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.     

Newly generated granule cells show rapid neuroplastic changes in the adult rat dentate gyrus during the first five days following pilocarpine-induced seizures

Long-term neuroplastic changes to dentate granule cells have been reported after seizures and were shown to contribute to recurrent excitatory circuitry. These changes include increased numbers of newborn granule cells, sprouted mossy fibers, granule cell layer dispersion, increased hilar ectopic granule cells and formation of hilar basal dendrites on granule cells. The goal of the current study was to determine the acute progression of neuroplastic changes involving newly generated granule cells after pilocarpine-induced seizures. Doublecortin (DCX) immunocytochemical preparations were used to examine the newly generated granule cells 1-5 days after seizures were induced. The results showed that there are rapid neuroplastic changes to the DCX-labeled cells. At 1 day after seizures were induced, there were significant increases in the percentage of DCX-labeled cells with hilar basal dendrites and in the progenitor cell population. At 2 days after seizures were induced, an increase in the thickness of the layer of DCX-labeled cells occurred. At 3 days after seizures were induced, the number of DCX-labeled cells was significantly increased. At 4 days after seizures were induced, developing synapses were observed on DCX-labeled hilar basal dendrites. Thus, newly generated granule cells in the adult dentate gyrus display neuroplastic changes by 1 day after pilocarpine-induced seizures and further changes occur to this population of cells in the subsequent 4 days. The presence of synapses, albeit developing ones, on hilar basal dendrites during this period indicates that newly generated granule cells become rapidly incorporated into dentate gyrus circuitry following seizures.     

Galanin expressed in the excitatory fibers attenuates synaptic strength and generalized seizures in the piriform cortex of mice

The neuropeptide galanin is considered to be an endogenous antiepileptic agent, presumably acting via inhibition of glutamate release. Previously, we have demonstrated that in mice ectopically overexpressing galanin in cortical and hippocampal neurons, particularly in granule cells and their axons, the mossy fibers, hippocampal kindling epileptogenesis is suppressed and is associated with attenuated frequency facilitation in mossy fiber-CA3 cell synapses. We hypothesized that changes in synaptic transmission might occur also in other excitatory synapses of the galanin overexpressing (GalOE) mouse, contributing to seizure suppression. Lateral olfactory tract (LOT) synapses, formed by axons of olfactory bulb (OB) mitral cells and targeting piriform cortex (PC) pyramidal cells, ectopically express galanin in GalOE mice. Using whole-cell patch-clamp recordings, we found that excitatory synaptic responses recorded in PC pyramidal cells during high frequency stimulation of the LOT were attenuated in GalOE mice as compared to wild-type controls. This effect was mimicked by bath application of galanin or its agonist galnon to wild-type slices, supporting the notion of ectopic galanin action. Since the high frequency activation induced in vitro resembles epileptic seizures in vivo, we asked whether the observed synaptic inhibition would result in altered epileptogenesis when animals were kindled via the same synapses. In male GalOE mice, we found that the latency to convulsions was prolonged, and once animals had experienced the first stage 5 seizure, generalized seizures were less sustainable. These data indicate that the PC is a possible target for epilepsy treatment by ectopically overexpressing galanin to modulate seizure activity.     

Integration of adult generated neurons during epileptogenesis

Adult generated neurons in the dentate gyrus become functionally integrated into the existing hippocampal circuit by forming synapses with mature neurons. It is now well established that seizure activity increases neural proliferation, but only recently has the fate of seizure-induced newborn neurons been examined. An emerging consensus proposes that newborn neurons are highly sensitive to their environment, such that synaptic integration is profoundly altered following insults such as seizures. Whether these changes contribute to or counteract epileptogenesis is a subject of great interest because neurogenesis provides a potential target for therapeutic intervention. In this review, we summarize the current understanding of the functional integration of adult generated granule cells in the normal rodent hippocampus, and describe how this process can be altered during epileptogenesis.     

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.     

Neuroprotective effects of a postischemic treatment with a bradykinin B2 receptor antagonist in a rat model of temporary focal cerebral ischemia

Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and developed a recurrent mossy fiber pathway were used to investigate this possibility. Actions of released zinc were inferred from the effects of chelation with 1 mM calcium disodium EDTA (CaEDTA). When granule cell population bursts were evoked by mossy fiber stimulation in the presence of 6 mM K(+) and 30 microM bicuculline, CaEDTA slowed the rate at which evoked bursting developed, but did not change the magnitude of the bursts once they had developed fully. The effects of CaEDTA were then studied on the pharmacologically isolated NMDA receptor- and AMPA/kainate receptor-mediated components of the fully developed bursts. CaEDTA increased the magnitude of NMDA receptor-mediated bursts and reduced the magnitude of AMPA/kainate receptor-mediated bursts. CaEDTA did not affect the granule cell bursts evoked in slices from untreated rats by stimulating the perforant path in the presence of bicuculline and 6 mM K(+). These results suggest that zinc released from the recurrent mossy fibers serves mainly to facilitate the recruitment of dentate granule cells into population bursts.     

Hippocampal granule cell activity and c-Fos expression during spontaneous seizures in awake, chronically epileptic, pilocarpine-treated rats: implications for hippocampal epileptogenesis

The process of postinjury hippocampal epileptogenesis may involve gradually developing dentate granule cell hyperexcitability caused by neuron loss and synaptic reorganization. We tested this hypothesis by repeatedly assessing granule cell excitability after pilocarpine-induced status epilepticus (SE) and monitoring granule cell behavior during 235 spontaneous seizures in awake, chronically implanted rats. During the first week post-SE, granule cells exhibited diminished paired-pulse suppression and decreased seizure discharge thresholds in response to afferent stimulation. Spontaneous seizures often began during the first week after SE, recruited granule cell discharges that followed behavioral seizure onsets, and evoked c-Fos expression in all hippocampal neurons. Paired-pulse suppression and epileptiform discharge thresholds increased gradually after SE, eventually becoming abnormally elevated. In the chronic epileptic state, interictal granule cell hyperinhibition extended to the ictal state; granule cells did not discharge synchronously before any of 191 chronic seizures. Instead, granule cells generated only low-frequency voltage fluctuations (presumed "field excitatory postsynaptic potentials") during 89% of chronic seizures. Granule cell epileptiform discharges were recruited during 11% of spontaneous seizures, but these occurred only at the end of each behavioral seizure. Hippocampal c-Fos after chronic seizures was expressed primarily by inhibitory interneurons. Thus, granule cells became progressively less excitable, rather than hyperexcitable, as mossy fiber sprouting progressed and did not initiate the spontaneous behavioral seizures. These findings raise doubts about dentate granule cells as a source of spontaneous seizures in rats subjected to prolonged SE and suggest that dentate gyrus neuron loss and mossy fiber sprouting are not primary epileptogenic mechanisms in this animal model.     

Parallel increases in the synaptic and surface areas of mossy fiber terminals following seizure induction

Seizure induction tends to be followed by the development of a predisposition to future seizure activity and the concurrent sprouting of the mossy fiber pathway into the inner molecular layer of the dentate gyrus, where recurrent excitatory synapses are formed. To determine whether synaptic remodeling of mossy fiber terminals within the hilus also occurs, rats were administered pentylenetetrazol and, 2 days later, control and experimental tissue was processed for the ultrastructural immunohistochemical identification of mossy fiber terminals. Examination of the structure of these terminals within random hilar fields indicated that selective changes had occurred, which were only observed in the ventral hilus, and which were specific to terminals forming synapses with mossy cell spines (vs. interneurons). This terminal population displayed significant parallel increases in both the total active zone area and the surface area of an average terminal (measured from random two-dimensional samples of terminal structure). Increases in total active zone area must reflect increases in the number and/or size of individual active zones. These findings suggest that changes in terminal size can subserve adjustments in the overall strength of a set of synaptic connections. In the context of the ventral hilus, a selective increase in the apparent strength of mossy fiber connections with mossy cells could support increases in excitability following seizure induction. Mossy cells form connections with granule cell proximal dendrites, providing another pathway for recurrent excitation.     

"Dormant basket cell" hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat

The "dormant basket cell" hypothesis suggests that postinjury hippocampal network hyperexcitability results from the loss of vulnerable neurons that normally excite insult-resistant inhibitory basket cells. We have reexamined the experimental basis of this hypothesis in light of reports that excitatory hilar mossy cells are not consistently vulnerable and inhibitory basket cells are not consistently seizure resistant. Prolonged afferent stimulation that reliably evoked granule cell discharges always produced extensive hilar neuron degeneration and immediate granule cell disinhibition. Conversely, kainic acid-induced status epilepticus in chronically implanted animals produced similarly extensive hilar cell loss and immediate granule cell disinhibition, but only when granule cells discharged continuously during status epilepticus. In both preparations, electron microscopy revealed degeneration of presynaptic terminals forming asymmetrical synapses in the mossy cell target zone, including some terminating on gamma-aminobutyric acid-immunoreactive elements, but no evidence of axosomatic or axoaxonic degeneration in the adjacent granule cell layer. Although parvalbumin immunocytochemistry and in situ hybridization revealed decreased staining, this apparently was due to altered parvalbumin expression rather than basket cell death, because substance P receptor-positive interneurons, some of which contained residual parvalbumin immunoreactivity, survived. These results confirm the inherent vulnerability of dendritically projecting hilar mossy cells and interneurons and the relative resistance of dentate inhibitory basket and chandelier cells that target granule cell somata. The variability of hippocampal cell loss after status epilepticus suggests that altered hippocampal structure and function cannot be assumed to cause the spontaneous seizures that develop in these animals and highlights the importance of confirming hippocampal pathology and pathophysiology in vivo in each case.     

The functional organization of the hippocampal dentate gyrus and its relevance to the pathogenesis of temporal lobe epilepsy

Temporal lobe seizures are frequently associated with a characteristic pattern of hippocampal pathology (hippocampal sclerosis), as well as pathology in other temporal lobe structures. Despite more than a century of study, the relationship between pathology and epileptogenesis remains unclear. Endfolium sclerosis, which is characterized by the loss of dentate hilar neurons that are presumed to govern dentate granule cell excitability, is evident whenever hippocampal sclerosis exists and is the only temporal lobe pathology in some patients. Because prolonged seizures or head trauma produce endfolium sclerosis and granule cell hyperexictability in experimental animals, hilar neuron loss may be the common pathological denominator and primary network defect underlying development of a hippocampal seizure “focus.” Physiological studies suggest that vulnerable hilar mossy cells normally excite neurons that mediate granule cell inhibition. Recent anatomical studies indicate that the axons of mossy cells project longitudinally, out of the lamellar plane in which their cell bodies lie. If mossy cells in one lamella excite inhibitory neurons in surrounding lamellae, neocortical excitation of one segment of the granule cell layer may produce lateral inhibition and limit neocortical excitation to the targeted lamella. In patients who have had status epilepticus, prolonged febrile seizures, head trauma, or encephalitis, loss of dentate mossy cells may deafferent inhibitory neurons, render them “dormant,” and thereby disinhibit an encephalitis, loss of dentate mossy cells may deafferent inhibitory neurons, render them “dormant,” and thereby disinhibit an enlarged expanse of the granule cell layer. The selective loss of neurons that normally govern lateral inhibition in the dentate gyrus may cause functional delamination of the granule cell layer and result in synchronous, multilamellar discharges in response to cortical stimuli. Repetitive seizures may ultimately produce the full pattern of hippocampal and mesial temporal sclerosis by destroying cells within the seizure circuit that were not injured irreversibly by the initial insult. Thus, hippocampal pathology may be both the cause and effect of seizures that originate in the temporal lobe.     

Neuroanatomical clues to altered neuronal activity in epilepsy: from ultrastructure to signaling pathways of dentate granule cells

The dynamic aspects of epilepsy, in which seizures occur sporadically and are interspersed with periods of relatively normal brain function, present special challenges for neuroanatomical studies. Although numerous morphologic changes can be identified during the chronic period, the relationship of many of these changes to seizure generation and propagation remains unclear. Mossy fiber sprouting is an example of a frequently observed morphologic change for which a functional role in epilepsy continues to be debated. This review focuses on neuroanatomically identified changes that would support high levels of activity in reorganized mossy fibers and potentially associated granule cell activation. Early ultrastructural studies of reorganized mossy fiber terminals in human temporal lobe epilepsy tissue have identified morphologic substrates for highly efficacious excitatory connections among granule cells. If similar connections in animal models contribute to seizure activity, activation of granule cells would be expected. Increased labeling with two activity-related markers, Fos and phosphorylated extracellular signal-regulated kinase, has suggested increased activity of dentate granule cells at the time of spontaneous seizures in a mouse model of epilepsy. However, neuroanatomical support for a direct link between activation of reorganized mossy fiber terminals and increased granule cell activity remains elusive. As novel activity-related markers are developed, it may yet be possible to demonstrate such functional links and allow mapping of seizure activity throughout the brain. Relating patterns of neuronal activity during seizures to the underlying morphologic changes could provide important new insights into the basic mechanisms of epilepsy and seizure generation.     

The cerebellum of the frog Rana ridibunda. An electron microscopic study

An electron microscopic study of neuronal types and different synaptic contacts has been made in the cerebellum of the frog Rana ridibunda. The Purkinje cells have a pear-shaped cell body and in their cytoplasm the organelles show a special arrangement because of the great amount of microtubules they contain. The granule cells are small, rounded neurons with a large nucleus surrounded by a thin rim of cytoplasm. The stellate cells are interneurons of the molecular layer whose large nuclei show a single finger-like invagination of its nuclear envelope. The afferent tracts to the cerebellum end either as climbing fibers or mossy fibers. The axon terminals of climbing fibers are large and the synaptic complexes exhibit all the features of a type-I Gray synapse. The mossy fibers reach the granular layer and synapses between them and granule cell dendrites are by far the most abundant. The parallel fibers establish synaptic contacts on the spines arising from the spiny branchlet units of the Purkinje cells and with the perikaryon and dendrites of stellate cells. The stellate cell axons cross the molecular layer and establish type-II Gray synapses on the Purkinje cells.     

Altered patterning of dentate granule cell mossy fiber inputs onto CA3 pyramidal cells in limbic epilepsy

Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell?CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP‐expressing mice and immunolabeling for the zinc transporter ZnT‐3 to reveal the pre‐ and postsynaptic components of granule cell?CA3 pyramidal cell synapses following pilocarpine‐epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity 1 month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and 3 months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT‐3 labeling of presynaptic giant boutons apposed to GFP‐expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus. © 2009 Wiley‐Liss, Inc.     

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。     

Reorganization of corticorubral synapses following cross-innervation of flexor and extensor nerves of adult cat: a quantitative electron microscopic study

A quantitative electron microscopic study of corticorubral synapses was performed in the red nucleus (RN) of adult cats to determine the morphological correlates for the changes in time course of corticorubral excitatory post-synaptic potentials, which occur following cross-innervation of forelimb extensor and flexor nerves. Corticorubral synaptic endings were identified by anterograde degeneration after lesions of the ipsilateral sensorimotor cortex. Rubrospinal neurons innervating upper spinal segments were electrophysiologically identified and filled with horseradish peroxidase (HRP). These cells were mainly situated in the dorsomedial part of RN. Electron micrographs of the degenerating corticorubral synaptic endings were taken in the region surrounding HRP-filled neurons and the diameter of the dendrites contacted by such terminals was measured.In the cross-innervated animals many degenerating terminals were found to synapse on dendrites with large diameter and the somata of neurons in RN. This is in contrast to the previous observations in normal cats, in which very few corticorubral synapses were found to synapse on proximal dendrites and somata of RN neurons. The diameter of HRP-filled neurons in cats which were cross-innervated was slightly smaller than those observed in normal animals. These results indicate that new corticorubral synapses were formed on proximal dendrites and somata of RN neurons as a consequence of cross-innervation.     

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.     

匹罗卡品致痫诱发大鼠齿状回颗粒细胞GAP-43 mRNA表达

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

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.     

A Golgi-electron microscopic study of fusiform neurons in the hilar region of the dentate gyrus

The fusiform cells of the dentate gyrus are located in a portion of the hilus within 100 micron of the granule cell layer. They have ovoid somata and bipolar dendrites that generally run parallel to the granule cell layer. The dendrites of these cells are either spiny or sparsely spiny. The spiny fusiform cell has numerous spines along its dendrites, which are contacted by terminals with the features of granule cell axon collaterals. This cell type also displays somal spines that are contacted by similar terminals. In contrast, the sparsely spiny fusiform cell displays only a few spines, which are contacted by multiple small axon terminals that synapse with both the stalk and end bulb of the spine. Most synaptic input for this cell type is made with the smooth surfaces of the soma and dendrites. A variety of terminals form synapses with the sparsely spiny fusiform cell, including terminals that resemble the fine axon collaterals of mossy fibers. The somata of these two cell types also display differences in the amount of Nissl bodies and the degree of nuclear infolding. The results indicate that spiny fusiform cells are similar to mossy cells, another hilar cell type that receives its major synaptic input from axon collaterals of mossy fibers from granule cells. The distribution of the dendrites of spiny fusiform cells and the pattern of granule cell axon collaterals suggest a high degree of convergence from granule cells. In contrast, the variety of axodendritic synapses for sparsely spiny fusiform cells suggests that more diverse inputs affect this cell's activity. Therefore, the structure and circuitry of these two hilar cell types are probably different. This study adds further evidence to indicate that the hilus contains a large variety of cell types with different neuronal connections.