The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus

Summary One of the most distinctive and common cell types in Golgi preparations of the hilus of the rat dentate gyrus is the mossy cell. We have used a variety of techniques including the Golgi method, the combined Golgi and electron microscopic (EM) method and the retrograde transport of horseradish peroxidase (HRP) to study the development, ultrastructure and synaptic connections of this cell type. The mossy cells identified in our light microscopic preparations are characterized by: (1) triangular or multipolar shaped somata; (2) three to four primary dendrites that arise from the soma and bifurcate once or more to produce an extensive dendritic arborization restricted, for the most part, to the hilus; (3) numerous thorny excrescences on their somata and proximal dendrites with typical spines on distal dendrites; and (4) axons that bifurcate and are directed toward the fimbria and the molecular layer of the dentate gyrus.The mossy cells have an immature appearance at birth and on subsequent days their maturation appears to lag somewhat behind that of the hippocampal pyramidal cells. On postnatal day 1, many of the dendrites bear growth cones primarily at their termini and have long, thin filipodia emanating from various points along their lengths. Many of the dendrites enter the molecular layer of the dentate gyrus, though this is rarely seen in the mature brain. Typical pedunculate spines are first commonly seen on the distal dendrites around postnatal day 7 while thorny excrescences are first commonly seen between postnatal days 11 and 14. By postnatal day 21, the dendrites have attained a mature appearance although the density of both typical spines and thorny excrescences is less than that found in adults.Two different retrograde transport methods were used to confirm that mossy cells give rise to the commissural projection to the contralateral dentate gyrus. The first method combined HRP histochemistry with a silver intensification procedure and the second method combined HRP histochemistry with Golgi staining. While the majority of commissurally projecting hilar neurons had the appearance of mossy cells, there were others that were smaller and either ovoid or fusiform.     

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.     

Hippocampal fields in the hedgehog tenrec. Their architecture and major intrinsic connections.

The Madagascan lesser hedgehog tenrec was investigated to get insight into the areal evolution of the hippocampal formation in mammals with poorly differentiated brains. The hippocampal subdivisions were analyzed using cyto- and chemoarchitectural criteria; long associational and commissural connections were demonstrated with tracer techniques. The hedgehog tenrec shows a well differentiated dentate gyrus, CA3 and CA1. Their major intrinsic connections lie within the band of variations known from other species. The dentate hilar region shows calretinin-positive mossy cells with extensive projections to the molecular layer. The calbindin- and enkephalin-positive granule mossy fibers form a distinct endbulb and do not invade the CA1 as reported in the erinaceous hedgehog. Isolated granule cells with basal dendrites were also noted. A CA2 region is hard to identify architecturally; its presence is suggested due to its contralateral connections. Subicular and perisubicular regions are clearly present along the dorsal aspects of the hemisphere, but we failed to identify them unequivocally along the caudal and ventral tip of the hippocampus. A temporal portion of the subiculum, if present, differs in its chemoarchitecture from its dorsal counterpart. The perisubicular region, located medially adjacent to the dorsal subiculum may be equivalent to the rat's presubiculum; evidence for the presence of a parasubiculum was rather weak.     

Enkephalin-like immunoreactivity in the mossy fiber pathway of the hippocampal formation of the tree shrew (Tupaia glis)

The distribution of enkephalin-like immunoreactivity within the hippocampal formation of the tree shrew (Tupaia glis) has been investigated. By far the most conspicuous immunoreactive structures within the hippocampal formation were components of the mossy fiber pathway. Immunoreactive granule cells within the dentate gyrus could often be seen sending dendrites into the molecular layer and axons into the hilus of the dentate gyrus. Within the regio inferior of Ammon's horn, massive immunoreactive terminal swellings associated with thin fibers were located in the zone just above the pyramidal cell layer. While the mossy fiber pathway was the most intensely labeled portion of the hippocampal formation, occasionally sparse terminals and lightly immunoreactive cell bodies were noted in regio superior.These results indicate that the mossy fiber system may be an important pathway for opiate modulation of hippocampal activity.     

Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatory postsynaptic currents (sEPSCs) >600 pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.     

Synaptic connections of dentate granule cells and hilar neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections

Simultaneous intracellular recordings were made in the dentate gyrus of rat hippocampal slices, from pairs of the following cell types: granule cells, interneurons located in the granule cell layer, hilar interneurons, and spiny hilar "mossy cells". Granule cells were found to have strong excitatory effects on mossy cells and interneurons. Interneurons inhibited granule cells as well as other interneurons. No synaptic connections from mossy cells onto other cell types were found, within the confines of the slice, using intracellular recording methods. However, at the ultrastructural level, axon terminals of horseradish peroxidase-filled mossy cells were found making synaptic contacts in the hilus on dendrites of interneurons. These studies provide the first step towards determining the functional interactions of the various cell types in the fascia dentata.     

Lesion-induced CA1 mossy fibers in the rat represent a neoinnervation

Summary The developmental pattern of hippocampal mossy fiber (dentate granule cell axon) innervation to the pyramidal cell layer was examined with anterograde transport methods. Injection of ³H-leucine into the dentate gyrus on PN 1 resulted in labeling of the incipient stratum lucidum extending to, but not beyond, the CA3 region on PN 3 and 5. Since destruction of CA3 pyramidal cells on PN 5 results in aberrant mossy fiber innervation to CA1 pyramidal cells (Cook and Crutcher 1985), these results suggest that the presence of mossy fibers in CA1 of the rat represents a neoinnervation (perhaps representing a more primitive pattern of connectivity) and not the persistence of a transient developmental projection.     

Postnatal development of the mouse dentate gyrus in organotypic cultures of the hippocampal formation

The hippocampal formation of newborn mice was explanted and maintained in Maximow culture assemblies for up to 35 days. At the time of explantation, only the suprapyramidal limb of the dentate gyrus was cytoarchitectonically distinct, and electron microscopy of newborn hippocampus revealed no definitive synapses. Histogenesis, as indicated by the development of the infrapyramidal limb of the dentate gyrus, and synaptogenesis, as indicated by the in vitro formation of mossy fiber synapses on the dendrites of hippocampal pyramidal neurons were studied by light and electron microscopy. At 12 days and thereafter in culture, mossy fiber terminals were found in synapsis with dendritic spines probably belonging to pyramidal cells of the hippocampal zone CA4. Near dentate granule cell somata a few axosomatic and many axospinous and axodendritic synapses were found. The data indicate that granule cells of the developing dentate gyrus are capable of differentiation in vitro into a structure essentially equivalent to that developed in vivo. The granule cells may become arranged into a recognizable granule cell layer, and develop dendritic processes which receive synapses virtually identical to those found in the intact organ. The differentiation of these features proceeds in the absence of the extrinsic afferents from the septum or from the contralateral hippocampal formation.     

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.     

Dentate hilar mossy cells and somatostatin-containing neurons are immunoreactive for the alpha8 integrin subunit: characterization in normal and kainic acid-treated rats

Integrins are heterodimeric cell surface receptors composed of different alpha and beta subunits that mediate cell-cell and cell-extracellular matrix interactions. They have been implicated in the regulation of neuronal migration, differentiation, process outgrowth, and plasticity. The alpha8 integrin subunit associates exclusively with the beta1 subunit to form a receptor (alpha8beta1) for fibronectin, vitronectin, tenascin, and osteopontin. In a previous study, we demonstrated that hippocampal dentate hilar neurons are immunoreactive for alpha8. The present study identifies the major types of alpha8-immunoreactive hilar neurons and characterizes the effects of kainic acid-induced seizures on alpha8-immunoreactivity in these cells. Examination of the hilus in normal rats revealed alpha8-immunoreactivity in the somatodendritic compartments of large hilar neurons identified as mossy cells, including a subset of dendritic thorny excrescences that were contacted by large mossy fiber terminals. alpha8-immunoreactivity also was found in approximately 71% of somatostatin-containing hilar cells. Kainic acid-induced seizures dramatically and rapidly altered the levels and distribution of alpha8-immunoreactivity in hilar neurons. After 1.5 h of seizures, alpha8-immunoreactivity in their dendrites was reduced greatly. One day after kainic acid treatment, labeling was diminished throughout the somatodendritic compartments of most hilar cells. This decrease appeared to be transient, since alpha8 labeling returned to normal levels in surviving hilar neurons within 2 weeks of treatment. In addition, many alpha8-immunoreactive hilar neurons, particularly in caudal dentate regions, were lost 3-5 weeks after kainic acid treatment.Our findings suggest that alpha8beta1 may mediate adhesive interactions of the dendritic processes of mossy cells and somatostatin-containing hilar neurons with other cellular elements or with extracellular matrix components. They also suggest that alpha8 may be susceptible to activity-dependent proteolysis that could modulate its function in the somatodendritic compartment of these cells.     

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。     

Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion

Immunohistochemical studies were performed to analyze the expressional changes in hippocampal synaptic vesicle protein 2A (SV2A) following pentylenetetrazole (PTZ) kindling. Repeated treatments of mice with sub-convulsive PTZ (40 mg/kg, i.p.) for 15 days progressively enhanced seizure susceptibility and induced clonic convulsions in most animals examined. Topographical analysis of hippocampal SV2A-immunoreactivity revealed that SV2A was densely expressed in the hilar region of the dentate gyrus, stratum lucidum of the CA3 field and around the periphery of CA3 pyramidal neurons. PTZ kindling region-specifically increased SV2A expression in the dentate hilus without affecting that in the stratum lucidum or the pyramidal cell layer of the CA3 field. Confocal laser microscopic analysis using PTZ-kindled mice illustrated that most SV2A was co-expressed with glutamic acid decarboxylase 67 in the cell bodies and dendrites of hilar interneurons. However, SV2A-immunoreactivity was negligibly observed in the hilar glutamatergic nerve terminals (mossy fibers) probed with the anti-vesicular glutamate transporter 1 antibody. The present study suggests that SV2A specifically regulates hilar GABAergic neurotransmission in the kindled hippocampus probably as a compensatory or prophylactic mechanism against kindling epileptogenesis.     

The persistent expression of a highly polysialylated NCAM in the dentate gyrus of the adult rat.

The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H), often termed 'embryonic NCAM', has been investigated in the hippocampal formation of developing and adult rats. To determine the immunohistochemical localization of NCAM-H, a monoclonal antibody that reacts with the polysialic acid portion of NCAM-H was used. In the late embryonic and early postnatal periods, immunoreactivity for NCAM-H was found throughout the hippocampal formation, except for the ventricular layer. Thereafter, the immunoreactivity gradually decreased and almost vanished in most parts in the adult. However, a strong immunoreactivity remained on a number of cells in the dentate gyrus of adult rats, particularly in the deepest part of the granular layer. The immunoreactive arborized dendrites, mostly arising from the primary apical pole of the granule cells, were found to enter the molecular layer. The mossy fibers also were positive. Electron-microscopic examination of the hilus portion showed that the immunoreactivity was detected on the plasma membrane of some axons in the mossy fiber bundles. Since postnatal neurogenesis is known to continue into adulthood in the deepest part of the granule cell layer of the dentate gyrus, these results suggest that, in the adult dentate gyrus, NCAM-H is expressed by newly generated granule cells, and that the NCAM-H-expressing new cells may participate in the formation of new neural circuits.     

Granule cells are the main source of excitatory input to a subpopulation of GABAergic hippocampal neurons as revealed by electron microscopic double staining for zinc histochemistry and parvalbumin immunocytochemistry

Immunocytochemistry was combined with a recent modification of Timm's method to evaluate semiquantitatively the mossy fiber innervation of dendrites and somata of parvalbumin-containing neurons of the hilus of the dentate gyrus and the CA3 area of Ammon's horn. Using this electron microscopic double staining technique, it was found that (1) the overwhelming majority (95%) of terminals forming asymmetric synapses with parvalbumin-positive dendrites in the dentate hilus, and the strata pyramidale and lucidum of the CA3 area of Ammon's horn, originated from granule cells; (2) two-thirds of the asymmetric axosomatic terminals of parvalbumin-positive neurons contained zinc; and (3) no zinc-containing axon terminals formed synapses with somata or main dendritic shafts of the granule cells.     

GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons

Immunocytochemical results indicate that GAD-positive neurons are found in the molecular and granule cell layers of the dentate gyrus as well as in the hilar region. GAD-positive cells in the molecular and granule cell layers are identified as various types of local circuit neurons. Most of the GAD-positive puncta found throughout the molecular layer and within the granule cell layer are interpreted as axon terminals of these neurons, including five types of basket cells. This interpretation is based on data that indicate the axons of basket cells form synapses with the somata and proximal dendrites of granule cells. The results in the hilus show that 60% of the hilar neurons are GAD-positive. Since previous studies have indicated that 80% of hilar neurons give rise to both associational and commissural pathways, many GABAergic neurons in the hilus are probably projection neurons. This finding is consistent with recent physiological data which suggest that commissural pathway stimulation directly inhibits granule cells. Therefore, GABAergic cells in the dentate gyrus appear to be both projection and local circuit neurons.     

Expression of synaptophysin in sprouting neurons after entorhinal lesion in the rat

Expression of the synaptic vesicle protein synaptophysin was studied in lesion-induced sprouting neurons of the contralateral entorhinal cortex and in the contralateral dentate gyrus using immunocytochemistry at the light- and electron-microscopic level. Perikaryal immunoreactivity for synaptophysin was found between 8 and 10 days postlesion. Light microscopy revealed that synaptophysin immunostaining was present in almost all neurons of layers II and III of the contralateral medial entorhinal cortex. These neurons give rise to the sprouting, crossed temporodentate pathway. In addition, some hilar neurons of the contralateral dentate gyrus, which are the parent cells of sprouting commissural fibers, were immunostained for synaptophysin. Transient immunostaining for synaptophysin was observed within cell bodies and dendrites. Additionally, the cell bodies were outlined by immunoreactive puncta, identified by electron microscopy as nerve terminals. Our results revealed that sprouting neurons express the major synaptic vesicle protein synaptophysin during reactive synaptogenesis in a pattern that reflects biosynthesis and sorting of this protein as seen in developing neurons during synapse formation. Received: 13 November 1996 / Accepted: 3 June 1997     

Neuropeptide-Y immunoreactivity in the pilocarpine model of temporal lobe epilepsy

 Neuropeptide-Y (NPY) is expressed by granule cells and mossy fibres of the hippocampal dentate gyrus during experimental temporal lobe epilepsy (TLE). This expression may represent an endogenous damping mechanism since NPY has been shown to block seizure-like events following high-frequency stimulation in hippocampal slices. The pilocarpine (PILO) model of epilepsy is characterized by an acute period of status epilepticus followed by spontaneous recurrent seizures and related brain damage. We report peroxidase-antiperoxidase immunostaining for NPY in several brain regions in this model. PILO-injected animals exhibited NPY immunoreactivity in the region of the mossy fibre terminals, in the dentate gyrus inner molecular layer and, in a few cases, within presumed granule cells. NPY immunoreactivity was also dramatically changed in the entorhinal cortex, amygdala and sensorimotor areas. In addition, PILO injected animals exhibited a reduction in the number of NPY-immunoreactive interneurons compared with controls. The results demonstrate that changes in NPY expression, including expression in the granule cells and mossy fibres and the loss of vulnerable NPY neurons, are present in the PILO model of TLE. However, the significance of this changed synthesis of NPY remains to be determined. Received: 19 August 1996 / Accepted: 21 March 1997     

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.     

Endocannabinoid-mediated depolarization-induced suppression of inhibition in hilar mossy cells of the rat dentate gyrus

Hilar mossy cells represent a unique population of local circuit neurons in the hippocampus and dentate gyrus. Here we use electrophysiological techniques in acute preparations of hippocampal slices to demonstrate that depolarization of a single hilar mossy cell can produce robust inhibition of local GABAergic afferents. This depolarization-induced suppression of inhibition (DSI) can be observed as a transient reduction in frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) or as a transient reduction in amplitude of evoked IPSCs (eIPSCs). We find that DSI of eIPSCs as observed in hilar mossy cells is enhanced by activation of muscarinic acetylcholine receptors, blocked by chelation of postsynaptic calcium, and critically dependent on retrograde activation of presynaptic cannabinoid type 1 (CB1) receptors. We further report that activation of CB1 receptors on GABAergic afferents to hilar mossy cells (by either endogenous or exogenous agonists) preferentially inhibits calcium-dependent exocytosis and that endocannabinoid-dependent retrograde signaling in this system is subject to tight spatial constraints.