Antiepileptic action of c-Jun N-terminal kinase (JNK) inhibition in an animal model of temporal lobe epilepsy.

Several phosphorylation signaling pathways have been implicated in the pathogenesis of epilepsy arising from both genetic causes and acquired insults to the brain. Identification of dysfunctional signaling pathways in epilepsy may provide novel targets for antiepileptic therapies. We previously described a deficit in phosphorylation signaling mediated by p38 mitogen-activated protein kinase (p38 MAPK) that occurs in an animal model of temporal lobe epilepsy, and that produces neuronal hyperexcitability measured in vitro. We asked whether in vivo pharmacological manipulation of p38 MAPK activity would influence seizure frequency in chronically epileptic animals. Administration of a p38 MAPK inhibitor, SB203580, markedly worsened spontaneous seizure frequency, consistent with prior in vitro results. However, anisomycin, a non-specific p38 MAPK activator, significantly increased seizure frequency. We hypothesized that this unexpected result was due to activation of a related MAPK, c-Jun N-terminal kinase (JNK). Administration of JNK inhibitor SP600125 significantly decreased seizure frequency in a dose-dependent manner without causing overt behavioral abnormalities. Biochemical analysis showed increased JNK expression and activity in untreated epileptic animals. These results show for the first time that JNK is hyperactivated in an animal model of epilepsy, and that phosphorylation signaling mediated by JNK may represent a novel antiepileptic target.

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

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.

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

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.

Reversed somatodendritic I(h) gradient in a class of rat hippocampal neurons with pyramidal morphology.

In CA1 and neocortical pyramidal neurons, I(h) is present primarily in the dendrites. We asked if all neurons of a pyramidal morphology have a similar density of I(h). We characterized a novel class of hippocampal neurons with pyramidal morphology found in the stratum radiatum, which we termed the 'pyramidal-like principal' (PLP) neuron. Morphological similarities to pyramidal neurons were verified by filling the neurons with biocytin. PLPs did not stain for markers associated with interneurons, and projected to both the septum and olfactory bulb. By using cell-attached patch-clamp recordings, we found that these neurons expressed a high density of I(h) in the soma that declined to a lower density in the dendrites, a pattern that is reversed compared to pyramidal neurons. The voltage-dependent activation and activation time constants of I(h) in the PLPs were similar to pyramidal neurons. Whole-cell patch-clamp recordings from the soma and dendrites of PLP neurons showed no significant differences in input resistance and local temporal summation between the two locations. Blockade of I(h) by ZD7288 increased the input resistance and temporal summation of simulated EPSPs, as in pyramidal neurons. When NMDA receptors were blocked, temporal summation at the soma of distal synaptic potentials was similar to that seen with current injections at the soma, suggesting a 'normalization' of temporal summation similar to that observed in pyramidal neurons. Thus, we have characterized a principal neuronal subtype in the hippocampus with a similar morphology but reversed I(h) somatodendritic gradient to that previously observed in CA1 hippocampal and neocortical pyramidal neurons.

Patch-clamp recording from neuronal dendrites.

Pyramidal neurons of the central nervous system have extensively arborized apical dendrites that contribute importantly to the signaling properties of the neuron. Recent advances in electrophysiological techniques have allowed recording from neuronal dendrites. These techniques depend on using infrared optics to visualize dendritic processes in the unstained brain slice preparation, on pipet positioning with high resolution micromanipulators, and on stringent techniques for brain slice preparation that preserved healthy dendritic processes, even in tissue from mature animals. The procedures underlying these techniques are described in this unit.