Abstracts

Rationale: Epilepsy is characterized by abnormal high synchronization discharge of neurons that can lead to central nervous system dysfunction. Frontal and temporal lobe epilepsy occupy a large proportion of refractory epilepsy. A recently developed chemogenetics technique not only allows cell-type specific inhibition or excitation of neuronal excitability, but also has the advantages of non-invasive. In this study, we used a virus to express a modified human M4 M4 muscarinic receptor (hM4Di) on pyramidal neurons and used hM4Di specific ligand clozapine-N-oxide (CNO) activating this receptors to achieve local or remote control the 4-aminopyridine (4-AP) induced seizures in rat. Methods: The Sprague-Dawley rats weighing 250-300g were used. A viral vector (rAAV2-CaMKIIa- hM4Di-mcherry) which expressed hM4Di in pyramidal cells was injected to either the right motor cortex or CA3 region of the right hippocampus for local control groups or to the bilateral anterior nucleus of thalamus (ANT) for remote control groups. One month later, the rotarod test was done in motor cortex transfected rats and the Morris water maze test was done in hippocampus and ANT transfected rats. To induce seizures, each rat was injected with 0.5µl (25mM) of 4-AP into the viral injection site for the local control group or into the bilateral ANT for the remote control group. Simultaneously, an intraperitoneal injection of CNO (2.5mg/kg) was given to the experimental group. Whether in the locally or remotely controlled group, we have set up the sham operation group to receive saline injections instead of viral vectors and then injected CNO, the viral transfection group consisted of rats were injected with viral vectors but not CNO,and the experimental group consisted of rats were injected with viral vectors and CNO. Virus transfection sites and range were confirmed by histological verification. Results: The results of rotarod test and Morris water maze test have no statistically significant between virus transfection group and sham operation group. On the neocortex seizure model, in locally controlled approach, the average duration of seizure (ADS) in the experimental group ( 30.3±3.2s, n=6) was significantly shorter than that in the control groups (sham operation group and only virus transfection group, 47.2±3.3s, n=5 and 45.7±2.5s, n=6, P<0.01). The interictal interval has no statistically significant between the three groups. In remotely controlled approach, the ADS has no statistically significant between the three groups. However, the interictal interval of seizure in the experimental group (181.5±26,3s) was statistically longer than that in two control groups (40.4±5.5s and 40.8±6.9s, P<0.001). On the hippocampal seizure model, the ADS in the locally controlled group (7.6±0.8s, n=6) was significantly shorter than that in the control groups (28.8±2.7s, n=6 and 27.4±1.3s, n=6, P<0.001). The ADS in the remotely controlled group (9.8±0.8s, n=6) was also shorter than that in two control groups (32.5±2.5s, n=6 and 31.6±2.0s, n=6, P<0.001). However, there was no statistical difference in the interictal interval between the three groups in both of approaches. Conclusions: Chemogenetics approach can not only locally control 4-aminopyridine-induced seizures, but also can reduce the seizures remotely in rat. Our results suggest that the use of chemogenetics techniques for local and remote control of seizures may open a new avenue for the precise treatment of epilepsy. Funding: This work was supported by the National Natural Science Foundation of China (81471391, 81671367, 81790653 XFY) and Beijing National Natural Science Foundation (11622308 XFY).