and FK506-treated neurons in the presence of HCN channel blocker, ZD7288 (ZD), were increased and similar to each other, implying that the reduction in IR by FK506 was HCN channel dependent. B, TS of five 20 Hz α-function current injections in pyramidal neuron dendrites was also significantly reduced in FK506-treated slices compared with control (**p < 0.01). Representative traces show dendritic current-clamp recordings at 160 μm in both control and FK506-treated neurons. TS from control and FK506-treated neurons in the presence of ZD7288 were increased and similar to each other, implying that the reduction in TS by FK506 was HCN channel dependent. C, AP firing from dendritic depolarizing current injections (500 ms) showed decreased excitability in pyramidal neuron dendrites from FK506-treated neurons compared with control (*p < 0.05). Representative traces show dendritic backpropagating AP firing from dendritic depolarizing current injection (500 pA) at recording distances in control and FK506-treated neurons of 170 and 160 μm, respectively.

Figure 3.

In vitro model replicates seizure-dependent downregulation of HCN channel gating. A, Representative trace shows a spontaneous SLE obtained with an extracellular recording in stratum pyramidale from the CA1 hippocampal area, in the presence of bath solution with 0 Mg²⁺ and 50 μm bicuculline at 35–37°C. Note prolonged inward current sink and superimposed brief spike events. B, I_h voltage-dependent activation following 1 h of in vitro seizure, control, and sham (1 h at 35–37°C bath temperature only) conditions. The in vitro seizure condition caused a significant hyperpolarization of I_h activation relative to control or sham conditions. C, I_h amplitude at maximal activation was similar across conditions.

Figure 4.

Increased neuronal excitability following in vitro SLEs. All measurements were performed with resting potential held at −65 mV. A, Dendritic current-clamp recordings in pyramidal neuron dendrites showed increased IR following in vitro SLEs compared with control. Representative current-clamp recordings in response to 300 pA hyperpolarizing current injection are shown. Dendritic recording distances in control and seizure conditions were 210 and 188 μm, respectively. B, TS of five 20 Hz α-function current injections in pyramidal neuron dendrites following the seizure-provoking conditions (thick trace) was not significantly different from TS in pyramidal neuron dendrites under control conditions (thin trace). Representative traces show dendritic current-clamp recordings at 240 and 188 μm, respectively. C, AP firing with depolarizing current injections (500 ms) showed increased excitability in pyramidal neuron dendrites following in vitro SLEs (**p < 0.01, *p < 0.05). Representative traces show increased dendritic backpropagating AP firing from dendritic depolarizing current injection (500 pA) in seizure conditions compared with control. The dendritic recording distances in control and seizure conditions were 188 and 220 μm, respectively.

Figure 5.

Downregulation of HCN channel gating following in vitro SLEs is partially reversed by p38 MAPK activation or CaN inhibition. A, SLEs (Sz) caused a hyperpolarized shift in I_h voltage-dependent activation. Application of CaN inhibitor FK506 following in vitro SLEs (Sz+FK506) reversed the hyperpolarized shift in I_h voltage-dependent activation to control levels. B, Application of p38 MAPK activator anisomycin following in vitro SLEs (Sz+ani) reversed the hyperpolarized shift in I_h voltage-dependent activation to control levels. C, Coapplication of FK506 and anisomycin following in vitro SLEs (Sz+ani+FK506) depolarized I_h voltage-dependent activation compared with both control and seizure conditions.

Figure 6.

CaN activity is upregulated and p38 MAPK activity is downregulated in the CA1 hippocampal area from epileptic animals. A, Application of FK506 (FK) to hippocampal slices from naive animals increased phosphorylation levels of DARPP-32 (p-D32) compared with untreated slices (control) (*p < 0.05), showing p-DARPP-32 levels are sensitive to dephosphorylation by CaN. p-DARPP-32 levels in CA1 hippocampal tissue were decreased in epileptic animals compared with control, implying increased CaN activity (*p < 0.05). The ratio of p-DARPP-32 to total DARPP-32 was further decreased (**p < 0.01). All p-DARPP-32 and total DARPP-32 levels were normalized to β-tubulin III (βT3) protein levels. Representative Western blots of p-DARPP-32, total DARPP-32 and βT3 protein levels are shown in each condition. Statistical significance compared with control levels. B, Anisomycin (ani) treatment of hippocampal slices from naive animals caused an increase in phosphorylated p38 MAPK (p-p38) levels in the CA1 hippocampal area compared with control (*p < 0.05). p-p38 MAPK levels in CA1 hippocampal tissue were decreased in epileptic animals compared with control (*p < 0.05), implying decreased p38 MAPK activity. The ratio of p-p38 MAPK to total p38 MAPK was further decreased (**p < 0.01). All p-p38 MAPK and total p38 MAPK levels were normalized to βT3 protein levels. Representative Western blots of p-p38 MAPK, total p38 MAPK and βT3 protein levels are shown in each condition. Statistical significance compared with control levels. C, Representative Western blots show that p-p38 MAPK levels following anisomycin treatment of hippocampal slices from both naive and epileptic animals are similar to each other in the CA1 hippocampal area, implying that activation of p38 MAPK cascade by anisomycin is intact in epileptic animals.

Figure 7.

CaN inhibition and p38 MAPK activation reverse downregulated HCN channel gating and neuronal hyperexcitability in epileptic animals. A, Application of either CaN inhibitor FK506 (epileptic+FK506) or p38 MAPK activator anisomycin (epileptic+ani) partially reversed the hyperpolarized shift in I_h voltage-dependent activation toward control levels in dendrites of CA1 hippocampal pyramidal neurons from chronically epileptic animals. Coapplication of anisomycin and FK506 (epileptic+ani+FK506) restored HCN channel gating nearly to control levels. B, Current-clamp recordings in pyramidal neuron dendrites from chronically epileptic animals showed decreased IR following coapplication of FK506 and anisomycin compared with untreated epileptic tissue. Representative dendritic current-clamp recordings in response to 100 pA hyperpolarizing current injection are shown. All recordings in B–D were performed with resting potential held at −65 mV, and dendritic recording distances in representative traces under drug-treated and untreated epileptic conditions were 180 and 170 μm, respectively. C, TS of five 20 Hz α-EPSP current injections in pyramidal neuron dendrites in epileptic tissue after treatment with anisomycin and FK506 was not significantly different from TS in pyramidal neuron dendrites under untreated epileptic conditions. D, AP firing from depolarizing dendritic current injections (500 ms) in pyramidal neurons from epileptic tissue following coapplication of FK506 and anisomycin showed significantly decreased excitability (*p < 0.05) that was similar to control levels. Representative traces show decreased dendritic backpropagating AP firing from dendritic current injection (700 pA) in drug-treated epileptic conditions compared with untreated epileptic conditions.