Mossy fiber synaptic reorganization induced by kindling: time course of development, progression, and permanence

Recent studies have revealed that mossy fiber axons of granule cells in the dentate gyrus undergo reorganization of their terminal projections in both animal models of epilepsy and human epilepsy. This synaptic reorganization has been demonstrated by the Timm method, a histochemical technique that selectively labels synaptic terminals of mossy fibers because of their high zinc content. It has been generally presumed that the reorganization of the terminal projections of the mossy fiber pathway is a consequence of axonal sprouting and synaptogenesis by mossy fibers. To evaluate this possibility further, the time course for development of Timm granules, which correspond ultrastructurally to mossy fiber synaptic terminals, was examined in the supragranular layer of the dentate gyrus at the initiation of kindling stimulation with an improved scoring method for assessment of alterations in Timm histochemistry. The progression and permanence of this histological alteration were similarly evaluated during the behavioral and electrographic evolution of kindling evoked by perforant path, amygdala, or olfactory bulb stimulation. Mossy fiber synaptic terminals developed in the supragranular region of the dentate gyrus by 4 d after initiation of kindling stimulation in a time course compatible with axon sprouting. The induced alterations in the terminal projections of the mossy fiber pathway progressed with the evolution of behavioral kindled seizures, became permanent in parallel with the development of longlasting susceptibility to evoked seizures, and were observed as long as 8 months after the last evoked kindled seizure. The results demonstrated a strong correlation between mossy fiber synaptic reorganization and the development, progression, and permanence of the kindling phenomenon.     

Long-term potentiation trains induce mossy fiber sprouting

It has been shown that both amygdaloid and hippocampal kindling induce sprouting of the mossy fibers in the dentate gyrus. In this study, we investigated whether non-epileptogenic stimulation could also induce mossy fiber sprouting. Long-term potentiation (LTP) was induced in the dentate gyrus by the application of brief, high-frequency trains to the perforant path. The potentiating stimulation was applied each day for 10 days, and the tissue was prepared for Timm labelling 7 days later. Sprouting was significantly increased in the LTP group compared to the implanted control rats. These results suggest that mossy fiber sprouting is not damage-induced and is dependent on neuronal activation.     

Mossy fiber synaptic reorganization in the epileptic human temporal lobe

The distribution of the mossy fiber synaptic terminals was examined using the Timm histochemical method in surgically excised hippocampus and dentate gyrus from patients who underwent lobectomy of the anterior part of the temporal lobe for refractory partial complex epilepsy. The dentate gyrus of epileptic patients demonstrated intense Timm granules and abundant mossy fiber synaptic terminals in the supragranular region and the inner molecular layer. In contrast, the dentate gyrus of presenescent nonepileptic primates demonstrated no Timm granules in the supragranular region. In nonepileptic senescent primates, occasional very sparse supragranular Timm granules were results are morphological evidence of mossy fiber synaptic reorganization in the temporal lobe of epileptic humans, and suggest the intriguing possibility that mossy fiber sprouting and synaptic reorganization induced by repeated partial complex seizures may play a role in human epilepsy.     

The expression of GABA in mossy fiber synaptosomes coincides with the seizure-induced expression of GABAergic transmission in the mossy fiber synapse

Although the granule cells of the dentate gyrus are glutamatergic, they contain the machinery for the synthesis and vesiculation of GABA. Furthermore, glutamic acid decarboxylase and the vesicular GABA transporter mRNA are expressed in the granule cells and mossy fibers in an activity-dependent manner, suggesting that these cells release GABA in addition to glutamate. Supporting this hypothesis, we found that seizures induce simultaneous glutamatergic and GABAergic transmission in the mossy fiber projection. To further explore this expression of inhibition, we looked for the presence and expression of endogenous GABA in a synaptosomal preparation enriched with mossy fiber nerve endings of kindled rats. We also studied the capacity of this preparation to capture and release [(3)H]GABA under control conditions and after kindling epilepsy. In accordance with our hypothesis we show that the mossy fiber synaptosomal preparation of the kindled rats has a significantly higher content of endogenous GABA than controls. We also found that the protein content in the mossy fiber synaptosomal preparation of kindled rats was significantly augmented, which is consistent with mossy fiber sprouting. Due to this, the total [(3)H]GABA incorporated in the synaptosomal preparation was also augmented. However, [(3)H]GABA uptake (expressed in % radioactivity/mg protein) and its evoked release were similar in both groups. With the present results, we provide further support for the hypothesis of the emergence of GABAergic transmission in the mossy fiber synapse that can constitute a protective mechanism in response to seizures.     

Intragranular mossy fibers in rats and gerbils form synapses with the somata and proximal dendrites of basket cells in the dentate gyrus.

Intragranular and supragranular mossy fibers arise from granule cells and are present in the dentate gyrus of hippocampi from kindled and epileptic animals. The intragranular fibers often appear as fibers perpendicular to the long axis of the granule cell layer at periodic intervals. Rats and gerbils were analyzed to determine whether such mossy fibers are also associated with nongranule cells (including the basket cells), which send their apical dendrites through this layer with a periodicity similar to that of mossy fibers. The results for rats and both epileptic and nonepileptic gerbils show that many intragranular mossy fibers are apposed to the surfaces of the somata and apical dendrites of basket cells where they form asymmetric synapses. This plexus of mossy fiber axons appears to follow the dendrites of these neurons into the inner molecular layer. Based on previous data indicating that basket cells are GABAergic inhibitory neurons, the present findings in normal rats and both types of gerbils suggest that intragranular and supragranular mossy fibers provide additional circuitry for feedback inhibition to granule cells. It is possible that under pathological conditions, such as denervation or kindling, these fibers sprout and form synapses with granule cells.     

Septotemporal variation of the supragranular projection of the mossy fiber pathway in the dentate gyrus of normal and kindled rats.

Previous studies have demonstrated regional variation in the anatomical organization and physiological properties of the hippocampus along its septotemporal (dorsoventral) axis. In this study, regional variation of the supragranular projection of the mossy fiber pathway in the dentate gyrus of normal and kindled rats was characterized with a scoring method for assessment of the distribution of mossy fiber synaptic terminals detected by Timm histochemistry. In normal rats, there was a sparse projection of the mossy fiber pathway into the supragranular region near the tips and crest of the dentate gyrus along the entire septotemporal axis, and a prominent projection into the supragranular region at the temporal pole. Kindling of the perforant path, amygdala, and olfactory bulb induced synaptic reorganization of the mossy fiber pathway into the supragranular region along the entire septotemporal axis of the dentate gyrus. There was regional variation of the seizure-induced synaptic reorganization along this axis, and distinct septotemporal patterns were observed as a function of the site of kindling stimulation. Kindling of the perforant path induced mossy fiber synaptic reorganization that was relatively more prominent in the septal pole than in the temporal pole of the dentate gyrus. In contrast, rats that received kindling stimulation of the amygdala had a more uniform distribution of synaptic reorganization along the septotemporal axis. As there is regional variation of the anatomical and physiological properties of the human epileptic hippocampus, these observations could be pertinent to human epilepsy.     

Vesicular GABA transporter mRNA expression in the dentate gyrus and in mossy fiber synaptosomes

In the normal granule cells of the dentate gyrus, glutamate and both gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) coexist. GAD expression is increased after seizures, and simultaneous glutamatergic and GABAergic neurotransmission from the mossy fibers to CA3 appears, supporting the hypothesis that GABA can be released from the mossy fibers. To sustain GABAergic neurotransmission, the amino acid must be transported into synaptic vesicles. To address this, using RT-PCR we looked for the presence and regulation of expression of the vesicular GABA transporter (VGAT) mRNA in the dentate gyrus and in mossy fiber synaptosomes of control and kindled rats. We found trace amounts of VGAT mRNA in the dentate gyrus and mossy fiber synaptosomes of control rats. In the dentate gyrus of kindled rats with several seizures and of control rats subject to one acute seizure, no changes were apparent either 1 or 24 h after the seizures. However, repetitive synaptic or antidromic activation of the granule cells in slices of control rats in vitro induces an activity-dependent enhancement of VGAT mRNA expression in the dentate. Surprisingly, in the mossy fiber synaptosomes of seizing rats, the levels of VGAT mRNA were significantly higher than in controls. These data show that the granule cells and their mossy fibers, besides containing machinery for the synthesis of GABA, also contain the elements that support its vesiculation. This further supports the notion that local synaptic molecular changes enable mossy fibers to release GABA in response to enhanced excitability.     

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.     

Mossy fiber sprouting in the dentate gyrus in a newly developed epileptic mutant, Ihara epileptic rat

We examined the correlation between seizure activity and development of mossy fiber sprouting in the hippocampal formation using Timm staining in a newly developed Ihara epileptic rat (IER). The sprouting of mossy fibers were clearly shown in the inner molecular portion of the dentate gyrus and in the stratum oriens of CA3 pyramidal cell layer with repeated seizures. A positive correlation between the frequency of generalized tonic and clonic convulsions and the Timm staining score in molecular layer of dentate gyrus was revealed. Sprouting of mossy fiber in IER seems to be linked with seizure activities resulting from epileptic bursts, not to the genetic mutation.     

The effect of amygdala kindling on hippocampal neurogenesis coincides with decreased reelin and DISC1 expression in the adult dentate gyrus

Temporal lobe seizures can induce the proliferation and abnormal migration of newly generated dentate granule cells, but little is known about the molecular mechanisms that govern these pathological events. Reelin and DISC1 (disrupted‐in‐schizophrenia 1) are proteins that play a regulatory role in the maturation and integration of new neurons in the developing and adult brain. In this study, we examined whether amygdala kindling results in aberrant neurogenesis and altered expression of reelin and DISC1 in the adult dentate gyrus. Using doublecortin immunohistochemistry, we found that short‐term kindling (i.e., 30 electrical stimulations) significantly increased the number of immature neurons in the dentate subgranular zone (SGZ), whereas long‐term kindling (i.e., 99 electrical stimulations) did not. However, doublecortin‐labeled neurons in long‐term kindled rats showed greater dendritic complexity than they did in short‐term kindled or control rats. We also found that long‐term kindling decreased the number of reelin‐positive cells and decreased DISC1 expression in the dentate granule cell layer and subgranular zone. Interestingly, kindling‐induced changes in reelin and DISC1 expression coincided with the appearance of ectopically located Prox1‐labeled granule cells in the hilus. These effects occurred independently of alterations in granule cell layer length, dentate volume, or the number of hilar neurons. Taken together, these findings suggest a novel role for DISC1 in the pathophysiology of temporal lobe epilepsy and further suggest that changes in reelin and DISC1 expression may contribute to aberrant neurogenesis in the kindling model. © 2009 Wiley‐Liss, Inc.     

Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats

The mossy fiber pathway in the dentate gyrus undergoes sprouting and synaptic reorganization in response to seizures. The types of new synapses, their location and number, and the identity of their postsynaptic targets determine the functional properties of the reorganized circuitry. The goal of this study was to characterize the types and proportions of sprouted mossy fiber synapses in kindled and kainic acid-treated rats. In normal rats, synapses labeled by Timm histochemistry or dynorphin immunohistochemistry were rarely observed in the supragranular region of the inner molecular layer when examined by electron microscopy. In epileptic rats, sprouted mossy fiber synaptic terminals were frequently observed. The ultrastructural analysis of the types of sprouted synapses revealed that 1) in the supragranular region, labeled synaptic profiles were more frequently axospinous than axodendritic, and many axospinous synapses were perforated; 2) sprouted mossy fiber synaptic terminals formed exclusively asymmetric, putatively excitatory synapses with dendritic spines and shafts in the supragranular region and with the soma of granule cells in the granule cell layer; 3) in contrast to the large sprouted mossy fiber synapses in resected human epileptic hippocampus, the synapses formed by sprouted mossy fibers in rats were smaller; and 4) in several cases, the postsynaptic targets of sprouted synapses were identified as granule cells, but, in one case, a sprouted synaptic terminal formed a synapse with an inhibitory interneuron. The results demonstrate that axospinous asymmetric synapses are the most common type of synapse formed by sprouted mossy fiber terminals, supporting the viewpoint that most sprouted mossy fibers contribute to recurrent excitation in epilepsy.     

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.     

法舒地尔对戊四氮点燃大鼠海马丝切蛋白及苔藓纤维出芽的影响

目的 探讨法舒地尔对戊四氮(PTZ)点燃大鼠海马组织中丝切蛋白(cofilin,非磷酸化形式)表达与苔藓纤维出芽程度关系的影响.方法 210只SD雄性大鼠分成戊四氮组、法舒地尔干预组和生理盐水对照组,采用PTZ慢性点燃癫癎模型,应用SABC法检测cofilin表达,用Timm染色检测苔鲜纤维出芽情况.结果 PTZ组大鼠点燃率、病死率与法舒地尔组比较差异无统计学意义.PTZ组和法舒地尔组CA3区苔藓纤维出芽评分差异无统计学意义,与对照组相比差异均有统计学意义(P＜0.05).PTZ组和法舒地尔组海马非磷酸化cofilin表达差异无统计学意义.结论 丝切蛋白可能通过苔藓纤维出芽与癫癎的发生相关.     

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.     

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.     

Kainic acid-induced recurrent mossy fiber innervation of dentate gyrus inhibitory interneurons: possible anatomical substrate of granule cell hyper-inhibition in chronically epileptic rats

Kainic acid-induced neuron loss in the hippocampal dentate gyrus may cause epileptogenic hyperexcitability by triggering the formation of recurrent excitatory connections among normally unconnected granule cells. We tested this hypothesis by assessing granule cell excitability repeatedly within the same awake rats at different stages of the synaptic reorganization process initiated by kainate-induced status epilepticus (SE). Granule cells were maximally hyperexcitable to afferent stimulation immediately after SE and became gradually less excitable during the first month post-SE. The chronic epileptic state was characterized by granule cell hyper-inhibition, i.e., abnormally increased paired-pulse suppression and an abnormally high resistance to generating epileptiform discharges in response to afferent stimulation. Focal application of the gamma-aminobutyric acid type A (GABA(A)) receptor antagonist bicuculline methiodide within the dentate gyrus abolished the abnormally increased paired-pulse suppression recorded in chronically hyper-inhibited rats. Combined Timm staining and parvalbumin immunocytochemistry revealed dense innervation of dentate inhibitory interneurons by newly formed, Timm-positive, mossy fiber terminals. Ultrastructural analysis by conventional and postembedding GABA immunocytochemical electron microscopy confirmed that abnormal mossy fiber terminals of the dentate inner molecular layer formed frequent asymmetrical synapses with inhibitory interneurons and with GABA-immunopositive dendrites as well as with GABA-immunonegative dendrites of presumed granule cells. These results in chronically epileptic rats demonstrate that dentate granule cells are maximally hyperexcitable immediately after SE, prior to mossy fiber sprouting, and that synaptic reorganization following kainate-induced injury is temporally associated with GABA(A) receptor-dependent granule cell hyper-inhibition rather than a hypothesized progressive hyperexcitability. The anatomical data provide evidence of a possible anatomical substrate for the chronically hyper-inhibited state.     

Stereological analysis of GluR2-immunoreactive hilar neurons in the pilocarpine model of temporal lobe epilepsy: correlation of cell loss with mossy fiber sprouting

Mossy fiber sprouting and the genesis of ectopic granule cells contribute to reverberating excitation in the dentate gyrus of epileptic brain. This study determined whether the extent of sprouting after status epilepticus in rats correlates with the seizure-induced degeneration of GluR2-immunoreactive (GluR2+) hilar neurons (presumptive mossy cells) and also quantitated granule cell-like GluR2-immunoreactive hilar neurons. Stereological cell counting indicated that GluR2+ neurons account for 57% of the total hilar neuron population. Prolonged pilocarpine-induced status epilepticus killed 95% of these cells. A smaller percentage of GluR2+ neurons (74%) was killed when status epilepticus was interrupted after 1-3.5 h with a single injection of phenobarbital, and the number of residual GluR2+ neurons varied among animals by a factor of 6.2. GluR2+ neurons were not necessarily more vulnerable than other hilar neurons. In rats administered phenobarbital, the extent of recurrent mossy fiber growth varied inversely and linearly with the number of GluR2+ hilar neurons that remained intact (P=0.0001). Thus the loss of each GluR2+ neuron was associated with roughly the same amount of sprouting. These findings support the hypothesis that mossy fiber sprouting is driven largely by the degeneration of and/or loss of innervation from mossy cells. Granule cell-like GluR2-immunoreactive neurons were rarely encountered in the hilus of control rats, but increased 6- to 140-fold after status epilepticus. Their number did not correlate with the extent of hilar cell death or mossy fiber sprouting in the same animal. The morphology, number, and distribution of these neurons suggested that they were hilar ectopic granule cells.     

Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression

Development of kindling and mossy fiber sprouting, and changes of gene expression were studied after 40 seizures produced during about 3 h by electrical stimulation every 5 min in the ventral hippocampus. As assessed by 5 test stimulations, enhanced responsiveness was present already after 6–24 h but from 1 week post-seizure increased gradually up to 4 weeks without additional stimuli. Sprouting of mossy fibers in the dentate gyrus was demonstrated only at 4 weeks with Timm's staining. In situ hybridization showed a transient increase (maximum at 2 h) of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), TrkB and TrkC mRNA levels and reduction (maximum at 12–24 h) of neurotrophin-3 (NT-3) mRNA expression in dentate granule cells after the seizures. In addition, BDNF mRNA levels were elevated in CAI and CA3 regions, amygdala and piriform cortex. Marked increases of mRNA for growth-associated protein (GAP-43), with maximum expression at 12–24 h, were observed in dentate granule cells and in amygdala-piri-form cortex. Dynorphin mRNA levels showed biphasic changes in dentate granule cells with an increase at 2 h followed by a decrease at 24 h. No long-term alterations of gene expression were observed. These findings indicate that increased responsiveness develops rapidly after recurring seizures but that the kindled state is reached gradually in about 4 weeks. Mossy fiber sprouting occurs in parallel to epileptogenesis and may play a causative role. Short-term changes of neurotrophin and Trk, GAP-43 and dynorphin mRNA levels and the assumed alterations of the corresponding proteins could trigger structural rearrangements underlying kindling but might also contribute to the initial increase of seizure susceptibility.     

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.     

Mossy fibre reorganization in the hippocampus of prion protein null mice

Mice lacking prion protein (PrP-null) are resistant to transmissible spongiform encephalopathies. However, the normal functions of this highly conserved protein remain controversial. This study examines whether PrP-null mice develop normal neuronal pathways, specifically the mossy fibre pathway, within the hippocampus. Timm stained hippocampal sections from the PrP-null group had more granules than the controls in: the granule cell layer, the inner molecular layer of the dentate gyrus, and the infrapyramidal region of CA3. This resembles the mossy fibre collateral and terminal sprouting seen in certain epilepsies. The abnormal connectivity might be predicted to promote epileptiform activity, but extracellular electrophysiological recordings from the granule cell layer revealed a reduced excitability in the PrP-null group, both with and without blockade of GABA_A receptor-mediated inhibition. We propose that reorganization of neuronal circuity is a feature of PrP-null mice.