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.     

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.     

Physiologic and Morphologic Characteristics of Granule Cell Circuitry in Human Epileptic Hippocampus

Summary: Morphological and electrophysiological techiques were used to examine granule cells and their mossy fiber axons in nine surgically resected hippocampal specimens from temporal lobe epilepsy (TLE) patients. Timm histochemistry showed mossy fiber sprouting into the inner molecular layer (IML) of the dentate in a subset of tissue samples. In slices from five tissue samples, stimulus-induced bursting activity could be induced with a low concentration (2.5 μM) of bicuculline; bursts were sensitive to the N -methyl- d -aspartate (NMDA) blocker, APV. There was a general correlation between such sprouting and experimentally induced yperexcit ability. Fourteen granule cells from five tissue samples were intracellularly stained [with lucifer yellow (LY) or neurobiotin]. Axons from a subset of these neurons showed axon collaterals reaching into the IML, but this axon projection pattern for single cells was not directly correlated with degree of mossy fiber sprouting shown grossly by Timm staining. Electron microscopic examination of intracellularly stained elements showed mossy fiber axon terminals making asymmetric synaptic contacts (including autapses on the granule cell dendrite) with dendritic shafts and spines in both apical and basal domains. These data are consistent with the hypothesis that mossy fiber sprouting provides a structural basis for recurrent excitation of granule cells, but does not provide direct support of the hypothesis that mossy fiber sprouting causes hyperexcitability. The data suggest that granule cell bursting activity is at least in part a function of compromised synaptic inhibition, since levels of γ-aminobutyric acid (GABA) blockade that are generally subthreshold for burst induction were epileptogenic in some tissue samples from human epileptic hippocampus.     

Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus.

This study examined the acute actions of brain-derived neurotrophic factor (BDNF) in the rat dentate gyrus after seizures, because previous studies have shown that BDNF has acute effects on dentate granule cell synaptic transmission, and other studies have demonstrated that BDNF expression increases in granule cells after seizures. Pilocarpine-treated rats were studied because they not only have seizures and increased BDNF expression in granule cells, but they also have reorganization of granule cell "mossy fiber" axons. This reorganization, referred to as "sprouting," involves collaterals that grow into novel areas, i.e., the inner molecular layer, where granule cell and interneuron dendrites are located. Thus, this animal model allowed us to address the effects of BDNF in the dentate gyrus after seizures, as well as the actions of BDNF on mossy fiber transmission after reorganization. In slices with sprouting, BDNF bath application enhanced responses recorded in the inner molecular layer to mossy fiber stimulation. Spontaneous bursts of granule cells occurred, and these were apparently generated at the site of the sprouted axon plexus. These effects were not accompanied by major changes in perforant path-evoked responses or paired-pulse inhibition, occurred only after prolonged (30-60 min) exposure to BDNF, and were blocked by K252a. The results suggest a preferential action of BDNF at mossy fiber synapses, even after substantial changes in the dentate gyrus network. Moreover, the results suggest that activation of trkB receptors could contribute to the hyperexcitability observed in animals with sprouting. Because human granule cells also express increased BDNF mRNA after seizures, and sprouting can occur in temporal lobe epileptics, the results may have implications for understanding temporal lobe epilepsy.     

Opioid modulation of recurrent excitation in the hippocampal dentate gyrus.

kappa opioid receptor activation inhibits granule cell-mediated excitatory neurotransmission in the hippocampal formation via a decrease in glutamate release from both perforant path and mossy fiber terminals. We now report a third, anatomically and pharmacologically distinct site of such kappa opioid inhibition within the hippocampus. Granule cell population responses to selective stimulation of an excitatory hilar pathway were decreased by the kappa(1) opioid receptor agonist U69,593, an effect blocked by the kappa(1) antagonist norbinaltorphimine. U69,593 also inhibited hilar path induced long-term potentiation (LTP) of granule cell responses. LTP in this pathway was also blocked by the NMDA receptor antagonist d-2-amino-5-phosphonovalerate, unlike granule cell mossy fiber LTP in CA3. The kappa opioid peptide dynorphin is present in hilar mossy fiber collaterals. Ultrastructural analysis of these collaterals demonstrated dynorphin-containing vesicles in asymmetric synapses formed between axon terminals and granule cell dendrites, suggesting direct granule cell-granule cell connections. Evoked release of endogenous dynorphin within the hilus was effective in reducing hilar excitation of granule cells, although this release, in contrast to the release of dynorphin in the dentate molecular layer, was not dependent on L-type calcium channels. No hilar path excitation was observed in the absence of bicuculline, suggesting a strong GABA(A)-mediated inhibition of this pathway. However, hilar path activity could be seen after LTP, with or without bicuculline. Thus, kappa opioids can inhibit granule cell recurrent excitation, likely via effects on excitatory mossy fiber collaterals. Such collaterals are thought to be important in mediating temporal lobe epilepsy.     

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.     

Seizure frequency correlates with loss of dentate gyrus GABAergic neurons in a mouse model of temporal lobe epilepsy

Epilepsy occurs in one of 26 people. Temporal lobe epilepsy is common and can be difficult to treat effectively. It can develop after brain injuries that damage the hippocampus. Multiple pathophysiological mechanisms involving the hippocampal dentate gyrus have been proposed. This study evaluated a mouse model of temporal lobe epilepsy to test which pathological changes in the dentate gyrus correlate with seizure frequency and help prioritize potential mechanisms for further study. FVB mice (n = 127) that had experienced status epilepticus after systemic treatment with pilocarpine 31–61 days earlier were video‐monitored for spontaneous, convulsive seizures 9 hr/day every day for 24–36 days. Over 4,060 seizures were observed. Seizure frequency ranged from an average of one every 3.6 days to one every 2.1 hr. Hippocampal sections were processed for Nissl stain, Prox1‐immunocytochemistry, GluR2‐immunocytochemistry, Timm stain, glial fibrillary acidic protein‐immunocytochemistry, glutamic acid decarboxylase in situ hybridization, and parvalbumin‐immunocytochemistry. Stereological methods were used to measure hilar ectopic granule cells, mossy cells, mossy fiber sprouting, astrogliosis, and GABAergic interneurons. Seizure frequency was not significantly correlated with the generation of hilar ectopic granule cells, the number of mossy cells, the extent of mossy fiber sprouting, the extent of astrogliosis, or the number of GABAergic interneurons in the molecular layer or hilus. Seizure frequency significantly correlated with the loss of GABAergic interneurons in or adjacent to the granule cell layer, but not with the loss of parvalbumin‐positive interneurons. These findings prioritize the loss of granule cell layer interneurons for further testing as a potential cause of temporal lobe epilepsy.     

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.     

Alumina gel injections into the temporal lobe of rhesus monkeys cause complex partial seizures and morphological changes found in human temporal lobe epilepsy

The goal of the present study was to determine whether alumina gel injections into temporal lobe structures cause complex partial seizures (CPS) and pathological changes observed in human temporal lobe epilepsy. Rhesus monkeys with alumina gel injections in the amygdala, perirhinal and entorhinal cortices, or Ammon's horn and dentate gyrus all initially displayed focal pathological electroencephalographic (EEG) slowing limited to the site of injection. After clinical seizures developed, they also displayed widespread pathological EEG slowing over both hemispheres, interictal and ictal epileptiform EEG abnormalities limited to the mesial–inferior temporal lobe on the side of injection, and different degrees of spread to other ipsilateral and contralateral structures. Noninjected control and nonepileptic monkeys with injections into the middle and inferior temporal gyri displayed no hippocampal neuronal loss or mossy fiber sprouting. When alumina gel was injected into the amygdala, CPS began within 3–6 weeks and degeneration of neurons and gliosis occurred in the perirhinal cortex or the hippocampus, with consequent sprouting of mossy fibers in the dentate gyrus. Dispersion of the granule cell layer was also observed. Other monkeys with alumina gel in the perirhinal and entorhinal cortices developed CPS within 2–3 weeks after the injections and displayed mossy fiber sprouting only after 4 weeks after the injections. Alumina gel in Ammon's horn and the dentate gyrus also induced CPS, but mossy fiber sprouting was limited to sites immediately adjacent to the injection, probably because none survived more than 4 weeks after the injections. This nonhuman primate model of CPS displayed similar anatomical, behavioral, and EEG features as observed in human temporal lobe epilepsy and provides opportunities to analyze the chronological sequence of epileptogenesis and to test potential therapies. J. Comp. Neurol. 401:266–290, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Association Between the Density of Mossy Fiber Sprouting and Seizure Frequency in Experimental and Human Temporal Lobe Epilepsy

Summary: Purpose : If the sprouting of granule cell axons or mossy fibers in the dentate gyrus is critical for the generation of spontaneous seizures in temporal lobe epilepsy (TLE), one could hypothesize that epileptic animals or humans with increased sprouting would have more frequent seizures. This hypothesis was tested by analyzing the data gathered from experimental and human epilepsy.
Methods : In experiment I (rats with "newly diagnosed" TLE), self-sustained status epilepticus was induced in rats by electrically stimulating the amygdala. Thereafter, the appearance of spontaneous seizures was monitored by continuous video-electroencephalography (EEG) until the animal developed two spontaneous seizures and for 11 d thereafter. Rats were perfused for histology, and mossy fibers were stained using the Timm method. In experiment II (rats with "recently diagnosed" TLE), status epilepticus was induced in rats and the development of seizures was monitored by video-EEG for 24 h/d every other day for 60 days. All animals were then perfused for histology. In experiment III (rats with "chronic" TLE), animals were monitored by video-EEG for 24 h/d every other day for 6 months before histologic analysis. To assess mossy fiber sprouting in human TLE, hippocampal sections from 31 patients who had undergone surgery for drug-refractory TLE were stained with an antibody raised against dynorphin.
Results and Conclusions : Our data indicate that the density of mossy fiber sprouting is not associated with the total number of lifetime seizures or the seizure frequency in experimental or human TLE.     

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.     

Strong paired pulse depression of dentate granule cells in slices from patients with temporal lobe epilepsy

Summary. Recurrent, feedback excitation by sprouted mossy fibers may contribute to the hyperexcitability observed in human temporal lobe epilepsy. Observations in rodent models of epilepsy mimic the findings in human tissue and reveal that dentate granule cells sprout axons which innervate fibers in their own dendritic layer. However, recent evidence in rodents suggest that these sprouted fibers may form connections which cause inhibition of dentate granule cells, not excitation. Thus, the net effect of sprouting in human epileptic tissue may not only be recurrent excitation. We analyzed paired pulse depression in dentate slices from 9 patients with temporal lobe epilepsy and found evidence for strong feedback inhibition. We also noted failure of high frequency stimulation induced inhibition in our human specimens. These data challenge the concept that human epileptic dentate granule cells are excited by recurrent mossy fiber sprouting. Accepted February 20, 1998; received December 1997     

Prolonged infusion of tetrodotoxin does not block mossy fiber sprouting in pilocarpine-treated rats

PURPOSE: Mossy fiber sprouting is a common abnormality found in patients and models of temporal lobe epilepsy. The role of mossy fiber sprouting in epileptogenesis is unclear, and its blockade would be useful experimentally and perhaps therapeutically. Results from previous attempts to block mossy fiber sprouting have been disappointing or controversial. In some brain regions, prolonged application of the sodium channel blocker tetrodotoxin prevents axon sprouting and posttrauma epileptogenesis. The present study tested the hypothesis that prolonged, focal infusion of tetrodotoxin would block mossy fiber sprouting after an epileptogenic treatment. METHODS: Adult rats were treated with pilocarpine to induce status epilepticus. Several hours to 3 days after pilocarpine treatment, a pump with a cannula directed toward the dentate gyrus was implanted to deliver 10 microM tetrodotoxin or vehicle alone at 0.25 microl/h. This method blocks local EEG activity in the hippocampus (Galvan et al. J Neurosci 2000; 20:2904-16). After 28 days of continuous infusion, rats were perfused with fixative, and their hippocampi analyzed anatomically with stereologic techniques. RESULTS: Tetrodotoxin infusion was verified immunocytochemically in tetrodotoxin-treated but not vehicle-treated hippocampi. Tetrodotoxin-infused and vehicle-infused hippocampi displayed similar levels of hilar neuron loss. The Timm stain revealed mossy fiber sprouting regardless of whether hippocampi were treated with tetrodotoxin infusion, vehicle infusion, or neither. CONCLUSIONS: Prolonged infusion of tetrodotoxin did not block mossy fiber sprouting. This finding suggests that sodium channel-mediated neuronal activity is not necessary for mossy fiber sprouting after an epileptogenic treatment.     

Mossy fiber sprouting interacts with sodium channel mutations to increase dentate gyrus excitability

Purpose:  Idiopathic epilepsy is caused by the complex interaction of genetic and environmental factors. The purpose of this study was to use computational approaches to explore the interaction between changes in sodium channel availability caused by mutations and mossy fiber sprouting.
Methods:  We used a previously published biophysically realistic computer model of dentate gyrus neurons and networks. A sensitivity analysis probed the effects of typical mutation-like changes in either single- or multiple-gating parameters. Isolated neuron models were stimulated with current injections, and networks were stimulated with perforant path synaptic input. The gene–environment interaction was studied by including mossy fiber sprouting into these networks.
Results:  Single neuron responses to current injections were complex, with increased sodium channel availability paradoxically reducing firing rates. In the absence of mossy fiber sprouting, control networks showed strong accommodation supporting the role of the dentate gyrus as a gate. Availability changes alone were not able to drive the networks into ictal states, even though they reduced the effectiveness of the dentate gyrus gate. Interestingly, the presence of electrophysiologic changes substantially increased the ability of mossy fiber sprouting to induce ictal activity.
Conclusion:  (1) Increased sodium channel availability does not necessarily lead to increased firing rates, (2) network excitability is most sensitive to changes in steady state activation of sodium channels, (3) mutation-induced changes in availability reduce the effectiveness of the dentate gyrus gate, and (4) mutations interact strongly with structural network changes to allow ictal-like activity in the dentate gyrus.     

癫痫大鼠海马出芽苔藓纤维突触的超微结构特征

目的:探讨匹罗卡品颞叶癫痫大鼠海马出芽苔藓纤维突触的超微结构特征及其在颞叶癫痫发病机制中的作用。方法:采用Timm组化染色标记出芽苔藓纤维突触末端,在电镜下观察新生突触的类型、比例、定位、以及突触后靶成分。结果:颞叶癫痫大鼠齿状回内分子层可见到银标记的突触末端,出芽苔藓纤维突触主要是轴棘型非对称性突触,其次是轴树型非对称性突触,偶可看到出芽轴突和颗粒细胞体形成突触联系。结论:轴棘型非对称性突触是颞叶癫痫大鼠海马出芽苔藓纤维突触的主要类型,出芽苔藓纤维突触的超微结构特性支持重组突触形成重复的兴奋性环路,而且形成的新的兴奋性环路可能在颞叶癫痫的发生与发展中起重要作用。     

Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus

This study determined in temporal lobe epilepsy patients if there were correlations among hippocampal granule cell expression of neurotrophin mRNAs, aberrant supragranular mossy fiber sprouting, and neuron losses. Consecutive surgically resected hippocampi (n=9) and comparison tissue from autopsies (n=3) were studied for:

1. Granule cell mRNA levels usingin situ hybridization for brainderived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3);
2. neo-Timm supragranular mossy fiber sprouting; and
3. Ammon’s horn neuron densities.

Clinically, patients were classified into those with hippocampal sclerosis (HS;n=7) and non-HS cases (i.e., mass lesions and autopsies;n=5). Results showed that compared to non-HS cases, HS patients showed increased granule cell mRNA levels for BDNF, NGF, and NT-3 (p=0.035,p=0.04,p=0.045 respectively; one-tail directional test). Moreover, granule cell BDNF mRNA levels correlated inversely with Ammon’s horn neuron densities (p=0.02) and correlated positively with greater supragranular mossy fiber sprouting (p=0.02). NGF mRNA levels correlated inversely with Ammon’s horn neuron densities (p=0.02), and TN-3 mRNA levels correlated inversely with age at surgery (p=0.04) and correlated positively with greater mossy fiber sprouting (p=0.026). These results indicate in the chronically damaged human hippocampus that granule cells express neurotrophin mRNAs, and mRNA levels correlate with either hippocampal neuron losses or aberrant supragranular mossy fiber sprouting. These data support the hypothesis that in the epileptic human hippocampus, there may be pathophysiologic associations among mossy fiber synaptic plasticity, hippocampal neuron damage, and granule cell mRNA neurotrophin levels.     

Detection of Increased Local Excitatory Circuits in the Hippocampus during Epileptogenesis Using Focal Flash Photolysis of Caged Glutamate

Summary:  Purpose: Local synaptic circuits, particularly recurrent excitation, are hypothesized to contribute to the generation and synchronization of epileptiform activity. The present study tested whether local excitatory circuits in the hippocampus are increased in an animal model of temporal lobe epilepsy, and thus may contribute to epileptic seizures.
Methods: Rats were given hourly injections of kainic acid to induce status epilepticus, which led to chronic epilepsy with spontaneous recurrent seizures. Whole-cell recording was performed in hippocampal slices, and focal flash photolysis of caged glutamate was used to detect local excitatory circuits.
Results: In the dentate gyrus of rats with kainate-induced epilepsy and mossy fiber sprouting, focal stimulations with caged glutamate at many different sites in the granule cell layer consistently evoked repetitive excitatory postsynaptic currents (EPSCs) in normal medium and prolonged bursts of action potentials in bicuculline; these responses were not observed in similarly treated slices from control rats. In CA1, focal flash photolysis of caged glutamate in stratum pyramidale revealed significantly more excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy than saline-treated control animals.
Conclusion: Focal flash photolysis of caged glutamate revealed that new local excitatory circuits are formed in both the dentate gyrus and CA1 area of rats with kainate-induced epilepsy, which supports the hypothesis that the progressive formation of new local excitatory circuits occurs in many locations during epileptogenesis.     

Rapid induction by kainic acid of both axonal growth and F1/GAP-43 protein in the adult rat hippocampal granule cells

Hippocampal granule cells do not normally express the axonal growth and plasticity-associated protein F1/GAP-43 in the adult rat. Using three different methods that lead to hypersynchronous activity in limbic circuits, expression of F1/GAP-43 mRNA can be induced in granule cells which is followed by sprouting in mossy fibers, the axons of granule cells. F1/GAP-43 mRNA expression in granule cells was induced in the temporal, but not septal, hippocampus beginning at 12 hours after kainic acid (KA) subcutaneous injection (10 mg/kg). Beginning 2 days after KA treatment, mossy fiber sprouts restricted to the temporal hippocampus were observed in the supragranular layer. In the same animal we also observed that levels of protein F1/GAP-43 immunoreactivity in this layer apparently increased at this same 2 day time point and same ventral hippocampal location. F1/GAP-43 protein levels and mossy fiber sprouting showed an increase up to 10 days after KA treatment. Sprouting was at a maximum at 40 days, the longest time point studied. These events parallel axonal regeneration with one critical difference: granule cell axons are not damaged by kainate. The rapid onset of axonal growth in the adult is striking and occurs earlier than reported previously (2 days vs. 12 days). Such growth closely associated with elevated levels of protein F1/GAP-43 may occur as a result of a) reactive synaptogenesis caused by the availability of post-synaptic surface on granule cell dendrites at the supragranular layer, b) Hebbian co-activation of the post-synaptic granule cells and their presynaptic afferents, and c) loss of target-derived inhibitory growth factor. © 1996 Wiley-Liss, Inc.     

Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: Visualization after retrograde transport of biocytin

In complex partial epilepsy and in animal models of epilepsy, hippocampal mossy fibers appear to develop recurrent collaterals, that invade the dentate molecular layer. Mossy fiber collaterals have been proposed to subserve recurrent excitation by forming granule cell-granule cell synapses. This hypothesis was tested by visualizing dentate granule cells and their mossy fibers after terminal uptake and retrograde transport of biocytin. Labeling studies were performed with transverse slices of the caudal rat hippocampal formation prepared 2.6–l70.0 weeks after pilocarpine-induced or kainic acid-induced status epilepticus. Light microscopy demonstrated the progressive growth of recurrent mossy fibers into the molecular layer; the densest innervation was observed in slices from pilocarpine-treated rats that had survived 10 weeks or longer after status epilepticus. Thin mossy fiber collaterals originated predominantly from deep within the hilar region, crossed the granule cell body layer, and formed an axonal plexus oriented parallel to the cell body layer within the inner one-third of the molecular layer. When sprouting was most robust, some recurrent mossy fibers at the apex of the dentate gyrus reached the outer two-thirds of the molecular layer. The distribution and density of mossy fiber-like Timm staining correlated with the biocytin labeling. When viewed with the electron microscope, the inner one-third of the dentate molecular layer contained numerous mossy fiber boutons. In some instances, biocytin-labeled mossy fiber boutons were engaged in synaptic contact with biocytin-labeled granule cell dendrites. Granule cell dendrites did not develop large complex spines (“thorny excrescences”) at the site of synapse formation, and they did not appear to have been permanently damaged by seizure activity. These results establish the validity of Timm staining as a marker for mossy fiber sprouting and support the view that status epilepticus provokes the formation of a novel recurrent excitatory circuit in the dentate gyrus. Retrograde labeling with biocytin showed that the recurrent mossy fiber projection often occupies a considerably greater fraction of the dendritic region than previous studies had suggested. © 1995 Wiley-Liss, Inc.