Abnormal Differentiation And Migration Of Newborn Neurons In Adult Hippocampus After Epileptic Seizures

It is among the most exciting and fasting progressing areas of neuroscience today, the study of adult neurogenesis, i.e. new neurons are continually being generated in adult mammalian brain. These newborn neurons eventually integrate into existing neuronal circuitries to exert functional effects. Mounting evidence suggests that ongoing neurogenesis in discrete brain regions not only occurs in normal adult mammalian animals, but also is modulated by multiple physiological factors and pathological events.Epilepsy is a common chronic neurological disorder characterized by recurrent unprovoked seizures, which are transient signs and/or symptoms of abnormal, excessive or synchronous neuronal activity in the brain. Epileptic seizures can lead to a drastic change of neurogenesis in hippocampal dentate gyrus. The phenomenon provides a brand new clue for investigating the plasticity of hippocampal structure and function after epileptic seizures, and also brings a new therapeutic strategy for treatment of epilepsy. However, the proliferation and differentiation of neural progenitor cells and the aberrant migration of newborn neurons after epileptic seizures, and its regulating mechanism in hippocampus remain largely unknown.In the study, we employed experimental epilepsy models in adult rodents, and performed the sequential three parts of the experiment by using multiple contemporary neurobiological techniques.1. Different Effects of Mild and Severe Seizures on the Migration of Hippocampal Newborn Neurons in Adult RatsRecent evidence shows that epileptic seizures can increase neurogenesis in the dentate gyrus and that mild and severe seizures induce different effects on hippocampal neural progenitors proliferation and differentiation. However, it is unknown whether different seizure severity has different effects on newborn neurons migration in the DG of adult rats.We established Li-PILO-induced mild and severe epileptic seizures models. Then we sought to examine the effects of different seizure severity on the migration of newborn cells in the dentate gyrus using immunohistochemistry, double immunefluorescence labeling and BrdU-labeling dividing cells methods. In addition, we further investigated the potential molecular mechanism underlying these processes. The results were as followings:⑴Most newborn neurons migrated into the granular cell layer in control and mild seizure groups, but severe seizures were associated with an aberrant migration of newborn neurons into the dentate hilus.⑵Severe epileptic seizures induced the formation of aberrant hilar basal dendrites and hilar-ectopic newborn neurons.⑶Severe seizures induced astrocyte activation and the expression of nestin, which was hardly seen in mild epileptic hippocampus.⑷Severe seizures induced the expression of the migration directional molecules netrin 1 and Sema-3A in the hilus, which were not present in the hilus of control and mild seizure-attacked rats,These results suggesting that severe seizures induce the expression of migration guiding molecules related to brain development, which may play a role in the aberrant migration of newborn neurons.2. Roles of Astrocytes and Microglia in Seizure-induced Aberrant Neurogenesis in the Hippocampus of Adult RatsIn this study, we established Li-PILO-induced SE model. By using hilar stereotaxic injection, immunohistochemistry and FJB staining methods, we examined activated patterns of astrocytes and microglia in epileptic dentate gyrus, then, observed the effects of activated astrocytes and microglia on aberrant migration of newborn neurons, respectively. The results were as followings:⑴SE induced a prominent activation of astrocytes and microglia in the dentate gyrus 3, 7, 14, and 20 days after the initial seizures.⑵By injecting fluorocitrate (FC) stereotaxicly into the hilus to inhibit astrocytic metabolism, we found that FC failed to prevent seizure-induced the formation of aberrant hilar basal dendrites, but instead promoted the degeneration of dentate granule cells after seizures.⑶In contrast, a selective inhibitor of microglia activation, minocycline (MC), inhibited the aberrant migration of newborn neurons at 14 days after status epilepticus.⑷By stereotaxic injection of lipopolysaccharide (LPS) into the intact dentate hilus to activate local microglia, we found that LPS promoted the development of aberrant hilar basal dendrites in the dentate gyrus.These results indicate that the activated microglia in the epileptic hilus may guide the aberrant migration of newborn neurons, and that MC could be a potential drug to impede seizure-induced aberrant migration of newborn neurons.3. No GAD67-Positive GABAergic Neurogenesis in the Dentate Gyrus of Adult Mice in Experimental Epilepsy ModelsMounting evidence shows that epileptic seizures influence normal migration pattern of hippocampal newborn neurons, which plays a role in the process of epileptogenesis and eventually contributes to the formation of chronic epilepsy. However, it is unknown whether the newborn neurons in the epileptic hippocampus can differentiate into inhibitory neurons, i.e.γ-aminobutyric acid (GABA) ergic interneurons. Here, we investigated the GABAergic neurogenesis in the epileptic hippocampus. By using glutamic acid decarboxylase (GAD) 67 -green fluorescence protein (GFP) knock-in mice, in which a GFP gene was introduced into the gene for GAD67 and thus all GABAergic neurons were fluorescent, we could easily observe green-fluorescent cells as GABAergic neurons containing GAD67. We employed pentylenetetrazol (PTZ)-induced chronic kindling model to mimic human generalized epilepsy, and lithium (Li) -pilocarpine (PILO) -induced status epilepticus (SE) model to reproduce human partial epilepsy. The results were as followings:⑴Both types of epilepsy significantly increased the number of newborn cells in the dentate gyrus at early time-point after seizures; however, there is a significant loss of newborn cells at 2 weeks after PTZ kindling and 8 weeks after Li-PILO-induced seizures.⑵About 80% of newborn dentate cells differentiated into neurons in control groups, whereas only 58% and 29% of newborn cells differentiated into neurons in the PTZ-kindling and Li-PILO models, respectively.⑶Double or triple immunofluorescence labeling did not reveal any newborn cells co-labeled with GFP in both intact and epileptic dentate gyrus.⑷A significant decrease in the total number of GABAergic neurons in the dentate gyrus was detected in the SE model but not in the PTZ-kindling model.These results indicate that epileptic seizures do not produce new GAD67-positive GABAergic interneurons in the dentate gyrus, but instead prolonged seizures result in the loss of GABAergic interneurons.