Stereologic estimation of hippocampal GluR2/3- and calretinin-immunoreactive hilar neurons (presumptive mossy cells) in two mouse models of temporal lobe epilepsy

Purpose: Hippocampal mossy cells receive dense innervation from dentate granule cells and, in turn, mossy cells innervate both granule cells and interneurons. Mossy cell loss is thought to trigger granule cell mossy fiber sprouting, which may affect granule cell excitability. The aim of this study was to quantify mossy cell loss in two animal models of temporal lobe epilepsy, and determine whether there exists a relationship between mossy cell loss, mossy fiber sprouting, and granule cell dispersion. Methods: Representative hippocampal sections from p35 knockout mice and mice with unilateral intrahippocampal kainate injection were immunolabeled for GluR2/3, two subunits of the amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor and calretinin to identify mossy cells. Mossy fibers were immunostained against synaptoporin. Key Findings: p35 Knockout mice showed no hilar cell death, but moderate mossy fiber sprouting and granule cell dispersion. In the kainate‐injected hippocampus, there was an 80% and 85% reduction of GluR2/3‐ and GluR2/3/calretinin‐positive hilar neurons, respectively, and dense mossy fiber sprouting and significant granule cell dispersion. In the contralateral hippocampus there was a 52% loss of GluR2/3‐, but only a 20% loss of GluR2/3‐calretinin‐immunoreactive presumptive mossy cells, and granule cell dispersion; no mossy fiber sprouting was observed. Significance: These results indicate a probable lack of causality between mossy cell death and mossy fiber sprouting.     

Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers.

Morphological data from humans with temporal lobe epilepsy and from animal models of epilepsy suggest that seizure-induced damage to dentate hilar neurons causes granule cells to sprout new axon collaterals that innervate other granule cells. This aberrant projection has been suggested to be an anatomical substrate for epileptogenesis. This hypothesis was tested in the present study with intra- and extracellular recordings from granule cells in hippocampal slices removed from rats 1-4 months after kainate treatment. In this animal model, hippocampal cell loss leads to sprouting of mossy fiber axons from the granule cells into the inner molecular layer of the dentate gyrus. Unexpectedly, when slices with mossy fiber sprouting were examined in normal medium, extracellular stimulation of the hilus or perforant path evoked relatively normal responses. However, in the presence of the GABAA-receptor antagonist, bicuculline, low-intensity hilar stimulation evoked delayed bursts of action potentials in about one-quarter of the slices. In one-third of the bicuculline-treated slices with mossy fiber sprouting, spontaneous bursts of synchronous spikes were superimposed on slow negative field potentials. Slices from normal rats or kainate-treated rats without mossy fiber sprouting never showed delayed bursts to weak hilar stimulation or spontaneous bursts in bicuculline. These data suggest that new local excitatory circuits may be suppressed normally, and then emerge functionally when synaptic inhibition is blocked. Therefore, after repeated seizures and excitotoxic damage in the hippocampus, synaptic reorganization of the mossy fibers is consistently associated with normal responses; however, in some preparations, the mossy fibers may form functional recurrent excitatory connections, but synaptic inhibition appears to mask these potentially epileptogenic alterations.     

匹罗卡品致痫诱发大鼠齿状回颗粒细胞GAP-43 mRNA表达

目的 研究边缘系统癫痫发作后海马颗粒细胞生长相关蛋白（GAP-43）基因表达变化。方法 建立匹罗卡品急、慢性癫痫模型,用原位杂交方法定量检测不同时间点海马颗粒细胞GAP-43mRNA表达。结果 对照组颗粒细胞几乎不表达GAP-43mRNA,匹罗卡品致病后6～12h颗粒细胞表达GAP-43mRNA增高,15～30d呈现第2次高峰。结论 成年大脑海马颗粒细胞在致痫后发生可塑性变化,GAP-43mRNA表达是癫痫大鼠大脑结构性重组（颗粒细胞苔藓纤维出芽）的重要分子机制。     

Synaptic connections of hilar basal dendrites of dentate granule cells in a neonatal hypoxia model of epilepsy

Numerous animal models of epileptogenesis demonstrate neuroplastic changes in the hippocampus. These changes occur not only for the mature neurons and glia, but also for the newly generated granule cells in the dentate gyrus. One of these changes, the sprouting of mossy fiber axons, is derived predominantly from newborn granule cells in adult rats with pilocarpine-induced temporal lobe epilepsy. Newborn granule cells also mainly contribute to another neuroplastic change, hilar basal dendrites (HBDs), which are synaptically targeted by mossy fibers in the hilus. Both sprouted mossy fibers and HBDs contribute to recurrent excitatory circuitry that is hypothesized to be involved in increased seizure susceptibility and the development of spontaneous recurrent seizures (SRS) that occur following the initial pilocarpine-induced status epilepticus. Considering the putative role of these neuroplastic changes in epileptogenesis, a critical question is whether similar anatomic phenomena occur after epileptogenic insults to the immature brain, where the proportion of recently born granule cells is higher due to ongoing maturation. The current study aimed to determine if such neuroplastic changes could be observed in a standardized model of neonatal seizure-inducing hypoxia that results in development of SRS. We used immunoelectron microscopy for the immature neuronal marker doublecortin to label newborn neurons and their HBDs following neonatal hypoxia. Our goal was to determine whether synapses form on HBDs from neurons born after neonatal hypoxia. Our results show a robust synapse formation on HBDs from animals that experienced neonatal hypoxia, regardless of whether the animals experienced tonic-clonic seizures during the hypoxic event. In both cases, the axon terminals that synapse onto HBDs were identified as mossy fiber terminals, based on the appearance of dense core vesicles. No such synapses were observed on HBDs from newborn granule cells obtained from sham animals analyzed at the same time points. This aberrant circuit formation may provide an anatomic substrate for increased seizure susceptibility and the development of epilepsy.     

Circuit Mechanisms of Seizures in the Pilocarpine Model of Chronic Epilepsy: Cell Loss and Mossy Fiber Sprouting

We used the pilocarpine model of chronic spontaneous recurrent seizures to evaluate the time course of supragranular dentate sprouting and to assess the relation between several changes that occur in epilep tic tissue with different behavioral manifestations of this experimental model of temporal lobe epilepsy. Pilo carpine-induced status epilepticus (SE) invariably led to cell loss in the hilus of the dentate gyrus (DG) and to spontaneous recurrent seizures. Cell loss was often also noted in the DG and in hippocampal subfields CA1 and CA3. The seizures began to appear at a mean of 15 days after SE induction (silent period), recurred at variable frequencies for each animal, and lasted for as long as the animals were allowed to survive (325 days). The granule cell layer of the DG was dispersed in epileptic animals, and neo-Timm stains showed supra-and intragranular mossy fiber sprouting. Supragranular mossy fiber sprout ing and dentate granule cell dispersion began to appear early after SE (as early as 4 and 9 days, respectively) and reached a plateau by 100 days. Animals with a greater degree of cell loss in hippocampal field CAS showed later onset of chronic epilepsy (r= 0.83, p < 0.0005), suggest ing that CA3 represents one of the routes for seizure spread. These results demonstrate that the pilocarpine model of chronic seizures replicates several of the fea tures of human temporal lobe epilepsy (hippocampal cell loss, suprar and intragranular mossy fiber sprouting, den tate granule cell dispersion, spontaneous recurrent sei zures) and that it may be a useful model for studying this human condition. The results also suggest that even though a certain amount of cell loss in specific areas may be essential for chronic seizures to occur, excessive cell loss may hinder epileptogenesis.     

Dentate granule cells in reeler mutants and VLDLR and ApoER2 knockout mice

We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3-CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that ( 1 ) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.     

Calretinin/PSA-NCAM immunoreactive granule cells after hippocampal damage produced by kainic acid and DEDTC treatment in mouse

There is a dramatic increase in the number of lightly immunoreactive calretinin cells in the granular layer of the dentate gyrus of the mouse hippocampus 1 day after excitotoxic injury using kainic acid combined with the zinc chelator diethyldithiocarbamate. At 7 days after treatment, these cells are strongly immunoreactive for calretinin and for the polysialated form of the glycoprotein neural cell adhesion molecule (PSA-NCAM). The reexpression of calretinin and PSA-NCAM after treatment corresponds well with the loss of input from the damaged hilar mossy cells. These cells could be considered immature granule cells since they are immunoreactive to markers for immature cells such as PSA-NCAM, and are not immunoreactive to calbindin D28k and neuronal nuclear specific protein NeuN (present in mature granule cells), or GABA (present in interneurons). Ultrastructural analysis of these cells indicates that they are immature. Labelling of cell proliferation with 5-bromo-2'-deoxyuridine (BrdU) shows that by day 1 no calretinin immunoreactive cell of the dentate gyrus corresponds to newly generated cells. By day 7 only 6% of the calretinin immunoreactive cells in the dentate gyrus are marked for BrdU. Our data indicate that the CR/PSA-NCAM immunoreactive cells of the dentate gyrus, in spite of their immature characteristics, are not the products of reactive neurogenesis. These cells could represent a reservoir of pre-existing not completely differentiated granule cells that react to damage.     

Cell damage and neurogenesis in the dentate granule cell layer of adult rats after pilocarpine- or kainate-induced status epilepticus

Dentate granule cells are generally considered to be relatively resistant to excitotoxicity and have been associated with robust synaptogenesis after neuronal damage. Synaptic reorganization of dentate granule cell axons, the mossy fibers, has been suggested to be relevant for hyperexcitability in human temporal lobe epilepsy and animal models. A recent hypothesis suggested that mossy-fiber sprouting is dependent on newly formed dentate granule cells. However, we recently demonstrated that cycloheximide (CHX) can block the mossy-fiber sprouting that would otherwise be induced by different epileptogenic agents and does not interfere with epileptogenesis in those models. Here, we investigated cell damage and neurogenesis in the dentate gyrus of pilocarpine- or kainate-treated animals with or without coadministration of CHX. Dentate granule cells were highly vulnerable to pilocarpine induced-status epilepticus (SE), but were hardly damaged by kainate-induced SE. CHX pretreatment markedly reduced the number of injured neurons after pilocarpine-induced SE. Induction of SE dramatically increased the mitotic rate of KA- and KA + CHX-treated animals. Induction of SE in animals injected with pilocarpine alone led to 2-7-fold increases in the mitotic rate of dentate granule cells as compared to 5- and 30-fold increases for pilocarpine + CHX animals. We suggest that such increased mitotic rates might be associated with a protection of a vulnerable precursor cell population that would otherwise degenerate after pilocarpine-induced SE. We further suggest that mossy-fiber sprouting and neurogenesis of granule cells are not necessarily linked to one another.     

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.     

The Role of Mossy Cell Death and Activation of Protein Synthesis in the Sprouting of Dentate Mossy Fibers: Evidence from Calretinin and Neo-Timm Staining in Pilocarpine-Epileptic Mice

Summary: Mossy fiber sprouting is a major anatomical reorganization seen in patients with temporal lobe epilepsy and animal models of epilepsy. The final outcome of this reorganization is viewed by many as epileptogenic. Yet, important and relevant data from both human and animal models of epilepsy challenge this prevailing view. Regardless of the outcome of this debate, understanding of the mechanisms that underlie mossy fiber sprouting (MFS) might contribute to our understanding of both the adaptive and maladaptive changes that take place in the nervous system after injury. Available evidence suggests that two events might be crucial for mossy fibers to sprout in epilepsy: the death of mossy cells and the synthesis of trophic factors. The availability of means that prevent MFS, which is normally triggered after induction of status epilepticus, allow for the testing of hypotheses regarding the need for and the sufficiency of specific events for mossy fibers to sprout. We present data on a specific marker for mossy cells, calretinin, in the pilocarpine model of epilepsy in mice. Our data suggest that in the presence of a protein synthesis inhibitor status epilepticus—induced death of mossy cells is not sufficient to trigger mossy fiber sprouting. We suggest that both events, mossy cell death and synthesis of trophic factors, might be necessary for robust MFS to ensue.     

Characterization of Target Cells for Aberrant Mossy Fiber Collaterals in the Dentate Gyrus of Epileptic Rat

Previous studies have demonstrated formation of recurrent excitatory circuits between sprouted mossy fibers and granule cell dendrites in the inner molecular layer of the dentate gyrus (9, 28, 30). In addition, there is evidence that inhibitory nonprincipal cells also receive an input from sprouted mossy fibers (39). This study was undertaken to further characterize possible target cells for sprouted mossy fibers, using immunofluorescent staining for different calcium-binding proteins in combination with Timm histochemical staining for mossy fibers. Rats were injected intraperitoneally with kainic acid in order to induce epileptic convulsions and mossy fiber sprouting. After 2 months survival, hippocampal sections were immunostained for parvalbumin, calbindin D28k, or calretinin followed by Timm-staining. Under a fluorescent microscope, zinc-positive mossy fibers in epileptic rats were found to surround parvalbumin-containing neurons in the granule cell layer and to follow their dendrites, which extended toward the molecular layer. In addition, dendrites of calbindin D28k-containing cells were covered by multiple mossy fiber terminals in the inner molecular layer. However, the calretinin-containing cell bodies in the granule cell layer did not receive any contacts from the sprouted fibers. Electron microscopic analysis revealed that typical Timm-positive mossy fiber terminals established several asymmetrical synapses with the soma and dendrites of nonpyramidal cells within the granule cell layer. These results provide direct evidence that, in addition to recurrent excitatory connections, inhibitory circuitries, especially those responsible for the perisomatic feedback inhibition, are formed as a result of mossy fiber sprouting in experimental epilepsy.     

Sprouting of mossy fibers and the vacating of postsynaptic targets in the inner molecular layer of the dentate gyrus

Aberrant mossy fiber sprouting, which presumably results from hilar mossy cell death after status epilepticus (SE), is a frequently studied feature of temporal lobe epilepsy. Although mossy fiber sprouting can be suppressed by the protein synthesis inhibitor cycloheximide, spontaneous seizures remain unaltered. We have investigated the mechanisms underlying the ability of cycloheximide to block SE-induced mossy fiber sprouting in the inner molecular layer of dentate gyrus (IML). Pilocarpine-induced SE in the presence of cycloheximide resulted in a reduced number of injured hilar cells compared to rats not pretreated with cycloheximide. Presumed mossy cells, identified by calcitonin gene related peptide (CGRP) immunohistochemistry, were not significantly reduced in either group 60 days after SE. Whereas controls had a strong band of CGRP-positive fibers (putative mossy cell axons) and no neo-Timm stained fibers in the IML, pilocarpine-treated rats had no CGRP fibers and strong neo-Timm staining. Cycloheximide-pilocarpine-treated animals, in contrast, had CGRP and neo-Timm staining similar to controls. Cycloheximide might protect hilar CGRP-positive cells during SE and, by allowing those cells to retain their normal axonal projection, prevent mossy fiber sprouting. The recently suggested "irritable" mossy cell hypothesis relies on the survival of mossy cells for network hyperexcitability. We hypothesized that CGRP may be a marker for a subpopulation of relatively resistant mossy cells in rats, which, if they survive injury, may become irritable and contribute to hyperexcitability. We suggest that cycloheximide prevents SE-induced mossy fiber sprouting by preventing the loss of hilar CGRP-positive cells (putative mossy cells).     

Consequences of neonatal seizures in the rat: Morphological and behavioral effects

Whereas neonatal seizures are a predictor of adverse neurological outcome, there is controversy regarding whether seizures simply reflect an underlying brain injury or can cause damage. We subjected neonatal rats to a series of 25 brief flurothyl-induced seizures. Once mature the rats were compared with control littermates for spatial learning and activity level. Short-term effects of recurrent seizures on hippocampal excitation were assessed by using the intact hippocampus formal preparation and long-term effects by assessing seizure threshold. Brains were analysed for neuronal loss, sprouting of granule cell axons (mossy fibers), and neurogenesis. Compared with controls, rats subjected to neonatal seizures had impaired learning and decreased activity levels. There were no differences in paired-pulse excitation or inhibition or duration of afterdischarges in the intact hippocampal preparation. However, when studied as adults, rats with recurrent flurothyl seizures had a significantly lower seizure threshold to pentylenetrazol than controls. Rats with recurrent seizures had greater numbers of dentate granule cells and more newly formed granule cells than the controls. Rats with recurrent seizures also had sprouting of mossy fibers in CA3 and the supragranular region. Recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development.     

Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.     

Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat

Aberrant reorganization of dentate granule cell axons, the mossy fibers, occurs in human temporal lobe epilepsy and rodent epilepsy models. Whether this plasticity results from the remodeling of preexisting mossy fibers or instead reflects an abnormality of developing dentate granule cells is unknown. Because these neurons continue to be generated in the adult rodent and their production increases after seizures, mossy fibers that arise from either developing or mature granule cells are potential substrates for this network plasticity. Therefore, to determine whether seizure-induced, mossy fiber synaptic reorganization arises from either developing or mature granule cell populations, we used low-dose, whole-brain x-irradiation to eliminate proliferating dentate granule cell progenitors in adult rats. A single dose of 5 Gy irradiation blocked cell proliferation and eliminated putative progenitor cells in the dentate subgranular proliferative zone. Irradiation 1 d before pilocarpine-induced status epilepticus significantly attenuated dentate granule cell neurogenesis after seizures. Two irradiations, 1 d before and 4 d after status epilepticus, essentially abolished dentate granule cell neurogenesis but failed to prevent mossy fiber reorganization in the dentate molecular layer. These results indicate that dentate granule cell neurogenesis in the mature hippocampal formation is vulnerable to the effects of low-dose ionizing irradiation. Furthermore, the development of aberrant mossy fiber remodeling in the absence of neurogenesis suggests that mature dentate granule cells contribute substantially to seizure-induced network reorganization.     

Newly born cells in the ageing dentate gyrus display normal migration, survival and neuronal fate choice but endure retarded early maturation

Addition of new granule cells to the dentate gyrus (DG) from stem or progenitor cells declines considerably during ageing. However, potential age-related alterations in migration, enduring survival and neuronal fate choice of newly born cells, and rate of maturation and dendritic growth of newly differentiated neurons are mostly unknown. We addressed these issues by analysing cells that are positive for 5'-bromodeoxyuridine (BrdU), doublecortin (DCX), BrdU and DCX, and BrdU and neuron-specific nuclear antigen (NeuN) in the DG of young adult, middle-aged and aged F344 rats treated with daily injections of BrdU for 12 consecutive days. Analyses performed at 24 h, 10 days and 5 months after BrdU injections reveal that the extent of new cell production decreases dramatically by middle age but exhibits no change thereafter. Interestingly, fractions of newly formed cells that exhibit appropriate migration and prolonged survival, and fractions of newly born cells that differentiate into neurons, remain stable during ageing. However, in newly formed neurons of the middle-aged and aged DG, the expression of mature neuronal marker NeuN is delayed and early dendritic growth is retarded. Thus, the presence of far fewer new granule cells in the aged DG is not due to alterations in the long term survival and phenotypic differentiation of newly generated cells but solely owing to diminished production of new cells. The results also underscore that the capability of the DG milieu to support neuronal fate choice, migration and enduring survival of newly born cells remains stable even during senescence but its ability to promote rapid neuronal maturation and dendritic growth is diminished as early as middle age.     

Mossy fiber sprouting as a potential therapeutic target for epilepsy

Hippocampal mossy fibers, axons of dentate granule cells, converge in the dentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. In the hippocampal formation of temporal lobe epilepsy patients, however, this stereotyped pattern of projection is often collapsed; the mossy fibers branch out of the dentate hilus and abnormally innervate the dentate inner molecular layer, a phenomenon that is termed mossy fiber sprouting. Experimental studies have replicated this sprouting in animal models of temporal lobe epilepsy, including kindling and pharmacological treatment with convulsants. Because these axon collaterals form recurrent excitatory inputs into dendrites of granule cells, the circuit reorganization is assumed to cause epileptiform activity in the hippocampus, whereas some recent studies indicate that the sprouting is not necessarily associated with early-life seizures. Here we review the mechanisms of mossy fiber sprouting and consider its potential contribution to epileptogenesis. Based on recent findings, we propose that the sprouting can be regarded as a result of disruption of the molecular mechanisms underlying the axon guidance. We finally focus on the possibility that prevention of the abnormal sprouting might be a new strategy for medical treatment with temporal lobe epilepsy.     

Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2‐specific markers Purkinje cell protein 4 (PCP4) and regulator of G‐Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin‐expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE‐associated pathological restructuring. © 2015 Wiley Periodicals, Inc.     

Dentate granule cell GABA(A) receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology

The dentate gyrus (DG) normally functions as a filter, preventing propagation of synchronized activity into the seizure-prone hippocampus. This filter or 'gatekeeper' attribute of the DG is compromised in various pathological states, including temporal lobe epilepsy (TLE). This study examines the role that altered inhibition may play in the deterioration of this crucial DG function. Using the pilocarpine animal model of TLE, we demonstrate that inhibitory synaptic function is altered in principal cells of the DG. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in dentate granule cells (DGCs) from epileptic animals were larger, more sensitive to blockade by zinc and less sensitive to augmentation by the benzodiazepine type site 1 modulator zolpidem. Furthermore, mIPSCs examined during a quiescent period following injury but preceding onset of epilepsy were significantly smaller than those present either in control or in TLE DGCs, and had already acquired sensitivity to blockade by zinc prior to the onset of spontaneous seizures. Rapid agonist application experiments demonstrated that prolonged (>35 ms) exposure to zinc is required to block GABAA receptors (GABAARs) in patches pulled from epileptic DGCs. Therefore, zinc must be tonically present to block DGC GABAARs and alter DG function. This would occur only during repetitive activation of mossy fibres. Thus, in the pilocarpine animal model of TLE, an early, de novo, expression of zinc-sensitive GABAARs is coupled with delayed, epilepsy-induced development of a zinc delivery system provided by aberrant sprouting of zinc-containing mossy fibre recurrent collaterals. The temporal and spatial juxtaposition of these pathophysiological alterations may compromise normal 'gatekeeper' function of the DG through dynamic zinc-induced failure of inhibition, predisposing the hippocampal circuit to generate seizures.     

Origin of microglia in the quail retina: Central-to-peripheral and vitreal-to-scleral migration of microglial precursors during development

Collateral sprouting of dentate granule cell axons, the mossy fibers, occurs in response to denervation, kindling, or excitotoxic damage to the hippocampus. Organotypic slice culture of rodent hippocampal tissue is a model system for the controlled study of collateral sprouting in vitro. Organotypic roller-tube cultures were prepared from hippocampal slices derived from postnatal day 7 mice. The Timm heavy metal stain and densitometry were used to assay the degree of mossy fiber collateral sprouting in the molecular layer of the hippocampal dentate gyrus. Factors influencing mossy fiber collateral sprouting were time in culture, positional origin of the slice culture along the septotemporal axis of the hippocampus, and presence of attached subicular-entorhinal cortical tissues. Collateral sprouting in the molecular layer was first detected after 6 days in culture and increased steadily thereafter. By 2 weeks considerable sprouting was apparent, and at 3 weeks intense sprouting was observed within the molecular layer. An intrinsic septal-to-temporal gradient of collateral sprouting was apparent at 14 days in culture. To determine whether differential damage to the mossy fibers was the basis for the differences in collateral sprouting along the septotemporal axis, we made complete transections of the mossy fiber projection as it exited the dentate hilus at various levels along the septotemporal axis; no differences were found on subsequent collateral sprouting in the dentate molecular layer. Timm-stained hippocampal cultures with an attached entorhinal cortex, a major source of afferent innervation to the dentate granule cells, displayed significantly less collateral sprouting at 10 days in culture compared to that in cultures from adjacent sections without attached subicular-entorhinal tissues present. Thus, time in culture, position along the septotemporal axis, and presence of afferent cortical tissues influence aberrant neurite collateral sprouting in organotypic slice cultures of neonatal mouse hippocampus. © Wiley-Liss, Inc.