Audiogenic kindling stimulates aberrant neurogenesis, synaptopodin expression, and mossy fiber sprouting in the hippocampus of rats genetically prone to audiogenic seizures

Highlights

• Audiogenic kindling enhances proliferation in the DG.

• Audiogenic kindling stimulates glutamatergic differentiation of newborn cells.

• Hilar cells differentiate faster than neurons of the DG granular layer.

• Audiogenic kindling increases sprouting of mossy fibers.

• Audiogenic kindling increases synaptopodin expression in the DG.

Abstract

Temporal lobe epilepsy is associated with considerable structural changes in the hippocampus. Pharmacological and electrical models of temporal lobe epilepsy in animals strongly suggest that hippocampal reorganization is based on seizure-stimulated aberrant neurogenesis but the data are often controversial and hard to interpret. The aim of the present study was to estimate neurogenesis and synaptic remodeling in the hippocampus of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). In our experiments we exposed KM rats to audiogenic kindling of different durations (4, 14, and 21 AGS) to model different stages of epilepsy development. Naïve KM rats were used as a control. Our results showed that even 4 AGS stimulated proliferation in the subgranular layer of the dentate gyrus (DG) accompanied with increase in number of doublecortin (DCX)-positive immature granular cells. Elevated number of proliferating cells was also observed in the hilus indicating the enhancement of abnormal migration of neural progenitors. In contrast to the DG, all DCX-positive cells in the hilus expressed VGLUT1/2 and their number was increased indicating that seizure activity accelerates glutamatergic differentiation of ectopic hilar cells. 14-day kindling further stimulated proliferation, abnormal migration, and glutamatergic differentiation of new neurons both in the DG granular and subgranular layers and in the hilus. However, after 21 AGS increased proliferation was observed only in the DG, while the numbers of immature neurons expressed VGLUT1/2 were still enhanced in both hippocampal areas. Audiogenic kindling also stimulated sprouting of mossy fibers and enhanced expression of synaptopodin in the hippocampus indicating generation of new synaptic contacts between granular cells, mossy cells, and CA3 pyramid neurons. Thus, our data suggest that epilepsy progression is associated with exacerbation of aberrant neurogenesis and reorganization of hippocampal neural circuits that contribute to the enhancement and spreading of epileptiform activity.

Introduction

Clinical and experimental studies strongly suggest the involvement of the hippocampus in the development of the limbic/mesial temporal lobe epilepsy. It was shown that recurrent excitation of the hippocampal neural circuits provides generalization of epileptiform activity and is associated with considerable structural abnormalities. In patients dispersion of granular cells in the dentate gyrus (DG), increased density of their basal dendrites, and sprouting of the mossy fibers were revealed [1], [2]. However, brain tissues are usually collected by surgery of pharmacoresistant patients or post-mortem. So, in humans it is impossible to determine early signs and trace the development of the structural aberrations. The most appropriate way to disclose the mechanisms underlying the changes in the hippocampal architecture is to use experimental models of epilepsy. Nowadays widely used experimental approaches imply administration of chemoconvulsants or electrical stimulation, and genetic models to study epilepsy development in animals. These models effectively reproduce main pathological processes described in the hippocampus of patients with epilepsy [3].

Observations of patients and results of experimental modeling of seizures indicate that epileptiform activity affects the hippocampal neurogenesis. Even single or rare seizures activate proliferation of neural stem cells in the subgranular layer of the DG and subsequent neuronal differentiation [4], [5], [6]. Long-term hyperexcitation in the hippocampus of rodents with epilepsy seems to have the same effects [4], [7], [8]. However, there are results indicating that prolonged and severe seizures significantly deplete pools of proliferating cells and neural progenitors [6], [9], [10]. Thus, correlation between alterations in hippocampal neurogenesis and epilepsy progression is still unclear.

Another hallmark of neurogenesis in the epileptic brain is aberrant migration of newborn cells. Under normal conditions, neural progenitors migrate mainly into the DG granular layer and differentiate into glutamatergic granular cells. In the epileptic hippocampus significant part of new cells migrate into the hilus and become ectopic glutamatergic neurons, which exhibit abnormally high activity and tend to synchronize with pyramidal cells of CA3 [11], [12].

New granular cells also contribute to structural reorganization in the hippocampus under the epileptiform activity. The studies showed that both newborn and mature granular neurons are characterized by increased sprouting of their axons, mossy fibers, which form new synapses with other granular cells and CA3 pyramidal neurons [13], [14]. In addition, they demonstrate increased amount of the basal dendrites that receive excitatory signals from mossy fibers [14], [15]. Thus, granular cells are involved in enhanced synaptogenesis that may contribute to the hyperactivation of hippocampal circuits and the extension of epileptiform activity. However, it was shown that some of new granular cells have reduced activity and may compensate for the hyperexcitability of other hippocampal cells [5], [16], [17]. These results, in opposite, suggest the neuroprotective role of neurogenesis during epilepsy development [5], [16], [17].

There is a plenty of data about alterations of neurogenesis in patients with limbic/mesial temporal lobe epilepsy and experimental models, but it is difficult to compare and to interpret these results. Pharmacological and electrical models have a number of limitations including high mortality of experimental animals, variable frequency and severity of spontaneous seizures, and extensive brain lesions. Furthermore, it is hard to distinguish neural damage associated with seizures and other systemic effects of stimulations [3]. Thus, more adequate experimental models to investigate the mechanisms of hippocampal abnormalities at different stages of epileptogenesis are rodents with innate susceptibility to audiogenic seizures (AGS) [18], [19], [20]. Today several strains of animals with inherited audiogenic sensitivity were designed including Krushinsky-Molodkina (KM) rats [18]. Single acoustic stimulations of audiogenic rats induce reflex seizures and mechanisms of its development are similar to the reflex epilepsy in human, with corresponding epileptiform activity arising in the brainstem [21]. Meanwhile, under repetitive stimulations of AGS according to audiogenic kindling protocol epileptiform activity extends to forebrain structures [22]. This process is accompanied with alterations in seizure pattern and electrophysiological characteristics of the cortex, the hippocampus, and the amygdala [22], [23], [24], [25]. Thus, rats genetically prone to AGS can be used as a model of limbic/mesial temporal lobe epilepsy in human [26]. We supposed that the study of structural reorganization of the hippocampus in KM rats exposed to audiogenic kindling could provide more detailed picture of aberrant neurogenesis and abnormalities in the formation of neural circuits at different stages of limbic epilepsy development.

In this work, we analyzed neurogenesis in the hippocampus of KM rats after audiogenic kindling of different durations. Our data showed that both short- and long-term audiogenic kindling stimulated proliferation, increased glutamatergic differentiation, and abnormal migration of newborn cells. Analysis of mossy fiber density and synaptopodin expression also revealed intensive reorganization of neural circuits in the hippocampus. Obtained results confirm that abnormal neurogenesis and synaptogenesis in the hippocampus make a significant contribution into the development of limbic epilepsy.