Acquired Dendritic Channelopathy in Temporal Lobe Epilepsy

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Science  23 Jul 2004:
Vol. 305, Issue 5683, pp. 532-535
DOI: 10.1126/science.1097065


Inherited channelopathies are at the origin of many neurological disorders. Here we report a form of channelopathy that is acquired in experimental temporal lobe epilepsy (TLE), the most common form of epilepsy in adults. The excitability of CA1 pyramidal neuron dendrites was increased in TLE because of decreased availability of A-type potassium ion channels due to transcriptional (loss of channels) and posttranslational (increased channel phosphorylation by extracellular signal-regulated kinase) mechanisms. Kinase inhibition partly reversed dendritic excitability to control levels. Such acquired channelopathy is likely to amplify neuronal activity and may contribute to the initiation and/or propagation of seizures in TLE.

Epileptic discharges correspond to highly synchronized and high-frequency activity of neurons. An augmented neuronal input-output relation is thought to be at the core of this increased excitability (1). Changes in the input-output relation of neurons may be caused by plastic changes on the synaptic level as reported in human and experimental epilepsy (24). Far less is known about changes of ion channels in the neuronal membrane, even though they control synaptic integration and intrinsic neuronal excitability. A number of rare genetic epilepsy syndromes are linked to mutations of ion channels (5), and acquired changes of somatic ion channels can occur after seizures (6, 7). The fate of dendritic ion channels, however, has remained an enigma. As the main site for synaptic integration, neuronal dendrites could play a key role in seizure initiation and propagation (810). Dendrites contain a very high density of ion channels that can be targeted by antiepileptic drugs (11, 12). Through their action on back-propagating action potentials (b-APs) (13, 14), A-type K+ channels are crucial modulators of information processing and synaptic plasticity in the dendrites. A reduction in A-type K+ channel activity increases b-AP amplitude and promotes burst firing in a location-dependent manner (13, 15). One useful measure of availability of A-type K+ channels, as well as of dendritic excitability, is thus the amplitude of the b-AP (16, 17).

With the use of whole-cell dendritic recordings in hippocampal CA1 pyramidal cells (18), we tested whether back-propagation was modified in pilocarpine-treated rats, an animal model for epilepsy that shares many pathophysiological similarities with TLE in humans (19). The amplitude of b-APs decreased with distance from the soma in sham (Fig. 1, A and B) (n = 41) as in control animals (16, 20). In the proximal part of the dendritic tree (<200 μm), back-propagation is mostly active and b-APs have a large amplitude (20). In the distal part (>250 μm), back-propagation is mostly passive and b-APs have a small amplitude. In epileptic animals (n = 62), b-AP amplitudes were significantly larger than those in sham (n = 50) at distances greater than 250 μm from the soma (Fig. 1, A and B). Other features of back-propagation, including the AP initiation site and resting membrane potentials (RMPs), were not modified in epileptic animals (figs. S1 and S2).

Fig. 1.

Large-amplitude b-APs invade the distal part of the dendritic tree in TLE because of a modification of the threshold that determines the transition from passive to active back-propagation. (A) b-AP amplitude was increased in the epileptic animal. Asterisks indicate a stimulus artifact. (B) Distribution of b-AP amplitudes measured at RMP as a function of distance from the soma (25 μm bins) in sham (n = 50) and epileptic (n = 62) animals. Whereas b-AP amplitude decreased progressively in sham animals, b-AP amplitudes remained large and constant at distances > 200 μm in epileptic animals (45 ± 2 mV, n = 53, and nostatistical difference between any bins). The difference with sham animals became significant >250 μm (double asterisks, P < 0.01; single asterisk, P < 0.05). (C) Variation of b-AP amplitude as a function of membrane potential [black dots, sham; red squares, pilo (epileptic); blue dotted trace, RMP of –59 mV for sham and –64 mV for pilo]. Arrows indicate θ, the threshold for the transition between passive and active back-propagation. Because θsham > RMPsham whereas θepileptic < RMPpilo, back-propagation at RMP was passive in the sham animal and active in the pilo animal. Insets display b-APs measured at various membrane potentials from hyperpolarized (–90 mV, bottom traces) to depolarized (–30 mV, top traces) potentials. As the membrane was depolarized, there was a sudden increase in amplitude (blue trace). Red trace, b-AP at RMP. (D) Difference between θ and RMP at each recording site as a function of the distance from the soma (black squares, sham, n = 29; red dots, pilo, n = 28). Above the blue line, where θx = RMPx at any distance x from the soma, b-APs have a small amplitude. Below the blue line, θx < RMPx and b-APs have a large amplitude. In the distal part of the dendritic tree (>250 μm), most values are above the blue line in the sham animals. By contrast, they are below the blue line in the epileptic animals.

What could be the mechanisms for the invasion of the dendritic tree by large-amplitude b-APs in TLE? Although back-propagation is decremental in control tissue, changing the availability of Na+ and/or K+ channels can switch back-propagation from passive to active (21, 22). The switch occurs when the membrane potential crosses a threshold potential θx (Fig. 2C), which takes into account Na+ and/or K+ channel availability at any given distance x from the soma (20). To measure θx, current pulses were injected to the dendrites to achieve various membrane potentials (Vm). At hyperpolarized levels, b-AP amplitude was small (Fig. 1C). As the membrane was further depolarized, there was a sudden increase in amplitude until the peak of the b-AP reached a maximum value (Fig. 1C). The transition threshold θx is the membrane potential where the second derivative of the b-AP as a function of Vm is maximum (20). In the example shown in Fig. 1C (320 μm from the soma), b-AP had a small amplitude at RMP in the sham animal because θ320 > RMP (blue dots). In contrast, at the same distance in the epileptic animal, θ320 < RMP and b-AP had a large amplitude at RMP. The distribution of θx along the dendritic tree in sham animals (Fig. 1D) (n = 29) was similar to that found in controls (20), i.e., θx > RMP for x > 260 μm and θx < RMP for x < 260 μm. However, the distribution of θx was shifted toward more negative values in epileptic animals (Fig. 1D) (n = 28), enabling active b-APs in TLE in the dendritic region that normally experienced passive b-APs in sham animals.

Fig. 2.

The control of back-propagation by A-type K+ channels was decreased in TLE. (A) Application of the K+ channel blocker 4-AP (5 mM) increased b-AP amplitude in sham and epileptic animals. Typical examples are displayed at the top. The amplitude at the peak of the b-AP was similar in sham (63 ± 6 mV, n = 10, black squares) and epileptic (61 ± 4 mV, n = 17, red dots) animals (P = 0.83 between sham and epileptic) in the presence of 4-AP (bottom). (B) The 4-AP–induced increase in amplitude was statistically greater in sham than in epileptic animals (double asterisks, P < 0.0003; single asterisk, P < 0.01; left). The presence of 4-AP shifted θ toward more negative membrane potentials, but the shift was statistically greater in sham than in epileptic (double asterisks, P < 0.0007; single asterisk, P < 0.01; right). These results indicate that fewer A-type K+ channels are available in epileptic animals in the absence of 4-AP. (C) Application of the PKC activator PDA (10 μM) increased b-AP amplitude by decreasing the availability of A-type K+ channels. The increase in amplitude was statistically smaller in epileptic (n = 5) as compared to sham (n = 5) animals (double asterisks, P < 0.01; single asterisk, P < 0.05; right), suggesting an increased basal level of phosphorylation of A-type K+ channels in epileptic animals.

What could be the mechanisms responsible for the modification of θ? Because b-AP amplitude and θ are very sensitive to A-type K+ channel availability (17), a decrease in A-type K+ function could result in increased back-propagation and a shift of θ (20). Although b-AP amplitude is smaller in sham than in epileptic animals at similar dendritic locations, blocking A-type K+ channels with 4-AP (5 mM) had a larger effect in sham (Fig. 2, A and B) (220 ± 61% of control for n = 10) than in epileptic (71 ± 24% for n = 17) animals. Blocking A-type K+ channels induced a shift of θ toward more negative values, which was significantly larger in epileptic (15.5 ± 2.5 mV for n = 5) than in sham (6.2 ± 3.8 mV for n = 5) animals (Fig. 2B). These results suggest that the control of b-AP amplitude by K+ channel is decreased in TLE. Interestingly, the relation between b-AP max dV/dt and b-AP amplitude in the presence of 4-AP was similar in sham (slope of 1.0, r = 0.8, and n = 10) and epileptic (slope of 0.9, r = 0.9, and n = 17) animals, suggesting that ion channels that control the rising phase of b-APs (e.g., Na+ channels) are not modified in TLE. Collectively, these results indicate that a decreased availability of 4-AP–sensitive K+ channels most likely accounts for the increased dendritic excitability found in TLE. The reduced availability of these channels may be caused by reduction in A-type K+ channel density, altered modulation of the channel protein by phosphorylation, or both.

A-type K+ channels in CA1 pyramidal cell dendrites are modulated by protein kinase C (PKC). This kinase causes phosphorylation of Kv4.2 channels (17) and shifts the activation curve of A-type K+ channels toward more positive values, thereby increasing b-AP amplitude (17, 23). If phosphorylation of A-type K+ channels is increased in TLE, PKC activation should have smaller effects on back-propagation in TLE as compared to sham. Indeed, application of 10 μM phorbol diacetate (PDA), a PKC activator, had a smaller effect in epileptic (15 ± 3% for n = 5) compared to sham (57 ± 15% for n = 5) animals (Fig. 2C). We next inhibited phosphorylation by application of the broad spectrum protein kinase inhibitor H7. In sham animals, bath application of 300 μM H7 decreased b-AP amplitude by 12 ± 4% (Fig. 3, A and C) (n = 5) and caused a shift of θ toward more positive values (Fig. 3, A and C) (7.4 ± 0.4 mV for n = 5), consistent with the basal level of phosphorylation of K+ channels reported previously (17). In epileptic animals, H7 decreased the amplitude of b-APs by 24 ± 7% (Fig. 3, B and C) (n = 8). The θ was shifted toward more positive values than in sham animals (Fig. 3, B and C) (14.5 ± 3.6 mV for n = 5). In epileptic animals, the effect of H7 on b-AP amplitude depended upon the initial value of θ before H7 application. If the difference between θ and RMP before H7 was greater than 14.5 mV (on average), back-propagation remained active (left of θ) (Fig. 3B, right). If the difference was smaller than 14.5 mV, back-propagation became weak (Fig. 3B, left). This suggests that additional modifications may be at play, such as a reduction in A-type K+ channel density.

Fig. 3.

Preventing A-type K+ channel phosphorylation partly restores back-propagation in TLE. (A) Application of H7 (300 μM) slightly decreased b-AP amplitude and shifted the transition threshold (arrows) toward more positive values in sham animals, consistent with a small basal level of phosphorylation of A-type K+ channels. (B) H7 had either a large or a small effect on b-AP amplitude, according to the initial value of θ (before H7 application). If H7 shifted θ at a potential greater than RMP, b-AP was considerably reduced (left) when back-propagation was switched back from active to passive. If the H7-induced shift left θ more negative than RMP, b-AP was slightly reduced (right), as in sham animals, and back-propagation remained active. (C) H7 significantly decreased b-AP amplitude in sham (n = 5) and epileptic (n = 8) animals (single asterisk, P < 0.05; top), but the difference in the decrease between sham and epileptic animals was not statistically significant (P = 0.07). The H7-induced shift of θ toward more positive values was significantly greater in epileptic than in sham animals (double asterisks, P < 0.002; single asterisk, P < 0.05; bottom), consistent with an increased level of phosphorylation of A-type K+ channels in TLE.

Kv4.2 channels are one of the final effectors of extracellular signal-regulated kinase (ERK), which is phosphorylated and activated downstream of PKC (17). We applied U0126, an inhibitor of mitogen-activated protein kinase (MEK), to block ERK activation and prevent the phosphorylation of Kv4.2 channels (17). Bath application of 10 μM U0126 for 30 min decreased b-AP amplitudes by 21.8 ± 5.9% (n = 7) and by 41.2 ± 10.2% (n = 11) in sham and epileptic animals, respectively (Fig. 4). The θ was shifted toward more positive values by 5.1 ± 2.0 mV (n = 7) and 19.1 ± 5.4 mV (n = 11) in sham and epileptic animals, respectively (Fig. 4). Targeting kinase inhibition more selectively in the ERK pathway thus resulted in a reduction of dendritic excitability comparable to that obtained with the kinase inhibitor H7 (fig. S3).

Fig. 4.

ERK inhibition partly restores back-propagation in TLE. Application of the MEK inhibitor U0126 (10 μM) shifted the transition threshold to more positive values and decreased b-AP amplitudes. The shift of θ was greater in epileptic (n = 11) than in sham (n = 7) animals (double asterisks, P < 0.0001; single asterisk, P < 0.05).

We next directly examined the expression of Kv4.2 channels as well as their phosphorylation status. With the use of an antibody that selectively recognizes Kv4.2 when phosphorylated at the ERK sites (24), we found a significant increase in the percent of ERK phosphorylated-Kv4.2 (fig. S4A) and a significant decrease in total Kv4.2 channel protein (fig. S4B) in epileptic as compared to sham animals. With the use of quantitative real-time reverse transcription polymerase chain reaction, we found a significant reduction in Kv4.2 mRNA amounts in epileptic compared to control animals (fig. S4, C and D).

Our results provide evidence that dendritic excitability in CA1 pyramidal neurons is increased in an animal model of TLE during the chronic phase of epilepsy. A decrease in availability of A-type K+ channels largely contributes to this phenomenon because of a partial loss of Kv4.2 K+ channels and increased ERK phosphorylation of the remaining ones, consistent with the increased amounts of PKC and ERK activation after seizures (25, 26). Other channel types may also contribute to enhanced dendritic excitability. A transcriptional up-regulation of T-type Ca2+ channels has been reported in the soma of CA1 pyramidal cells in epileptic animals (7). Although our results suggest that the reported increased dendritic excitability is not due to a change of availability of transient Na+ channels, a contribution of persistent Na+ channels cannot be ruled out (27). Modifications of other somatic channels such as Ih have been reported in seizure models (6), but these channels do not appear to contribute much to b-AP amplitude (28).

Down-regulation of A-type K+ channel function in pyramidal neuron dendrites will increase neuronal excitability in multiple ways. The increased amplitude of back-propagating APs will lead to greater calcium influx through voltage-gated channels, boosting dendritic excitatory postsynaptic potentials (EPSPs), particularly those arriving distally from the entorhinal cortex (3), and increasing EPSP-spike coupling (29) through inactivation of dendritic K+ channels. This increase in excitability is likely to lower the threshold for seizure initiation in the hippocampus, a frequent site of focal seizure onset in human temporal lobe epilepsy.

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