The Specific Effects of OD-1, a Peptide Activator, on Voltage-Gated Sodium Current and Seizure Susceptibility

Abstract

OD-1, a scorpion toxin, has been previously recognized as an activator of voltage-gated Na⁺ currents. To what extent this agent can alter hippocampal neuronal Na⁺ currents and network excitability and how it can be applied to neuronal hyperexcitability research remains unclear. With the aid of patch-clamp technology, it was revealed that, in mHippoE-14 hippocampal neurons, OD-1 produced a concentration-, time-, and state-dependent rise in the peak amplitude of I_Na. It shifted the I_Na inactivation curve to a less negative potential and increased the frequency of spontaneous action currents. Further characterization of neuronal excitability revealed higher excitability in the hippocampal slices treated with OD-1 as compared with the control slices. A stereotaxic intrahippocampal injection of OD-1 generated a significantly higher frequency of spontaneous seizures and epileptiform discharges compared with intraperitoneal injection of lithium-pilocarpine- or kainic acid-induced epilepsy, with comparable pathological changes. Carbamazepine significantly attenuated OD-1 induced seizures and epileptiform discharges. The OD-1-mediated modifications of I_Na altered the electrical activity of neurons in vivo and OD-1 could potentially serve as a novel seizure and excitotoxicity model.

Keywords: OD-1; action current; neuronal excitability; scorpion toxin; seizure; voltage-gated Na+ current.

Figure 1

Stimulatory effect of OD-1 on voltage-gated Na⁺ current (I_Na). In these experiments, cells were bathed in Ca²⁺-free Tyrode’s solution, and the recording pipette was filled with a Cs⁺-containing solution. (A) Representative I_Na traces were obtained in the control (control) and during exposure to 0.3 μM OD-1 and 3 μM OD-1. The inset indicates the voltage protocol used. (B) Concentration-dependent effect of OD-1 on the peak amplitude of I_Na (mean ± SEM; n = 9–11 for each point). The peak amplitude of I_Na in response to brief membrane depolarization from −80 to −10 mV was measured. The peak I_Na during exposure to 30 μM OD-1 was taken to be 100%. The continuous line represents the best fit of the data points to the sigmoidal Hill equation, as detailed in the Materials and Methods section. The EC₅₀ values and Hill coefficient were 0.71 μM and 1.3, respectively. (C) Time courses of the relative increase in I_Na following treatment with 0.3 μM OD-1 (a) and 3 μM OD-1 (b). The trajectory in the presence of 0.3 μM OD-1 and 3 μM OD-1 was fitted using a single exponential (as indicated by smooth curves) with a value of 4.62 and 3.43 msec, respectively. The relative increase (i.e., (I_control-I_OD-1)/I_control) was evaluated by dividing the OD-1-sensitive current by the current obtained in the control. (D) The relationship of the reciprocal to the time constant (i.e., 1/τ) of the relative increase versus the OD-1 concentration was plotted (mean ± SEM; n = 9–12 for each point). The data points shown in the open circles were fitted using a linear regression and indicated that there was a molecularity of one. On the basis of the binding scheme, the forward (k₊₁*) and backward (k₋₁) rate constants for OD-1-induced stimulation of I_Na were calculated to be 0.148 msec⁻¹μM⁻¹ and 0.135 msec⁻¹, respectively. (E) Effect of OD-1 and OD-1 plus tetrodotoxin (TTX) on voltage-gated Na⁺ current in mHippoE-14 neurons. Cells were bathed in Ca²⁺-free, Tyrode’s solution, and the pipette was filled with a Cs⁺-containing solution. The current trace labeled “a” was obtained in the presence of 1 μM OD-1, while that labeled “b” was obtained after further addition of 1 μM TTX, but still in the presence of 1 μM OD-1. The upper part shows the applied voltage-clamp protocol. (F) Effect of OD-1 and OD-1 plus carbamazepine (CBZ) on voltage-gated Na⁺ current in mHippoE-14 neurons. Cells were bathed in Ca²⁺-free, Tyrode’s solution, and the pipette was filled with a Cs⁺-containing solution. The current trace labeled “a” was obtained in the presence of 1 μM OD-1, while that labeled “b” was obtained after further addition of 10 μM CBZ, but still in the presence of 1 μM OD-1. The upper part shows the voltage-clamp protocol applied. Data were analyzed using a one-way ANOVA with the least-significant difference post hoc comparisons.

Figure 2

Average conductance–voltage relationship (A,B) and steady-state inactivation curves (C) of peak I_Na obtained with or without the addition of OD-1. The experiments were conducted in cells bathed with Ca²⁺-free Tyrode’s solution, and the recording pipette was filled with a Cs⁺-containing solution. (A) Representative I_Na traces evoked by different voltage steps are shown in the uppermost part. The upper part indicates current traces obtained in the control, while the lower part is those taken during exposure to 3 μM OD-1. (B) Average conductance–voltage relationship of peak I_Na in the absence (■) and presence (□) of 3 μM OD-1 (mean ± SEM; n = 9–11 for each point). Notably, the presence of OD-1 increased the peak conductance of I_Na with no change in the overall conductance-versus-voltage relationship to the current. (C) Steady-state inactivation curve of I_Na obtained with and without the addition of 3 μM OD-1 (mean ± SEM; n = 9 for each point; absence (■) and presence (□) of 3 μM OD-1). Data were analyzed using a one-way ANOVA with the least-significant difference post hoc comparisons.

Figure 3

Stimulatory effect of OD-1 on AC frequency and amplitude. Cell-attached voltage-clamp current recordings were made in these experiments. The recording pipette was filled with a K⁺-containing solution, and once the cell-attached model was clearly achieved, the potential was maintained at the resting potential of the cell (−70 mV). (A) Representative current traces obtained in the absence (upper) and presence of 1 μM OD-1 (middle) and 3 μM OD-1 (lower). The results regarding the addition of OD-1 were obtained 2 min after the cells were exposed to the compound (1 or 3 μM). Note that the trace displaying inward deflections reflects the emergence of ACs. Summary bar graphs showing the effect of OD-1 on the frequency and amplitude of spontaneous ACs are shown in (B) and (C), respectively, (mean ± SEM; n = 11). * Significantly different from the control group (p < 0.05). ** Significantly different from OD-1 (1 μM; p < 0.05). Data were analyzed using a one-way ANOVA with the least-significant difference post hoc comparisons.

Figure 4

Comparison of hippocampal CA1 neuronal excitability in Sprague Dawley (SD) rats with OD-1 treatment (OD-1 group) and without OD-1 treatment (control group). The OD-1 concentration was 1 μM. (A) The I_Na was significantly higher in the brain slices in the OD-1 group, as compared with the control group. (B) The threshold current required one to elicit an AP in the presence and absence of OD-1. (C) Representative traces of AP firing in the presence and absence of OD-1. Depolarizing current injection from 0 to 140 pA in 20 pA increments with a duration of 500 msec. Current injection yielded a significantly higher number of APs in the OD-1 group, as compared to the control group. The horizontal lines indicate mean values. (D) The number of APs elicited by threshold current injection in the control and with OD-1. The horizontal lines indicate mean values. * Significantly different from the control group (p < 0.05). Data were analyzed using an ANOVA Kruskal–Wallis H test, followed by Dunn’s multiple comparison tests (n = 7 in each group).

Figure 5

The spontaneous recurrent seizure (SRS) and epileptiform discharges on EEG during the chronic stage following status epilepticus. (A) The OD-1 group had a significantly higher total SRS count compared with the naïve, Li-Pi, KA (all * p < 0.05), and OD-1 + CBZ groups (^## p < 0.01). The total SRS count in the KA group was higher than that in the OD-1 + CBZ group (p < 0.05). The control group (NS + OD-1) also had a significantly higher total SRS count than the CBZ + OD-1 group. (B) and (C) Representative figures of the epileptiform discharges on EEG showing that the high frequency epileptiform bursts in rats with SRS were more frequently seen in the OD-1 group and NS + OD-1 group compared with the naïve, Li-Pi, and KA groups (all * p < 0.05) and CBZ + OD-1 group (^## p < 0.01). Similarly, the epileptiform discharges in the KA group was more frequent than that of the CBZ + OD-1 group (p < 0.05; * p < 0.05, ^## p < 0.01, n = 7 in each group). Data were analyzed using ANOVA followed by Fisher’s least significant difference tests.

Figure 6

There were similar amounts of cresyl violet stained hippocampal neurons in the Li-Pi, KA, and OD-1 models. Scale bar = 200 μm. The semiquantitative neuronal damage scores were similar across the three groups (p = 0.20) although the OD-1 group had a slightly higher overall score. The naïve control (1.4 ± 0.01) showed significantly less damage than the other three groups (* p < 0.05). Data were analyzed using an ANOVA Kruskal–Wallis H test followed by Dunn’s multiple comparison tests (n = 7 in each group).

Figure 7

There were similar amounts of cresyl violet stained neurons in the Li-Pi, KA, and OD-1 models. Scale bar = 200 μm. The semiquantitative neuronal damage scores were similar across the three groups (p = 0.20) although the OD-1 group had a slightly higher overall score. The naïve control (1.9 ± 0.1) showed significantly less damage than the other three groups (* p < 0.05). Data were analyzed using an ANOVA Kruskal–Wallis H test followed by Dunn’s multiple comparison tests (n = 7 in each group).