Synaptic connections from multiple subfields contribute to granule cell hyperexcitability in hippocampal slice cultures

Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term (postnatal day 11; 40-60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABA(A) receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.     

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.     

移植的齿状回颗粒细胞在培养的大鼠海马组织上移行特征

为探讨齿状回颗粒细胞在培养的海马组织表面上的移行过程以及苔状纤维的投射特征,本实验从生后3d的绿色荧光蛋白(GFP)基因导入的SD大鼠海马组织切片中分离出齿状回颗粒细胞层,移植到培养的SD大鼠海马组织切片表面上,共同培养3～4d后,激光共聚焦显微镜观察。结果显示,97.6%的细胞移行到齿状回和CA3区,仅有2.4%细胞移行到CA2和CA1区。移行到齿状回颗粒细胞层的颗粒细胞投射苔状纤维到齿状回门区和CA3区的透明层,却不到CA2和CA1区。上述结果提示,在海马组织培养中移植的齿状回颗粒细胞的移行以及苔状纤维的投射方向明显受宿主的影响。     

Role of mossy fiber sprouting and mossy cell loss in hyperexcitability: a network model of the dentate gyrus incorporating cell types and axonal topography

Mossy cell loss and mossy fiber sprouting are two characteristic consequences of repeated seizures and head trauma. However, their precise contributions to the hyperexcitable state are not well understood. Because it is difficult, and frequently impossible, to independently examine using experimental techniques whether it is the loss of mossy cells or the sprouting of mossy fibers that leads to dentate hyperexcitability, we built a biophysically realistic and anatomically representative computational model of the dentate gyrus to examine this question. The 527-cell model, containing granule, mossy, basket, and hilar cells with axonal projections to the perforant-path termination zone, showed that even weak mossy fiber sprouting (10-15% of the strong sprouting observed in the pilocarpine model of epilepsy) resulted in the spread of seizure-like activity to the adjacent model hippocampal laminae after focal stimulation of the perforant path. The simulations also indicated that the spatially restricted, lamellar distribution of the sprouted mossy fiber contacts reported in in vivo studies was an important factor in sustaining seizure-like activity in the network. In contrast to the robust hyperexcitability-inducing effects of mossy fiber sprouting, removal of mossy cells resulted in decreased granule cell responses to perforant-path activation in agreement with recent experimental data. These results indicate the crucial role of mossy fiber sprouting even in situations where there is only relatively weak mossy fiber sprouting as is the case after moderate concussive experimental head injury.     

Expression of synaptopodin, an actin-associated protein, in the rat hippocampus after limbic epilepsy

Synaptopodin, a 100 kD protein, associated with the actin cytoskeleton of the postsynaptic density and dendritic spines, is thought to play a role in modulating actin-based shape and motility of dendritic spines during formation or elimination of synaptic contacts. Temporal lobe epilepsy in humans and in rats shows neuronal damage, aberrant sprouting of hippocampal mossy fibers and subsequent synaptic remodeling processes. Using kainic acid (KA) induced epilepsy in rats, the postictal hippocampal expression of synaptopodin was analyzed by in situ hybridization (ISH) and immunohistochemistry. Sprouting of mossy fibers was visualized by a modified Timm's staining. ISH showed elevated levels of Synaptopodin mRNA in perikarya of CA3 principal neurons, dentate granule cells and in surviving hilar neurons these levels persisted up to 8 weeks after seizure induction. Synaptopodin immunoreactivity in the dendritic layers of CA3, in the hilus and in the inner molecular layer of the dentate gyrus (DG) was initially reduced. Eight weeks after KA treatment Synaptopodin protein expression returned to control levels in dendritic layers of CA3 and in the entire molecular layer of the DG. The recovery of protein expression was accompanied by simultaneous supra- and infragranular mossy fiber sprouting. Postictal upregulation of Synaptopodin mRNA levels in target cell populations of limbic epilepsy-elicited damage and subsequent Synaptopodin protein expression largely co-localized with remodeling processes as demonstrated by mossy fiber sprouting. It may thus represent a novel postsynaptic molecular correlate of hippocampal neuroplasticity.     

癫痫发作敏感大鼠齿状回苔状纤维侧枝发芽

目的　探讨海马齿状回苔状纤维侧枝发芽与癫痫发作敏感性形成之间的关系。方法　在颈部皮下注射惊厥剂量的海人酸 (KA ,10mg/kg)诱发大鼠出现癫痫发作后 ,采用Timm’s染色法 ,分别在注射KA后3d、7d和 1个月 3个时间点观察致痫大鼠海马齿状回内苔状纤维发芽的情况。结果　Timm’s染色发现 ,注射KA后 7d ,海马齿状回分子层内带和颗粒细胞上层出现苔状纤维的异常发芽 ,注射KA后 1个月海马齿状回内Timm’s染色颗粒颜色加深 ,范围增大。提示海马苔状纤维发芽形成的时间过程与癫痫发作敏感性形成的时间过程一致。结论　海马齿状回分子层内带和颗粒细胞上层出现异常的苔状纤维发芽可能与癫痫发作敏感性形成有关。     

Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures

Sprouting of the mossy fiber axons of the dentate granule cells is a structural neuronal plasticity found in the mature brain of epileptic humans and experimental animals. Mossy fiber sprouting typically arises in experimental animals after repeated seizures and may contribute to the hyperexcitability of the epileptic brain. Investigation of the molecular triggers and spatial cues involved in mossy fiber sprouting has been hampered by the lack of an optimal in vitro model for studying this rearrangement. For an in vitro model to be feasible, the circuitry and receptors involved in convulsant-induced mossy fiber sprouting would have to be localized near the granule cells, rather than being dependent on long-range brain interconnections. However, it is not known whether this is the case. We report here that that application of the convulsant, kainic acid, to organotypic hippocampal explant cultures induces seizures, neuronal cell death, and subsequent dramatic mossy fiber sprouting with a similar laminar preference and time-course to that seen in intact animals. Prolonged (48 h) but not transient (4 h) kainic acid treatment caused regionally selective neuronal cell death. Cultures treated with kainic acid for a prolonged period displayed a time- and dose-dependent increase in supragranular Timm staining reflective of increased mossy fiber innervation to this area. Direct visualization of mossy fiber axons with neurobiotin-labeling revealed that mossy fibers in kainic acid-treated cultures exhibited a dramatic increase in supragranular axonal branch points and synaptic boutons. The cellular and molecular determinants required for kainic acid-induced cell death and subsequent mossy fiber reorganization thus appear to be intrinsic to the hippocampal slice preparation, and are preserved in culture. Given the ease with which functional inhibitors or pharmacological agents may be utilized in this system, slice cultures may provide a powerful model in which to study the molecular components involved in triggering mossy fiber outgrowth and underlying its laminar specificity. Elucidation of these molecular pathways will likely have both specific utility in clarifying the functional consequences of mossy fiber sprouting, as well as general utility in understanding of synaptic reorganization in the mature central nervous system.     

Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.     

Hippocampal mossy fiber sprouting is not impaired in brain-derived neurotrophic factor-deficient mice

In human temporal lobe epilepsy, a loss of hilar neurons followed by the sprouting of recurrent mossy fiber collaterals and the reinnervation of free synaptic sites on granule cell dendrites are discussed as possible mechanisms underlying hippocampal hyperexcitability. Dentate granule cells have been shown to upregulate brain-derived neurotrophic factor (BDNF) as well as TrkB, the high-affinity receptor for BDNF, in response to limbic seizures. This raised the possibility that BDNF is an important factor in hippocampal mossy fiber sprouting. Here we have used slice cultures of hippocampus, in which mossy fibers sprout and form a supragranular plexus in response to granule cell deafferentation, and have compared cultures from early postnatal BDNF-deficient mice and wild-type mice. We demonstrate that there is sprouting of supragranular mossy fibers in cultured slices from both BDNF knock-out and wild-type mice. We conclude that BDNF is not an essential factor for mossy fiber sprouting. However, our data do not exclude a role for BDNF in mossy fiber sprouting in wild-type mice, as compensatory mechanisms might have become effective in the mutant. Received: 6 January 1998 / Accepted: 23 February 1998     

Involvement of highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive granule cells in the amygdaloid-kindling-induced sprouting of a hippocampal mossy fiber trajectory

The mossy fiber system in the hippocampus of amygdaloid-kindled rats was examined by using highly polysialylated neural cell adhesion molecule (PSA-NCAM) as a marker for immunohistochemical detection of immature dentate granule cells and mossy fibers in combination with bromodeoxyuridine (BrdU) labeling of newly generated granule cells. Statistically significant increases in BrdU-labeled cells and PSA-NCAM-positive cells occurred in the dentate gyrus following kindling. The increase in PSA-NCAM-immunoreactive neurites was confined to the entire stratum lucidum of CA3. Immunoelectron-microscopic examination also revealed that PSA-NCAM-positive immature synaptic terminals of the sprouting mossy fibers increased in the stratum lucidum of CA3 in the kindled rats. The increase in the numbers of PSA-NCAM-positive granule cells correlated well with the increase in the immunopositive neurites and synaptic terminals on the mossy fiber trajectory. The increase in these PSA-NCAM-immunopositive structures is thought to reflect the enhancement of sprouting and synaptogenesis of mossy fibers by a subset of granule cells newly generated during amygdaloid-kindling and suggests that the reorganization of the mossy fiber system on the normal trajectory at least in part contributes to the acquisition and maintenance of an epileptogenic state.     

Potential roles for hyaluronan and CD44 in kainic acid-induced mossy fiber sprouting in organotypic hippocampal slice cultures

The most well-documented synaptic rearrangement associated with temporal lobe epilepsy is mossy fiber sprouting (MFS). MFS is a pronounced expansion of granule cell mossy fiber axons into the inner dentate molecular layer. The recurrent excitatory network formed by MFS is hypothesized to play a critical role in epileptogenesis, which is the transformation of the normal brain into one that is prone to recurrent spontaneous seizures. While many studies have focused on the functional consequences of MFS, relatively few have investigated the molecular mechanisms underlying the increased propensity of mossy fibers to invade the inner molecular layer. We hypothesized that changes in two components of the extracellular matrix, hyaluronan and its primary receptor, CD44, contribute to MFS. Hyaluronan contributes to laminar-specificity in the hippocampus and increases in hyaluronan and CD44 are associated with temporal lobe epilepsy. We tested our hypothesis in an in vitro model of MFS using a combination of histological and biochemical approaches. Application of kainic acid (KA) to organotypic hippocampal slice cultures induced robust MFS into the inner dentate molecular layer compared with vehicle-treated controls. Degradation of hyaluronan with hyaluronidase significantly reduced but did not eliminate KA-induced MFS, suggesting that hyaluronan played a permissive role in MFS, but that loss of hyaluronan signaling alone was not sufficient to block mossy fiber reorganization. Comparison of CD44 expression with MFS revealed that when CD44 expression in the molecular layers was high, MFS was minimal and when CD44 expression/function was reduced following KA treatment or with function blocking antibodies, MFS was increased. The time course of KA-induced reductions in CD44 expression was identical to the temporal progression of KA-induced MFS reported previously in hippocampal slice cultures, suggesting that reduced CD44 expression may help promote MFS. Understanding the molecular mechanisms underlying MFS may lead to therapeutic interventions that limit epileptogenesis.     

海人酸诱导癫痫大鼠齿状回神经肽Y能苔状纤维侧枝发芽

目的：探讨海马苔状纤维侧枝发芽与癫痫发作敏感性形成的关系。材料与方法：在海人酸诱发大鼠出现癫痫发作后,采用免疫组织化学染色法观察大鼠海马齿状回内神经肽Ｙ能纤维的异常发芽。结果：首次证实在癫痫发作后７天时,在海马齿状回内分子层就已出现神经肽Ｙ能纤维的异常增生。结论：这可能是对癫痫发作后齿状回门区神经肽Ｙ能神经元缺失的一种代偿性变化。     

Blockade of excitation reveals inhibition of dentate spiny hilar neurons recorded in rat hippocampal slices.

1. Extracellular and intracellular recordings in rat hippocampal slices were used to compare the synaptic responses to perforant path stimulation of granule cells of the dentate gyrus, spiny "mossy" cells of the hilus, and area CA3c pyramidal cells of hippocampus. Specifically, we asked whether aspects of the local circuitry could explain the relative vulnerability of spiny hilar neurons to various insults to the hippocampus. 2. Spiny hilar cells demonstrated a surprising lack of inhibition after perforant path activation, despite robust paired-pulse inhibition and inhibitory postsynaptic potentials (IPSPs) in adjacent granule cells and area CA3c pyramidal cells in response to the same stimulus in the same slice. However, when the slice was perfused with excitatory amino acid antagonists [6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), or CNQX with 2-amino-5-phosphonovaleric acid (APV)], IPSPs could be observed in spiny hilar cells in response to perforant path stimulation. 3. The IPSPs evoked in spiny hilar cells in the presence of CNQX were similar in their reversal potentials and bicuculline sensitivity to IPSPs recorded in dentate granule cells or hippocampal pyramidal cells in the absence of CNQX. 4. These results demonstrate that, at least in slices, perforant path stimulation of spiny hilar cells is primarily excitatory and, when excitation is blocked, underlying inhibition can be revealed. This contrasts to the situation for dentate and hippocampal principal cells, which are ordinarily dominated by inhibition, and only when inhibition is compromised can the full extent of excitation be appreciated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Excitatory synaptic input to granule cells increases with time after kainate treatment

Temporal lobe epilepsy is usually associated with a latent period and an increased seizure frequency following a precipitating insult. After kainate treatment, the mossy fibers of the dentate gyrus are hypothesized to form recurrent excitatory circuits between granule cells, thus leading to a progressive increase in the excitatory input to granule cells. Three groups of animals were studied as a function of time after kainate treatment: 1-2 wk, 2-4 wk, and 10-51 wk. All the animals studied 10-51 wk after kainate treatment were observed to have repetitive spontaneous seizures. Whole cell patch-clamp recordings in hippocampal slices showed that the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) in granule cells increased with time after kainate treatment. This increased excitatory synaptic input was correlated with the intensity of the Timm stain in the inner molecular layer (IML). Flash photolysis of caged glutamate applied in the granule cell layer evoked repetitive EPSCs in 10, 32, and 66% of the granule cells at the different times after kainate treatment. When inhibition was reduced with bicuculline, photostimulation of the granule cell layer evoked epileptiform bursts of action potentials only in granule cells from rats 10-51 wk after kainate treatment. These data support the hypothesis that kainate-induced mossy fiber sprouting in the IML results in the progressive formation of aberrant excitatory connections between granule cells. They also suggest that the probability of occurrence of electrographic seizures in the dentate gyrus increases with time after kainate treatment.     

Metallothioneins and zinc dysregulation contribute to neurodevelopmental damage in a model of perinatal viral infection

Neonatal Borna disease (NBD) virus infection in the Lewis rat results in life-long viral persistence and causes behavioral and neurodevelopmental abnormalities. A hallmark of the disorder is progressive loss of cerebellar Purkinje and dentate gyrus granule cells. Findings of increased brain metallothionein-I and -II (MT-I/-II) mRNA expression in cDNA microarray experiments led us to investigate MT isoforms and their relationship to brain zinc metabolism, cellular toxicity, and neurodevelopmental abnormalities in this model. Real-time PCR confirmed marked induction of MT-I/-II mRNA expression in the brains of NBD rats (40.5-fold increase in cerebellum, p<0.0001; 6.8-fold increase in hippocampus, p=0.003; and 9.5-fold increase in striatum, p=0.0012), whereas a trend toward decreased MT-III mRNA was found in hippocampus (1.25-fold decrease, p=0.0841). Double label immunofluorescence revealed prominent MT-I/-II expression in astrocytes throughout the brain; MT-III protein was decreased in granule cell neurons and increased in astrocytes, with differential subcellular distribution from cytoplasmic to nuclear compartments in NBD rat hippocampus. Modified Timm staining of hippocampus revealed reduced zinc in mossy fiber projections to the hilus and CA3, accumulation of zinc in glial cells and degenerating granule cell somata, and robust mossy fiber sprouting into the inner molecular layer of the dentate gyrus. Zinc Transporter 3 (ZnT-3) mRNA expression was decreased in hippocampus (2.3-fold decrease, p= 0.0065); staining for its correlate protein was reduced in hippocampal mossy fibers. Furthermore, 2 molecules implicated in axonal pathfinding and mossy fiber sprouting, the extracellular matrix glycoprotein, tenascin-R (TN-R), and the hyaluronan receptor CD44, were increased in NBD hippocampal neuropil. Abnormal zinc metabolism and mechanisms of neuroplasticity may contribute to the pathogenesis of disease in this model, raising more general implications for neurodevelopmental damage following viral infections in early life.     

AMPA receptor alterations precede mossy fiber sprouting in young children with temporal lobe epilepsy

Following neurological injury early in life numerous events, including excitotoxicity, neural degeneration, gliosis, neosynaptogenesis, and circuitry reorganization, may alone or in concert contribute to hyperexcitability and recurrent seizures in temporal lobe epilepsy. Our studies provide new evidence regarding the temporal sequence of key elements of hippocampal reorganization, mossy fiber sprouting and glutamate receptor subunit up-regulation, in a subset of young temporal lobe epileptic patients. Without evidence of mossy fiber sprouting, the youngest age group (3-10 years old) of mesial temporal lobe epileptic patients demonstrated enhanced glutamate receptor subunit profiles, suggesting that the dendritic change precedes axonal sprouting. However, sclerotic hippocampal specimens from epileptic patients ages 12-15 years old had the characteristic features of glutamate receptor up-regulation and mossy fiber sprouting first identified in the adult, indicating that reconstructed circuits appear early in the course of the disease. Non-sclerotic hippocampal specimens from lesion associated temporal lobe epileptic patients of all age groups showed minimal cell loss, sparse staining of glutamate receptor subunits in the dentate gyrus, and little or no mossy fiber sprouting. These compelling findings suggest a progressive sequence of events in the reorganization of the dentate gyrus of sclerotic hippocampal specimens. We suggest that cell loss and up-regulation of glutamate receptor subunits appear early in temporal lobe epilepsy and contribute to the synaptic plasticity that may facilitate the subsequent sprouting of mossy fiber collaterals which compound an already precipitous state of decline. The combination of pre-synaptic and post-synaptic changes serves as a potential substrate for hyperexcitability.     

Electrophysiological evidence of monosynaptic excitatory transmission between granule cells after seizure-induced mossy fiber sprouting

Mossy fiber sprouting is a form of synaptic reorganization in the dentate gyrus that occurs in human temporal lobe epilepsy and animal models of epilepsy. The axons of dentate gyrus granule cells, called mossy fibers, develop collaterals that grow into an abnormal location, the inner third of the dentate gyrus molecular layer. Electron microscopy has shown that sprouted fibers from synapses on both spines and dendritic shafts in the inner molecular layer, which are likely to represent the dendrites of granule cells and inhibitory neurons. One of the controversies about this phenomenon is whether mossy fiber sprouting contributes to seizures by forming novel recurrent excitatory circuits among granule cells. To date, there is a great deal of indirect evidence that suggests this is the case, but there are also counterarguments. The purpose of this study was to determine whether functional monosynaptic connections exist between granule cells after mossy fiber sprouting. Using simultaneous recordings from granule cells, we obtained direct evidence that granule cells in epileptic rats have monosynaptic excitatory connections with other granule cells. Such connections were not obtained when age-matched, saline control rats were examined. The results suggest that indeed mossy fiber sprouting provides a substrate for monosynaptic recurrent excitation among granule cells in the dentate gyrus. Interestingly, the characteristics of the excitatory connections that were found indicate that the pathway is only weakly excitatory. These characteristics may contribute to the empirical observation that the sprouted dentate gyrus does not normally generate epileptiform discharges.     

Enhanced but fragile inhibition in the dentate gyrus in vivo in the kainic acid model of temporal lobe epilepsy: a study using current source density analysis

Temporal lobe epilepsy is related to many structural and physiological changes in the brain. We used kainic acid in rats as an animal model of temporal lobe epilepsy, and studied the neural interactions of the dentate gyrus in urethane-anesthetized rats in vivo. Our initial hypothesis was that sprouting of mossy fibers, the axons of the granule cells, increases proximal dendritic excitatory currents in the inner molecular layer of the dentate gyrus. Extracellular currents were detected in vivo using current source density analysis. Backfiring the mossy fibers in CA3 or orthodromic excitation of the granule cells through the medial perforant path induced a current sink at the inner molecular layer. However, the sink or inferred excitation at the inner molecular layer was not increased in kainic acid-treated rats and the sink actually correlated negatively with the degree of mossy fiber sprouting. It is inferred that the latter sink was mediated mainly by association fibers and not by recurrent mossy fibers. After kainic acid treatment, paired-pulse inhibition of the population spikes in the dentate gyrus was increased. In contrast, reverberant activity that involved looping around an entorhinal-hippocampal circuit was increased in kainic acid-treated rats, compared to control rats. The increase of inhibition in kainic acid-treated rats was readily blocked by a small dose of GABA(A) receptor antagonist bicuculline. The latter dose of bicuculline induced paroxsymal spike bursts in kainic acid-treated but not control rats, demonstrating that the increased inhibition in dentate gyrus was fragile.In conclusion, after kainic acid induced seizures, the dentate gyrus in vivo showed an increase in inhibition that appeared to be fragile. The hypothesized increase in proximal dendritic excitation due to mossy fiber sprouting was not detected. However, the fragile inhibition could explain the seizure susceptibility in patients with temporal lobe epilepsy.     

The C5a anaphylatoxin receptor CD88 is expressed in presynaptic terminals of hippocampal mossy fibres

Background  

In the periphery, C5a acts through the G-protein coupled receptor CD88 to enhance/maintain inflammatory responses. In the brain, CD88 can be expressed on astrocytes, microglia and neurons. Previous studies have shown that the hippocampal CA3 region displays CD88-immunolabelling, and CD88 mRNA is present within dentate gyrus granule cells. As granule cells send dense axonal projections (mossy fibres) to CA3 pyramidal neurons, CD88 expression could be expressed on mossy fibres. However, the cellular location of CD88 within the hippocampal CA3 region is unknown.