d-Leucine: Evaluation in an epilepsy model

Abstract

Background: Current medicines do not provide sufficient seizure control for nearly one-third of patients with epilepsy. New options are needed to address this treatment gap. We recently found that the atypical amino acid d-leucine protected against acutely-induced seizures in mice, but its effect in chronic seizures has not been explored. We hypothesized that d-leucine would protect against spontaneous recurrent seizures. We also investigated whether mice lacking a previously-described d-leucine receptor (Tas1R2/R3) would be protected against acutely-induced seizures.

Methods: Male FVB/NJ mice were subjected to kainic acid-induced status epilepticus and monitored by video-electroencephalography (EEG) (surgically implanted electrodes) for 4weeks before, during, and after treatment with d-leucine. Tas1R2/R3 knockout mice and controls underwent the maximal electroshock threshold (MES-T) and 6-Hz tests.

Results: There was no difference in number of calendar days with seizures or seizure frequency with d-leucine treatment. In an exploratory analysis, mice treated with d-leucine had a lower number of dark cycles with seizures. Tas1R2/R3 knockout mice had elevated seizure thresholds in the MES-T test but not the 6-Hz test.

Conclusions: d-Leucine treatment was ineffective against chronic seizures after kainic acid-induced status epilepticus, but there was some efficacy during the dark cycle. Because d-leucine is highly concentrated in the pineal gland, these data suggest that d-leucine may be useful as a tool for studying circadian patterns in epilepsy. Deletion of the Tas1R2/R3 receptor protected against seizures in the MES-T test and, therefore, may be a novel target for treating seizures.

Keywords: Epilepsy; Kainic acid; Sleep; Taste receptors; d-Amino acid.

Figure 1. EEG tracing from a typical kainic acid-induced seizure

(A) EEG was recorded in a male FVB/NJ mouse during a seizure using epidural screw electrodes (cross-hemisphere derivation shown; high pass filter 0.5 Hz, low pass filter 40 Hz). Sections of EEG tracing in (A) are shown on a longer time scale to demonstrate further EEG detail after seizure onset (B), mid-seizure (C), and during periods of brief suppression at the end of the seizure (D).

Figure 2. Seizures per hour in mice before, during, and after treatment with D-leucine

Mice were monitored by continuous video EEG (A–E). Seizures per hour are indicated during light and dark cycles. Shaded boxes represent the treatment period with D-leucine (1.5% w/v in drinking water). Mouse #5 died after the treatment period ended and therefore, the posttreatment period was truncated. Note that seizure frequency scales differ between panels.

Figure 3. Duration of sleep activity during light and dark cycles

(A) Total sleep time during pretreatment, treatment, and posttreatment phases (P = 0.22 for pretreatment vs. treatment periods, paired t-test). (B) Sleep time during light cycle hours during each of the treatment phases (P = 0.46 for pretreatment vs. treatment periods, paired t-test). (C) Sleep time during dark cycle hours during each of the treatment phases (P = 0.42 for pretreatment vs. treatment periods, paired t-test). In all panels, the solid line indicates the period used for statistical comparison; data from the dashed lines were not included in the comparison because mouse #5 did not complete the full posttreatment period.

Figure 4. Acute seizure testing in Tas1R2/R3 double knockout mice

(A) Probability of mice having a tonic hindlimb extension seizure (THLE) at the indicated MES-T stimulus currents for Tas1R2/R3 knockout mice or C57BL/6 controls. Data are from the same cohort used in Fig. 4B, retested in 3 sessions separated by one week; each mouse received a given current stimulus only once. Data points represent the percentage of mice from all cohorts that had seizures at a given current. Probit analysis curves represent a probability function, therefore not all points lie on the curve (N = 19 trials for 7 Tas1R2/R3 knockout mice, with 3 fatalities over the course of testing; N = 12 trials for 6 wildtype mice, with 4 fatalities over the course of testing; CC₅₀ = 11.2 mA (95% CI = 10.0–12.4 mA) for Tas1R2/R3 knockout, CC₅₀ = 8.5 mA for wildtype mA (95% CI = 7.1 – 9.9 mA) (P = 0.03, probit analysis). (B) Probability of mice having a seizure at the indicated 6 Hz stimulus currents for Tas1R2/R3 knockout mice or C57BL/6 controls (this test was performed prior to the MES-T test). Data are from the same cohort used in Fig. 4A, retested in 3 sessions separated by one week; each mouse received a given current stimulus only once. Data points represent the percentage of mice from all cohorts that had seizures at a given current. Probit analysis curves represent a probability function, therefore not all points lie on the curve (N = 21 trials including 7 Tas1R2/knockout mice and 6 wildtype mice, repeated multiple times; CC₅₀ = 16.1 mA (95% CI = 13.7–18.4 mA) for Tas1R2/R3 knockout, CC₅₀ = 15.7 mA for wildtype mA (95% CI = 13.2 – 18.2 mA) (P = 0.83, probit analysis). All mice survived this testing.