Gender differences in febrile seizure-induced proliferation and survival in the rat dentate gyrus

PURPOSE: Febrile seizures are fever-associated early-life seizures that are thought play a role in the development of epilepsy. Seizure-induced proliferation of dentate granule cells has been demonstrated in several adult animal models and is thought to be an integral part of epileptogenesis. The aim of the present study was to investigate proliferation and survival of dentate gyrus (DG) cells born after early-life hyperthermia (HT)-induced seizures in male and female rats. METHODS: At postnatal day (PN) 10, male and female rats were exposed to heated air to induce seizures. Littermates were used as normothermia controls. Convulsive behavior was observed by two researchers. From PN11 to PN16, rats were injected with bromodeoxyuridine (BrdU) to label dividing cells. The number of BrdU-immunoreactive cells in the DG was counted at PN17 and PN66. RESULTS: At PN17, male as well as female HT rats had the same amount of BrdU-positive cells compared with controls. At PN66, significantly more BrdU-positive cells were left in HT females (53%) than in controls (44%, percentage of BrdU-positive cells at PN17), whereas no difference was found between HT males and male controls. The net result of proliferation and survival at PN66 was that female HT rats had the same number of BrdU-immunoreactive cells as controls, whereas male HT rats had 25% more BrdU-immunoreactive cells than did controls (p < 0.05). CONCLUSIONS: Early-life seizures cause a sexually dimorphic cytogenic response that results in an increased population of newborn DG cells in young adult males, while leaving that of young adult females unaltered.     

Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy

PURPOSE: Mitogenic effects of seizures on granule cell progenitors in the dentate gyrus were studied in two rat models of epilepsy. We investigated which stage of epileptogenesis is critical for eliciting progenitor cell division and whether seizure-induced neuronal degeneration is responsible for the enhancement of progenitor cell division. METHODS: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neurogenesis of dentate granule cells was evaluated using the bromodeoxyuridine (BrdU) labeling method, and neuronal degeneration was assessed by in situ DNA fragmentation analysis. RESULTS: After injection of KA, the number of BrdU-positive granule cells began to increase at day 3 after the treatment, peaked at day 5, and returned to baseline at day 10. By day 13, the values were lower than control. After kindling, the number of BrdU-positive cells began to increase after five consecutive experiences of stage I seizures. The increase occurred from day 1 to day 3 after the last electrical stimulation, but returned to baseline by day 7. After generalized seizures were well established, repeated stimulation did not facilitate division of granule cell progenitors. DNA fragmentation was noted in pyramidal neurons in the CA1, CA3, and hilus regions at 18 h after KA injection, but not in the kindling model. CONCLUSIONS: These observations indicate that a mechanism in epileptogenesis boosts dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. Pyramidal neuronal degeneration is not necessary for triggering the upregulation. It is suggested that newly born granule cells may play a role in the network reorganization that occurs during epileptogenesis.     

Activation of the dentate gyrus by pentylenetetrazol evoked seizures induces mossy fiber synaptic reorganization

Kindled seizures evoked by electrical stimulation of limbic pathways in the rat induce sprouting and synaptic reorganization of the mossy fiber pathway in the dentate gyrus (DG). To investigate whether seizures evoked by different methods also induce reorganization of this pathway, the distribution of mossy fiber terminals in the DG was examined with Timm histochemistry after systemic administration of pentylenetetrazol, a chemoconvulsant that reduces Cl- mediated GABAergic inhibition. Myoclonic seizures evoked by subconvulsant doses of pentylenetetrazol (24 mg/kg i.p.) were not accompanied by electrographic seizures in the DG, and did not induce mossy fiber sprouting. Generalized tonic-clonic seizures evoked by repeated administration of PTZ (24 mg/kg i.p.) were consistently accompanied by electrographic seizure activity in the DG, and induced sprouting and synaptic reorganization of the mossy fiber pathway. The results demonstrated that repeated generalized tonic-clonic seizures evoked by pentylenetetrazol induced mossy fiber synaptic reorganization when ictal electrographic discharges activated the circuitry of the DG.     

Disambiguating the similar: the dentate gyrus and pattern separation

The human hippocampus supports the formation of episodic memory without confusing new memories with old ones. To accomplish this, the brain must disambiguate memories (i.e., accentuate the differences between experiences). There is convergent evidence linking pattern separation to the dentate gyrus. Damage to the dentate gyrus reduces an organism's ability to differentiate between similar objects. The dentate gyrus has tenfold more principle cells than its cortical input, allowing for a divergence in information flow. Dentate gyrus granule neurons also show a very different pattern of representing the environment than “classic” place cells in CA1 and CA3, or grid cells in the entorhinal cortex.More recently immediate early genes have been used to “timestamp” activity of individual cells throughout the dentate gyrus. These data indicate that the dentate gyrus robustly differentiates similar situations. The degree of differentiation is non-linear, with even small changes in input inducing a near maximal response in the dentate. Furthermore this differentiation occurs throughout the dentate gyrus longitudinal (dorsal-ventral) axis. Conversely, the data point to a divergence in information processing between the dentate gyrus suprapyramidal and infrapyramidal blades possibly related to differences in organization within these regions.The accumulated evidence from different approaches converges to support a role for the dentate gyrus in pattern separation. There are however inconsistencies that may require incorporation of neurogenesis and hippocampal microcircuits into the currents models. They also suggest different roles for the dentate gyrus suprapyramidal and infrapyramidal blades, and the responsiveness of CA3 to dentate input.     

The prototypic mineralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of the adrenalectomized rat

Glucocorticoid receptor activation inhibits granule cell proliferation in the hippocampus, but little is known about the role of mineralocorticoid receptors in this process. Here we administered aldosterone to adrenalectomized (ADX) rats, and monitored neurogenesis by BrdU immunohistochemistry. ADX significantly increased the number of BrdU-positive cells and aldosterone replacement further augmented BrdU-positivity. Our results indicate that aldosterone, most probably acting through mineralocorticoid receptors, may positively influence the proliferation and survival of newly-generated granule cells.     

Response of seizure-induced newborn neurons in the dentate gyrus of adult rats to second episode of seizures

In this study we examined the unknown issue of whether seizure-induced newborn hippocampal neurons in freely moving adult rats are able to respond to pathophysiological stimuli in the same way as their neighboring neurons do. Three days after pentylenetrazol (PTZ)-induced generalized seizures, rats received 5-bromodeoxyuridine (BrdU) injections to label dividing cells, followed 4 weeks later by the second PTZ injection to induce second episode of generalized seizures. We observed that the first episode of PTZ-induced seizures resulted in a significant increase in the number of newborn neurons in the adult hippocampal dentate gyrus. In comparison with vehicle-injected control rats that exhibited no Fos immunoreactivity and mild glutamic acid decarboxylase 67 (GAD67) expression in the dentate granule cells, rats killed 2-6 h following the second PTZ injection showed intensive Fos and GAD67 expression in virtually all granule cells with or without BrdU double-labeling. These findings provide important evidence indicating that seizure-induced newborn neurons in freely moving adult rats are able to respond to pathophysiological stimuli in the same way as neighboring neurons do.     

NDRG2通过Hes1参与癫痫发作后海马齿状回的神经发生

目的 探讨N-Myc下游调节基因2(N-Myc downstream regulated gene 2,NDRG2)与癫痫发作后海马齿状回神经发生的关系。方法 C57BL/6小鼠20只,随机分为癫痫组和对照组,每组又分为癫痫造模后1和7 d两个时间点,每个时间点5只,通过蛋白免疫印迹检测癫痫后海马齿状回NDRG2蛋白相对表达水平和mRNA相对表达水平变化; 使用双皮质素(DCX)染色标记未成熟神经元,神经巢蛋白(Nestin)标记神经干细胞,神经核蛋白(NeuN)标记成熟神经元,观察NDRG2对海马齿状回神经干细胞增殖影响; 采用RT-PCR检测发状分裂相关增强子1(hairy and enhancer of split 1,Hes 1)、NDRG2 mRNA相对表达表达水平,并分析两者之间的相关性; 观察NDRG2参与癫痫发作后神经发生的可能机制。结果 癫痫组与对照组比较,DCX、Nestin、NeuN、Hes1、NDRG2蛋白相对表达水平在1和7 d这2个时间点有显著性增高,并随时间逐渐递增。结论 癫痫发作后海马NDRG2蛋白相对表达水平增高,与癫痫发作后海马齿状回的神经细胞增值时间具有一致性和相关性,NDRG2可能参与癫痫发作后海马齿状回的神经发生过程; 同时发现海马NDRG2表达增加和Hes1分子表达增加具有相关性,故推测NDRG2可能通过Hes1参与癫痫发作后海马齿状回的神经发生。     

Increased granule cell neurogenesis in the adult dentate gyrus following mossy fiber stimulation sufficient to induce long-term potentiation

Neurons are continually added at a low rate to the granule cell layer of the dentate gyrus during adulthood in rats. The functional significance of this unusual feature is not completely understood, although recent studies suggest continued granule cell neurogenesis is essential for normal learning and memory. We report here that, in the adult rat, stimulation of the granule cell mossy fibers sufficient to induce long-term potentiation (LTP) increases the number of newly formed granule cells in the dentate gyrus, indicating that granule cell neurogenesis is regulated by efferent activity and, possibly, the induction of LTP.     

Early-life status epilepticus induces ectopic granule cells in adult mice dentate gyrus

A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these “ectopic” GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs.     

The development and decay of kindling-induced increases in paired-pulse depression in the dentate gyrus

Epileptogenic stimulation (kindling) leads to an increase in recurrent inhibition in the dentate gyrus, possibly due to an increase in benzodiazepine receptors. A second, late inhibitory component also potentiates as a result of kindling. In the present experiments, the time course of the development and decay of this kindling-induced increase in inhibition was studied. Rats were kindled by stimulation of the perforant path or by direct stimulation of the dentate gyrus. Paired-pulse stimulation was applied to the perforant path and field potential measures were taken within the dentate gyrus. These procedures allowed the monitoring of inhibitory events in chronic preparations over prolonged periods. Input/output measures of the baseline responses were also monitored during the course of, and after the completion of kindling. The increase in the early component (about 20-50+ ms) of paired-pulse depression was seen after the first kindling stimulation. The increase in the late component of depression (about 100-500+ ms) did not develop until after about 10 stimulations had been delivered. The late component then decayed more rapidly than the early component after the completion of kindling. The baseline response also showed some indication of depression, particularly in the dentate gyrus kindled group, raising the possibility that feedforward inhibition had also been potentiated.     

Radiation of the rat brain suppresses seizure-induced neurogenesis and transiently enhances excitability during kindling acquisition

PURPOSE: Adult hippocampal neurogenesis is enhanced in several models for temporal lobe epilepsy (TLE). In this study, we used low-dose whole brain radiation to suppress hippocampal neurogenesis and then studied the effect of this treatment on epileptogenesis in a kindling model for TLE. METHODS: Half of the rats were exposed to a radiation dose of 8 Gy one day before the initiation of a rapid kindling protocol. Afterdischarge threshold (ADT), afterdischarge duration (ADD), clinical seizure severity, and inflammation were compared between groups. On the first and third day after radiation, rats were injected with 5'-bromo-2'-deoxyuridine (BrdU) to evaluate neurogenesis. Seven and 21 days after radiation, numbers of doublecortin (DCX) positive neuroblasts in subgranular zone and granule cell layer were compared between groups. RESULTS: We showed that radiation significantly suppressed neurogenesis and neuroblast production during kindling acquisition. Radiation prevented an increase in ADT that became significantly lower in radiated rats. On the third and fourth kindling acquisition day radiated rats developed more severe seizures more rapidly, which resulted in a significantly higher mean severity score on these days. Differences in ADD could not be demonstrated. DISCUSSION: Our results demonstrate that brain radiation with a relatively low dose effectively suppressed the generation of new granule cells and transiently enhanced excitability during kindling acquisition. Although seizure-induced neurogenesis was lower in the radiated rats we could not detect a strong effect on the final establishment of the permanent fully kindled state, which argues against a prominent role of seizure-induced neurogenesis in epileptogenesis.     

Reversal learning of the rabbit nictitating membrane response following kindling-induced potentiation within the hippocampal dentate gyrus

The following experiment examined the effects of kindled seizures on reversal learning and the effects of both kindling and classical conditioning of the rabbit nictitating membrane on granule cell responsivity to perforant path input. Kindling resulted in significant potentiation of the population spike, the excitatory postsynaptic potential (EPSP) and the magnitude of twin pulse inhibition. Following kindling, rabbits were trained in a discrimination-reversal paradigm with either a tone or light paired with a corneal airpuff. Kindling did not affect acquisition of the initial discriminative response but did retard the rate of reversal learning. Kindling-induced potentiation, within dentate excitatory and inhibitory circuits, persisted for the duration of training. Thus, these results do not distinguish between the contribution of kindling-induced potentiation within dentate excitatory and inhibitory circuits to discrimination-reversal training. Spikes evoked during tone presentations were of reduced amplitude compared to spikes evoked either between trials or during light trials. The EPSP was not affected by stimulus conditions. In control rabbits, the magnitude of both the spike and EPSP increased across training. Training-related potentiation, in kindled rabbits, could not be separated from kindling-induced potentiation. These results demonstrate that an LTP-like effect of both the population spike and EPSP occurs with discrimination-reversal training.     

GABAergic signaling in young granule cells in the adult rat and mouse dentate gyrus

Throughout most of the developing brain, including the hippocampus, GABAergic synapses are the first to become functional. Several features of GABAergic signaling change across development, suggesting that this signaling in the immature brain may play important roles in the growth of young neurons and the establishment of networks. To determine whether GABA(A) receptor (GABA(A)R)-containing synapses in new neurons born in the adult dentate gyrus have similar immature features, we examined spontaneous and evoked GABA(A)R-mediated synaptic currents in young (POMC-EGFP or doublecortin-immunostained) granule cells in acute slice preparations from adult mice and rats. Spontaneous inhibitory postsynaptic currents (IPSCs) were observed in nearly all immature granule cells, but their frequency was considerably lower and their decay time constant was nearly two times longer than in neighboring mature (doublecortin-non-immunoreactive or EGFP-non-expressing) granule cells within the sub-granular zone. Evoked IPSCs (eIPSCs) in mature granule cells, but not immature granule cells, were sensitive to zolpidem, suggesting a maturational increase in GABA(A)R alpha1-subunit expression. Perforated-patch recording revealed that eIPSCs depolarized young neurons, but hyperpolarized mature neurons. The early establishment of synaptic GABAergic inputs slow IPSC decay time, and depolarizing action of eIPSCs are remarkably similar to features previously seen in neurons during development, suggesting that they are intrinsic features of immature neurons and not functions of the surrounding circuitry. These developmental features in adult-born granule cells could play a role in maturational processes such as developmental cell death. However, treatment of adult mice with GABA(A)R agonists and an inverse agonist did not significantly alter the number of 4- to 14-day-old BrdU-labeled cells.     

Activation of ERK by spontaneous seizures in neural progenitors of the dentate gyrus in a mouse model of epilepsy

Cellular changes that are associated with spontaneous seizures in temporal lobe epilepsy are not well understood but could influence ongoing epilepsy-related processes. In order to identify cell signaling events that could occur at the time of spontaneous seizures, the localization of phosphorylated extracellular signal-regulated kinase (pERK) was studied in a pilocarpine mouse model of epilepsy at very short intervals (1.5-2.5 min) after detection of a spontaneous seizure. Within the hippocampal formation, immunolabeling of pERK was evident in a subpopulation of cells in the subgranular zone (SGZ) of the dentate gyrus at these short intervals. Many of these cells had a long vertical process and resembled radial glia, while others had short processes and were oriented horizontally. Labeling with a series of developmental markers demonstrated that virtually all pERK-labeled cells were neural progenitor cells (NPCs). A high percentage (∼ 80%) of the pERK-labeled cells was labeled with either glial fibrillary acidic protein or brain lipid binding protein, indicating that these cells were radial glia-like NPCs. A smaller percentage of labeled cells expressed NeuroD, suggesting that they were later-developing NPCs that were assuming a neuronal identity. Early expression of pERK was not detected in immature neurons. Double labeling with proliferation markers demonstrated that approximately 30% of pERK-labeled NPCs expressed Mcm2, indicating that they were actively proliferating. Furthermore, virtually all radial glia-like NPCs that were in the proliferative cycle expressed pERK. These findings suggest that spontaneous seizures and associated ERK activation could contribute to the proliferation of radial glia-like NPCs in this epilepsy model.     

A theoretical network model to analyse neurogenesis and synaptogenesis in the dentate gyrus

We describe a strongly biologically motivated artificial neural network approach to model neurogenesis and synaptic turnover as it naturally occurs for example in the hippocampal dentate gyrus (DG) of the developing and adult mammalian and human brain. The results suggest that cell proliferation (CP) has not only a functional meaning for computational tasks and learning but is also relevant for maintaining homeostatic stability of the neural activity. Moderate rates of CP buffer disturbances in input activity more effectively than networks without or very high CP. Up to a critical mark an increase of CP enhances synaptogenesis which might be beneficial for learning. However, higher rates of CP are rather ineffective as they destabilize the network: high CP rates and a disturbing input activity effect a reduced cell survival. By these results the simulation model sheds light on the recurrent interdependence of structure and function in biological neural networks especially in hippocampal circuits and the interacting morphogenetic effects of neurogenesis and synaptogenesis.     

Kainic acid increases the proliferation of granule cell progenitors in the dentate gyrus of the adult rat

Granule cell progenitors in the dentate gyrus of the hippocampal formation have the unusual capacity to be able to divide in the brains of adult rats and primates. The basal proliferation rate of granule cell progenitors in the adult rat is low compared with development, however, it is possible that this rate may become significantly altered under pathological conditions such as epilepsy. We have investigated whether the proliferation of granule cell progenitors is increased in adult rats in a model of temporal lobe epilepsy, by using systemic bromodeoxyuridine injections to label dividing cells. We report here for the first time that granule cell neurogenesis is increased bilaterally 1 week after a single unilateral intracerebroventricular injection of kainic acid. Bromodeoxyuridine labeled neurons increased at least 6-fold on the side ipsilateral to the kainic acid injection compared to controls, but significantly, were also increased, by at least 3-fold on the side contralateral to the injection. The dividing cells in the subgranular zone were identified as neurons since they expressed Class III beta tubulin but not glial fibrillary acidic protein.     

An early decrease in cell proliferation after pentylenetetrazole-induced seizures

There are increasing data on the influence of seizures on neurogenesis in the adult brain. However, data on cell proliferation and differentiation during the early stages of kindling are scarce. We have used pentylenetetrazole (PTZ)-induced kindling to investigate the temporal profile of cytogenesis in the germinative zones of adult rat brain. For comparison, we also used a single PTZ-induced generalized tonic–clonic seizure. During kindling development, the density of 5-bromo-2'-deoxyuridine-positive cells demonstrated similar changes in all germinative zones: a dramatic decrease after the first subthreshold PTZ injection, and a gradual increase to the control level following repeated PTZ administration. On the contrary, a single PTZ-induced generalized tonic–clonic seizure was followed by an increase in the number of proliferating cells in both the dentate gyrus and the subventricular zone. These results may indicate the existence of global mechanisms affecting cellular proliferation in adult brain during seizures. Different temporal profiles of neuronal damage and proliferation changes suggest that neurodegeneration is unlikely to be a global proliferation-regulating factor. The data may contribute to better understanding of the initial phase of kindling development and epileptogenesis.     

Down-regulation of norepinephrine sensitivity after induction of long-term neuronal plasticity (kindling) in the rat dentate gyrus

Actions of norepinephrine (NE) in the dentate gyrus were examined before and after kindling-induced epilepsy, neuronal plasticity produced by daily high-frequency stimulation. NE, acting on β₁-receptors, depolarized granule cells, increased input resistance, firing and influx of Ca²⁺ in response to repetitive stimulation, and elicited long-lasting potentiation of synaptic potentials. In addition, NE acting via α₁-receptors, attenuated Ca²⁺-dependent regenerative potentials. After kindling-induced plasticity, there were marked reductions in all these effects of NE on granule cells, changes likely to influencing kindling-induced seizures, protecting against further enhancement of excitability once plasticity is in place.     

Time course for kindling-induced changes in the hilar area of the dentate gyrus: reactive gliosis as a potential mechanism

Recurrent seizure activity induced during kindling has been reported to cause an increase in the hilar area of the dentate gyrus of the hippocampus. To date, very little is known about the mechanism of this increase. This study investigated the time course for kindling-induced changes in the hilar area of the dentate gyrus at seven days, one month, and two months post-kindling. Hilar area of the dentate gyrus was significantly increased by approximately 46% at seven days and remained elevated at one month, but declined back to control levels by two months. Glial fibrillary acidic protein (GFAP) immunostaining was also evaluated at the same time points to determine whether kindling-induced changes in the hilar area of the dentate gyrus are related to kindling-induced glial cell changes. Increases in hilar GFAP immunostaining by approximately 57% were observed at seven days and at one month post-kindling, but not at two months post-kindling. These findings indicate that kindling-induced changes in the hilar area of the dentate gyrus and kindling-induced glial cell changes follow a similar time course, and that kindling-induced glial cell changes may mediate the observed changes in the hilar area of the dentate gyrus.