Seizure Generation Via Aberrant Integration Of New Neurons Into The Dentate Circuits In Stroke

BackgroundEpileptic seizures are well-known neurological complications following stroke. Epidemiological surveys have found that the incidence of post-stroke epilepsy was more than10%, accounting for one third the newly diagnosed cases of epilepsy in adults. In the newly diagnosed patients of epilepsy over60years old, about45%of the etiology is cerebrovascular disease. As patients with cerebrovascular diseases live longer and longer, the prevalence of post-stroke epilepsy is gradually increasing. Despite the high prevalence of this disease, scientists still don’t well understand the mechanism of post-stroke epilepsy and there is no effective treatment. Neural progenitor cells, which mainly locate in sub-granule zones of dentate gyrus and the subventricular zones of lateral ventricles in all adult mammalian, can proliferate and differentiate into new neurons. In the process of neuronal development, they gradually extend axons and dendrites, form excitatory synapses with their goals, then integrate into the existing neural circuits and play relevant roles. Neurogenesis is regulated by a variety of factors. Research has shown that newborn rats after stroke showed cognitive dysfunction and a highly excited state accompanied by epilepsy. Another related reports indicated that stroke could promote activation of neural progenitor cell to proliferate and migrate to the injury sites, participating in brain repair. Newborn hippocampal neurons are morphological abnormal after stroke, but whether these abnormal newborn neurons are related to the development of epilepsy after stroke is unknown.ObjectiveTo observe the morphologies and functions of newborn neurons in the dentate gyrus of stroke mice, and explore whether they are related to the post-stroke epilepsy.Methods4-month-old C57BL/6J mice, Nestin-Cre mice, ChR2mice, TeTX mice were used in the following experiments. Middle cerebral artery occlusion(MCAO) was used to construct focal cerebral ischemia or sham control. magnetic resonance images(MRI) and Fluoro-Jade C staining were used to detect the brain dameges. Infection of retroviruses(RV), AAV and rabies virus and microinjection of fluorescent dyes were used to label newborn neurons. BrdU labeling was applied to label newborn cells. RT-PCR was performed to detect the mRNA levels of GAD67, GFAP and Calbindin-D28K in interneurons, glial cells and granule neurons. The whole-cell patch clamp electrophysiology recordings were used to detect the electrophysiology characteristics of newborn granule neurons and existed granule neurons. Kainic Acid (KA) was used to induce acute seizures. The epileptic status of mice was monitored by the video/EEG (electroencephalogram).ResultsSpontaneous recurrent seizures are generated in MCAO mice. The vedio/EEG recordings was used to monitor the EEG and behavior of C57-MCAO/sham mice which received MCAO/sham process for3continuously months. The results showed that C57-MCAO mice began to exhibit the recurrent epileptic seizures about1month after the MCAO surgery, accompanied with abnormal EEG.New neurons were morphologically abnormal in MCAO mice.4-month-old male C57BL/6mice were undergone MCAO/sham surgeries and were stereotaxically injected with the retrovirus paticals(RV-EGFP) to label newborn neurons.2and4weeks after the sugeris, the number and morphologies of the GFP+cells were observed. The results showed that the number of GFP+cells and the proportion of bipolar cells increased significantly in MCAO mice, and newborn neruons had multiple basal dendrites.New neurons showd aberrant synaptic connections in MCAO mice. Method one:4-month-old male C57BL/6mice were undergone MCAO/sham surgeries and were stereotaxically injected with the retrovirus paticals(RV-EGFP) to label newborn neurons.4weeks later, the GFP+cells and GFP-cell were microinjected with green and red bio-dyes respectively. The results showed that the axons of newborn neurons in C57-MCAO mice stretched deeply into the molecular layer. Method two:4-month-old male C57BL/6mice were undergone MCAO/sham surgeries and were intraperitoneally injected with BrdU to label newborn cells and stereotaxically injected with the AAV paticals(AAV-EGFP-G).3weeks later,△Rabies virus paticles(△Rabies-mCherry) were injected in the same positions to label AAV-infected neurons and their superior neurons.1weeks later, mice were sacrificed to observed the number and morphology and distribution of EGFP+/mCherry+, EGFP+/mCherry-and EGFP-/mCherry+cells and whether these cells were colocated with BrdU+cells. The results showd that most of EGFP-/mCherry+cells were also BrdU+cells.New neurons aberrantly integrate into the dentate circuits in C57-MCAO mice. Method one:4-month-old male Nes-Cre mice firstly received MCAO surgery (Nes-Cre-MCAO mice).1week later, the Nes-Cre-MCAO mice were injected with AAV (AAV-ChR2-EGFP) at DG, and at the same time, they were intraperitoneally injected with BrdU for5consecutive days.4weeks later, the brain of the Nes-Cre-MCAO mice were stained with NeuN and BrdU.The number, morphology and distribution of GFP+/NeuN+cells and GFP+/NeuN+/BrdU+cells and the proportion of NeuN+/BrdU+cells in GFP+cells were observed. The results showd that the number of total newborn neurons increased but the number of newborn cells differentiated into neurons did not change in Nes-Cre-MCAO mice. Method two:4-month-old male ChR2mice received MCAO surgery (ChR2-MCAO mice), then tamoxifen (TAM) was given to induce the expression of ChR2and EGFP.4weeks later, the synaptic connections between ChR2+/EGFP+cells at DG and ChR2-/EGFP-cells at DG/CA3were recorded. The results showd that the EPSCs of ChR2-/EGFP-cells at CA3in MCAO/sham mice could be recorded, and the EPSCs of ChR2-/EGFP-cells at DG in MCAO mice could be recorded, but the EPSCs of ChR2-/EGFP" cells at CA3in sham mice could not be recorded.New neurons form functional synapses with exisiting granule neurons in MCAO mice.4weeks after ChR2-mice received MCAO/sham surgery (ChR2and EGFP expression have been induced), the synaptic connections between ChR2+/EGFP+cells in DG and ChR2-/EGFP-cells in DG and CA3were recorded. The results showed that ChR2-/EGFP" cells in CA3of ChR2-MCAO/sham-mice, ChR2-/EGFP-cells in DG of ChR2-MCAO-mice could record EPSCs. The EPSCs was linearly positive correlated with the intensity of blue light, and they were sensitivity to AMPA receptor antagonist.The expression of TeTX had no effect on the survival and electrophysiological characteristics of newborn neurons in MCAO-mice and could block the abnormal synaptic transmission of newborn neurons in MCAO-mice.4-month-old male TeTX mice received MCAO surgery (TeTX-MCAO-mice), and half of them received TAM (TeTX+-MCAO-mice) and the others (TeTX--MCAO-mice) did not.4weeks later, Fluoro-Jade C staining was used to detect the effect of TeTX on cell survival. The results showed the number of F-J+cells were similar in two groups. Also, electrophysiological recordings of GFP+cells and GFP" cells in two groups showed that the EPSCs and IPSCs were similar. TeTX-MCAO-mice were injected AAV (AAV-ChR2)1week after the focal ischemia.4weeks later, animals drank water with doxycycline (DOX)(TeTX+-ChR2+-MCAO-mice) or vehicle (TeTX"-ChR2+-MCAO-mice) for1week. Then the EPSCsNMDA of DG and CA3by stimulating newborn neurons with light and mossy fibers with electric was recorded for the first time. Then all animals drank normal water for1week and then the EPSCsNMDA was recorded for the second time by using the same way. The results showed that the EPSCsNMDA could be recorded in DG and CA3of the TeTX--ChR2+-MCAO/sham-mice by light and electric stimulation and in CA3of TeTX+-ChR2+-MCAO-mice stimulated by electric for the first record. For the second time, the EPSCsNMDA could be recorded in DG of the TeTX--ChR2+-MCAO/sham-mice by light and electric stimulation.Specific blockade of synaptic transmission of newborn neurons in hippocampus can reduce the frequency and strength of seizures in MCAO-mice.4-month-old male TeTX mice received MCAO/sham surgery (TeTX-MCAO/sham-mice).3weeks later, animals drank water with DOX (TeTX+-MCAO/sham-mice) or vehicle (TeTX"-MCAO/sham-mice) for1week. Then EEG and behavior were recorded.We found sham-mice had no seizure. TeTX--MCAO-mice had seizures and the number of seizures increased as the recovery time. TeTX+-MCAO-mice had less seizures compared with TeTX--MCAO-mice.4-month-old male TeTX4+/-mice received MCAO/sham surgery. Then kainic acid (KA,10-30mg/kg) was injected to induce acute seizures. EEQ behavior and motality were recorded. The results showed that the motality increased with the increase of the doses of KA, mainly TeTX--MCAO-mice. The EEG of sham-mice and TeTX+-MCAO/sham-mice were mainly normal, while TeTX--MCAO-mice were obviously abnormal and the seizure index was higher.4-month-old male TeTX mice received MCAO/sham surgery (TeTX-MCAO/sham-mice).4weeks later, their behavior was continuously recorded for4weeks for the first time (R1). Then mice were given DOX to induce the expression of TeTX for5weeks, and the behavior was continuously recorded for the last4weeks for the second time (R2). Then mice were given normal water to deduce the expression of TeTX for5weeks, and the behavior was continuously recorded for the last4weeks for the third time (R3). It was shown that TeTX+/--sham-mice had no seizure in the entire duration. TeTX--MCAO-mice had similar seizure index in Rl and R3. TeTX+-MCAO-mice had lower seizure index in R2.ConclusionsNewborn neurons integrate into the existing neural circuits abnormally, and it is the cellular basis of spontaneous recurrent seizures after stroke. Blocking the abnormal synaptic connections can reduce the intensity and frequency of seizures. The results provide new therapeutic interventions and ideas for treatment of patients who suffer post-stroke epilepsy.