Optogenetic intervention of seizures improves spatial memory in a mouse model of chronic temporal lobe epilepsy

Abstract

Objective: To determine if closed-loop optogenetic seizure intervention, previously shown to reduce seizure duration in a well-established mouse model chronic temporal lobe epilepsy (TLE), also improves the associated comorbidity of impaired spatial memory.

Methods: Mice with chronic, spontaneous seizures in the unilateral intrahippocampal kainic acid model of TLE, expressing channelrhodopsin in parvalbumin-expressing interneurons, were implanted with optical fibers and electrodes, and tested for response to closed-loop light intervention of seizures. Animals that responded to closed-loop optogenetic curtailment of seizures were tested in the object location memory test and then given closed-loop optogenetic intervention on all detected seizures for 2 weeks. Following this, they were tested with a second object location memory test, with different objects and contexts than used previously, to assess if seizure suppression can improve deficits in spatial memory.

Results: Animals that received closed-loop optogenetic intervention performed significantly better in the second object location memory test compared to the first test. Epileptic controls with no intervention showed stable frequency and duration of seizures, as well as stable spatial memory deficits, for several months after the precipitating insult.

Significance: Many currently available treatments for epilepsy target seizures but not the associated comorbidities, therefore there is a need to investigate new potential therapies that may be able to improve both seizure burden and associated comorbidities of epilepsy. In this study, we showed that optogenetic intervention may be able to both shorten seizure duration and improve cognitive outcomes of spatial memory.

Keywords: behavior; closed-loop; cognitive impairment; comorbidity; temporal lobe epilepsy.

Figure 1.

Spatial learning and memory deficits in the intrahippocampal kainic acid model. (A) In the object location memory test animals are exposed to identical objects for 10 minutes, then brought back to the testing chamber 24 hours later, where one object is moved to a new location and the animals are allowed to investigate for 5 minutes. Animals are timed for how long they explore each object and a discrimination index is calculated. (B) Comparison of OLM performance between IHKA animals and non-IHKA injected animals in three different cohorts at three timepoints after IHKA treatment. Animals given IHKA treatment had significantly lower discrimination indexes than controls in all three tests, whether they were tested 10 days post IHKA (control: n=6; IHKA: n=10; p<0.001, Z=−3.09, Mann-Whitney Test), 3–4 months post IHKA (control: n=5; IHKA: n=9; p=0.002, Z=2.8), or 6–7 months post IHKA (control: n=14; IHKA: n=9; p<0.001, Z=3.24). (C) Comparison of the total time that animals spent exploring both objects in each test between IHKA and non-IHKA animals. There were no significant differences between exploration times in control and IHKA animals within each time point (p=0.26, p=0.70, p=0.37 respectively, Mann-Whitney Test). (D) Correlation of the time from IHKA injection compared to OLM performance in animals. Linear regression analysis showed no correlation between time from IHKA and OLM performance (Pearson’s R = 0.09). (E) Comparison of OLM performance between female and male animals in both control and IHKA categories (control female: n=16, control male: n=12; IHKA female: n=23, IHKA male: n=28) showed no significant differences (control: p=0.24; IHKA: p=0.56, Mann-Whitney Test), and therefore both sexes were combined in all cohorts for analysis. (F) Video tracking analysis revealed that IHKA animals ran significantly faster than controls in OLM tests (IHKA: n=50; control: n=34; p<0.001, Student’s t-test).

Figure 2.

Quantification of electrographic seizures in the IHKA model over time. Animals given IHKA showed both (A) electrographic seizures with no behavioral manifestation and (B) and behavioral seizures. Scale bars are (A) 1 mV, 5 sec, and (B) 2 mV, 5 sec. Asterisks in (A) indicate events classified as electrographic seizures. (C, D) Seizure duration and seizure frequency were quantified in a semi-automated manner in IHKA treated animals (n=4) over the course of 24 hours and no significant differences were found in (C) seizure frequency (p=0.34, Degrees of freedom (DF)=23, Kruskal-Wallis ANOVA followed by Dunn’s Test) but a significant difference was found within the (D) seizure duration (p<0.001, DF=23, 9 am seizure duration significantly different compared to 4 am, 5 am, 7 am, p<0.05, and 9 am seizure duration significantly different compared to 6 pm, 10 pm, 11 pm, p<0.01). Shaded areas indicate light-off phase of light cycle. Following this, seizure duration and seizure frequency were analyzed for one day a month over the course of 6 months (E, F) which was limited to the same 5–6 hour period of time each day, approximately 7 am until 12 pm, due to the fluctuations seen in (C, D). No significant changes were seen in seizure frequency (p=0.76, DF=5, Kruskal-Wallis ANOVA) and seizure duration (p=0.24, DF=5) in these animals (n=4) during this time.

Figure 3.

Effects of optogenetic intervention of seizures on learning and memory. (A) Behavioral paradigm for performing optogenetic intervention in animals using two OLM tests with closed-loop optogenetic seizure intervention provided in between. (B) Example of seizure suppression when closed-loop optogenetic intervention system responds with laser light directed to the dorsal hippocampus after seizure detection (pink line, lower trace) to activate PV interneurons in PV-ChR2, compared to when no laser light is provided. (C) By programming the closed-loop system to randomly respond to 50% of detected seizures with laser light, seizures with light treatment were significantly shorter than seizures without light intervention (n=8 animals, at least 50 seizure events analyzed for each animal, p<0.001, Z=3.1, Mann-Whitney Test). (D) When animals were given closed-loop optogenetic intervention for two weeks prior to and leading up to the testing day of a second OLM test, animals that received closed-loop intervention performed significantly better during the 2^nd test compared to performance in the first test (left panel: n=8, p=0.04, Z=−2.03, W=3, Wilcoxon Signed Rank Test). Implanted epileptic animals that did not receive closed-loop stimulation did not improve performance in the second test compared to the first (center panel: n=18, p=0.58, Z=0.55, W=144) while naïve control animals showed normal learning in both tests (DI>20), though they performed significantly lower in the second test compared to the first test (right panel: n=34, p=0.008, Z=2.65, W=362). (E) No significant differences were observed between the exploration time of animals between the first and second OLM tests in all three groups (p=0.94, Z=−0.07, W=17; p=0.87, Z=0.16, W=121; p=0.1, Z=1.63, W=312.5, respectively, Wilcoxon Signed Rank Test).