Progressive dendritic HCN channelopathy during epileptogenesis in the rat pilocarpine model of epilepsy

Abstract

Ion channelopathy plays an important role in human epilepsy with a genetic cause and has been hypothesized to occur in epilepsy after acquired insults to the CNS as well. Acquired alterations of ion channel function occur after induction of status epilepticus (SE) in animal models of epilepsy, but it is unclear how they correlate with the onset of spontaneous seizures. We examined the properties of hyperpolarization-activated cation (HCN) channels in CA1 hippocampal pyramidal neurons in conjunction with video-EEG (VEEG) recordings to monitor the development of spontaneous seizures in the rat pilocarpine model of epilepsy. Our results showed that dendritic HCN channels were significantly downregulated at an acute time point 1 week postpilocarpine, with loss of channel expression and hyperpolarization of voltage-dependent activation. This downregulation progressively increased when epilepsy was established in the chronic period. Surprisingly, VEEG recordings during the acute period showed that a substantial fraction of animals were already experiencing recurrent seizures. Suppression of these seizures with phenobarbital reversed the change in the voltage dependence of I(h), the current produced by HCN channels, but did not affect the loss of HCN channel expression. These results suggest two mechanisms of HCN channel downregulation after SE, one dependent on and one independent of recurrent seizures. This early and progressive downregulation of dendritic HCN channel function increases neuronal excitability and may be associated with both the process of epileptogenesis and maintenance of the epileptic state.

Figure 1.

Chronology of procedures and characteristic EEG events. A, Timeline of procedures. B, EEG trace of an electrographic seizure corresponding to a Racine class 0 event associated with behavioral arrest lasting ∼13 s. Note the abrupt onset and end of the seizure, as well as generally uniform spiking amplitude three times that of baseline. Predominant spike frequency was 5 Hz. Trace labels indicate electrode position. C, EEG trace of an electrographic seizure corresponding to a class 5 event with a generalized convulsion ∼40 s in duration. The seizure discharge progressively grows in amplitude to five times over baseline, with frequency that increases up to 10 Hz and then slows as the seizure progresses. EEG amplitude is greatly attenuated after the event, indicative of postictal depression.

Figure 2.

Spontaneous seizures begin during the acute period 1 week postpilocarpine. A, Histogram showing the number of seizures per all animals monitored during the acute period. Both class 0–2 and class 3–5 seizures were evident in the first week postpilocarpine. Seizure activity was reduced in animals also receiving PB during the first week after pilocarpine compared with animals that received pilocarpine alone. B, Cumulative percentage of animals that experienced seizures during the first week postpilocarpine. Forty percent of pilocarpine-only-treated animals experienced seizures, whereas 38% of pilocarpine-treated animals that received PB in the first week postpilocarpine had seizures, although all of these seizures were of class 0–2. C, Number of seizures per animal monitored during the chronic period. At week three, class 0–2 events had declined in frequency, and by 5 weeks postpilocarpine, ictal activity had mostly shifted to class 3–5 events. D, Cumulative percentage of animals developing seizures during the chronic period. By 5 weeks postpilocarpine, 91% of animals had experienced a spontaneous seizure.

Figure 3.

Downregulation of I_h at 1 week postpilocarpine (acute period). A, Voltage-dependent activation of I_h in dendrites of CA1 hippocampal pyramidal neurons from naive animals (control; circles) was similar to that from age-matched sham-injected animals (acute sham; squares). B, I_h voltage-dependent activation from pilocarpine-treated animals in the acute period (acute pilo; solid squares) was hyperpolarized compared with sham-injected animals (acute sham; open squares; *p < 0.05). C, I_h density obtained from postpilocarpine animals in the acute period was significantly reduced compared with sham-injected animals (**p < 0.01). Representative current traces shown are from voltage commands of −152 and −157 mV in pyramidal neuron dendrites from sham-injected and pilocarpine-treated animals, respectively. The dendritic recording distances were 180 and 200 μm, respectively. D, The RMP in pyramidal neuron dendrites from pilocarpine-treated animals showed a hyperpolarized shift compared with the RMP of sham-injected animals (*p < 0.05), consistent with I_h downregulation. E, I_h activation time constants (fast and slow) measured near V_1/2 in pyramidal neuron dendrites from pilocarpine-treated animals were increased compared with those of sham-injected animals (*p < 0.05). I_h deactivation time constants were similar in sham-injected and pilocarpine-treated animals.

Figure 4.

Downregulation of I_h at 3–5 weeks postpilocarpine (chronic period). A, Voltage-dependent activation of I_h in dendrites of CA1 hippocampal pyramidal neurons from naive animals (control; circles) was similar to that from age-matched sham-injected animals (chronic sham; squares). B, I_h voltage-dependent activation from pilocarpine-treated animals in the chronic period (chronic pilo; solid squares) was hyperpolarized compared with sham-injected animals (chronic sham; open squares; *p < 0.05). C, I_h density obtained from pilocarpine-treated animals was significantly reduced compared with sham-injected animals (**p < 0.01). Current traces shown are from voltage commands of −150 and −153 mV in pyramidal neuron dendrites from sham-injected and pilocarpine-treated animals, respectively. The dendritic recording distance in both sham-injected and pilocarpine-treated animals was 160 μm. D, The RMP in pyramidal neuron dendrites from pilocarpine-treated animals was significantly hyperpolarized compared with that of sham-injected animals (*p < 0.05), consistent with I_h downregulation. E, Slow but not fast I_h activation time constants of pilocarpine-treated animals were increased compared with those of sham-injected animals (*p < 0.05). I_h deactivation time constants were similar in both sham-injected and pilocarpine-treated animals.

Figure 5.

Decreased HCN protein expression at acute and chronic periods after pilocarpine treatment. A, HCN1 and HCN2 protein expression in CA1 hippocampal tissue was decreased in pilocarpine-treated animals compared with sham-injected animals at the acute period (**p < 0.01 and ***p < 0.001). Representative blots of HCN1, HCN2, and actin protein expression are shown in each condition. B, HCN1 but not HCN2 protein expression remained reduced during the chronic period compared with age-matched sham-injected tissue (*p < 0.05). C, I_h density obtained from animals treated with PB during the acute period postpilocarpine (acute pilo+PB) was significantly reduced compared with control (**p < 0.01), similar to rats treated with pilocarpine alone (acute pilo; **p < 0.01, one-way ANOVA with Tukey's post hoc test). Unlike I_h density, V_1/2 in pilocarpine-treated animals with PB was unchanged from control and different from rats treated with pilocarpine alone (*p < 0.05, one-way ANOVA with Tukey's post hoc test). D, HCN1 and HCN2 protein expression in animals receiving PB during the acute period postpilocarpine was decreased compared with sham-injected animals treated with PB only (*p < 0.05). Loss of HCN1/2 expression was similar in pilocarpine-treated animals with PB as in those treated with pilocarpine alone.

Figure 6.

Increased IR and dendritic AP firing in the chronic period postpilocarpine. A, Representative current-clamp recordings in response to 100 pA hyperpolarizing current injection demonstrated increased IR in pyramidal neuron dendrites from pilocarpine-treated animals. All measurements were performed with resting potential held at −65 mV, and dendritic recording distances in naive and pilocarpine-treated animals were 160 and 180 μm, respectively. B, Representative traces show unchanged TS in dendritic current-clamp recordings at 160 and 170 μm in naive and pilocarpine-treated animals, respectively. All measurements were performed with resting potential held at −65 mV. C, AP firing with depolarizing current injections (500 ms) showed increased excitability in pyramidal neuron dendrites from pilocarpine-treated animals in the chronic period compared with naive animals (**p < 0.01). Representative traces show enhanced dendritic AP firing with current injection of 700 pA in pyramidal neuron dendrites from epileptic animals compared with that from naive animals. The dendritic recording distances in naive and pilocarpine-treated animals were 180 and 170 μm, respectively.