Temporal lobe epilepsy induces intrinsic alterations in Na channel gating in layer II medial entorhinal cortex neurons

Abstract

Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) channels play a critical role in AP generation and conduction we sought to determine if Na channel gating parameters and expression levels were altered in TLE. Na channel currents recorded from isolated mEC TLE neurons revealed increased Na channel conductances, depolarizing shifts in inactivation parameters and larger persistent (I_NaP) and resurgent (I_NaR) Na currents. Immunofluorescence experiments revealed increased staining of Na_v1.6 within the axon initial segment and Na_v1.2 within the cell bodies of mEC TLE neurons.

These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.

Introduction

Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy that involves the limbic structures of the temporal lobe including the entorhinal cortex (EC). The EC receives input from the parahippocampus, prefrontal cortex, and frontal cortex (Apergis-Schoute et al., 2006). This activity is then sent to the hippocampus via the perforant path and the temporoammonic path (TAP) (Burwell, 2000). The EC is subdivided into five main cortical layers with layers I-III superficial and layers IV-V deep layers. Layer II consists of non-stellate and stellate neurons and receives excitatory input from the perirhinal cortex, parasubiculum, olfactory structures as well as structures of the EC (Witter et al., 1989) and form the major excitatory input into the dentate gyrus (DG) and CA3 via the perforant path and the TAP.

In TLE, both animal models and patient studies have shown a decreased volume of the EC (Jutila et al., 2001, Bartolomei et al., 2005), corresponding to substantial loss of layer III neurons within the superficial layers of the mEC (Du et al., 1993). Although mEC layer II neurons are spared, they become hyper-excitable, displaying prolonged excitatory synaptic responses to stimulation of the EC deep layers (Bear et al., 1996). This increase in neuronal activity ultimately leads to an excessive excitatory input onto DG neurons of the hippocampus, further exciting the hippocampal–EC circuit (Kobayashi et al., 2003). Potential mechanisms for the hyper-excitability include reduced inhibitory input onto mEC layer II neurons (Kumar and Buckmaster, 2006), hyper-excitability of remaining mEC layer III neurons, providing enhanced synaptic activity via stimulation of the TAP (Ang et al., 2006), and synaptic re-organization within mEC layer II, although the latter has been recently suggested not to exist (Kumar et al., 2007). In addition to altered synaptic networks, changes intrinsic to the neuron, including modulations in ion channel activity, could also be involved.

In hippocampal neurons from animal seizure models, Na channel gating and expression levels are altered in a manner that would favor an increase in neuronal excitability (Ketelaars et al., 2001, Agrawal et al., 2003, Aronica et al., 2001, Whitaker et al., 2001, Vreugdenhil et al., 1998). Na channels are comprised of an α-subunit, and a variable number of auxiliary β-subunits (Catterall, 2000). Neurons are known to express multiple Na channel isoforms (Kress and Mennerick, 2008, Candenas et al., 2006) with the highest expression density along the axonal initial segment (AIS), a specific region near the start of the axon and the site for action potential (AP) initiation. In view of the importance of Na channels in initiating and propagating APs it is not surprising that altered activity and expression of Na channels could be pro-excitatory.

In this study we show that mEC layer II neurons from TLE animals continue to be hyper-excitable when devoid of synaptic input. We hypothesize that alteration's in Na channel activity and expression in TLE neurons account, in part, for this continued hyper-excitability. We propose that the changes in Na channel behavior, together with synaptic network changes, contribute to the hyper-excitability of mEC layer II neurons, altering the threshold for seizure initiation and spread throughout the EC-hippocampal circuit.