Inhibition of T-Type calcium channels in mEC layer II stellate neurons reduces neuronal hyperexcitability associated with epilepsy

Abstract

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca²⁺ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca²⁺ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca²⁺ channel antagonist, TTA-P2 (1 μM), was applied to determine the role of T-type Ca²⁺ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca²⁺ channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 μM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP's in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Ca_v3.1 mRNA compared to controls. Ca_v3.2 or Ca_v3.3 levels were unchanged. These findings support a role for T-type Ca²⁺ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Ca_v3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

Keywords: Entorhinal cortex; Stellate neurons; T-type calcium channel; Temporal lobe epilepsy.

Figure 1.. TTA-P2 ameliorates the hyper-excitability of mEC layer II stellate cells in TLE.

Action potentials were elicited using depolarizing current steps ranging from −20 to 280 pA. Firing frequency was increased and onset to firing was earlier in TLE (n = 13) compared to control (A; n = 16). Application of the selective T-type Ca²⁺ channel blocker, TTA-P2 (1 μM), reduced firing rates of control (n = 7) and TLE (n = 8) neurons to similar levels (B and C). Representative traces for control (D) and TLE (E) before and after application of TTA-P2, at current injection steps of 60, 140, 220, and 280pA. Action potentials were also evoked synaptically by stimulation of mEC layer III neurons. Representative traces for control (F) and TLE (G) before and after application of TTA-P2 (1 μM). Application of TTA-P2 reduced evoked action potentials in TLE neurons but had no effect on control neurons (right hand side F and G). Values represent means ± S.E.M. * P<0.05 (Students t-test or one-way ANOVA).

Figure 2.. TTA-P2 strongly inhibits rebound action potentials in mEC layer II neurons.

Sustained hyperpolarizing pulses of increasing intensity were used to evoke rebound action potentials. Firing frequency for control (n = 6) and TLE (n = 8) neurons was not significantly different (A, B - top traces). Application of the selective T-type Ca²⁺ channel blocker, TTA-P2 (1 μM), reduced firing rates of control (n = 6) but completely abolished APs in TLE (n = 8) neurons. C. Bar chart showing effect of TTA-P2 (1 μM) on control and TLE neurons. Values represent means ± S.E.M. * P<0.05 (Students t-test).

Figure 3.. Cav3.1 mRNA is increased in TLE but somatic current density is unchanged.

(A) Cav3.1 mRNA expression is increased in TLE mEC layer II compared to control. (B) Somatic T-type Ca²⁺ channel current density recorded from mEC layer II neurons. Right panel shows example traces of T current family of currents. Values represent means ± S.E.M. * P<0.0001 (one-way ANOVA).