Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro

Adult neurogenesis in the dentate gyrus is assuming an increasingly important role in supporting hippocampal-dependent learning and the modulation of mood and anxiety. Moreover, injury to the developing postnatal dentate gyrus has profound effects on neurogenesis and hippocampal learning throughout life. Organotypic hippocampal slice cultures represent an attractive model for studying neurogenesis both in the early postnatal and adult hippocampus, as they retain much of their anatomical and functional circuitry in vitro. Ongoing neurogenesis has been recently demonstrated in organotypic hippocampal slice cultures. However, cell proliferation, one of the critical components of neurogenesis, has yet to be characterized in this culture system. We examined single-pulse S-phase bromo-deoxyuridine (BrdU) labeling in the dentate granule layer with respect to the septotemporal position of origin of the slice culture, the medium the cultures were grown in, and the time the cultures were maintained in vitro up to 14 days, when they are believed to have matured to a near adult state. Using single 10-microm sections through a culture as our reference volume, we report significant effects of septotemporal position on the number of granule layer cells and the number of cells in S-phase, as estimated by short-survival (2 hours) BrdU studies. We report a declining rate of BrdU incorporation, evidence of significant structural changes within the granule cell layer, and differences in cell death between culture media over the first 14 days in vitro. We report caution with the use of BrdU cell density and changes in cell number to indirectly estimate proliferation.     

The organization and development of the hippocampal mossy fiber system

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat.The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus.During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.     

Synaptic and axonal remodeling of mossy fibers in the hilus and supragranular region of the dentate gyrus in kainate-treated rats

Seizures evoked by kainic acid and a variety of experimental methods induce sprouting of the mossy fiber pathway in the dentate gyrus. In this study, the morphological features and spatial distribution of sprouted mossy fiber axons in the dorsal dentate gyrus of kainate-treated rats were directly shown in granule cells filled in vitro with biocytin and in vivo with the anterograde lectin tracer Phaseolus vulgaris leucoagglutinin (PHAL). Sprouted axon collaterals of biocytin-filled granule cells projected from the hilus of the dentate gyrus into the supragranular layer in both transverse and longitudinal directions in kainate-treated rats but were not observed in normal rats. The sprouted axon collaterals projected into the supragranular region for 600–700 μm along the septotemporal axis. Collaterals from granule cells in the infrapyramidal blade crossed the hilus and sprouted into the supragranular layer of the suprapyramidal blade. Sprouted axon segments in the supragranular layer had more terminal boutons per unit length than the axon segments in the hilus of both normal and kainate-treated rats but did not form giant boutons, which are characteristic of mossy fiber axons in the hilus and CA3. Mossy fiber axons in the hilus of kainate-treated rats had more small terminal boutons, fewer giant boutons, and there was a trend toward greater axon length compared with mossy fibers in the hilus of normal rats. With the additional length of supragranular sprouted collaterals, there was an overall increase in the length of mossy fiber axons in kainate-treated rats. The synaptic and axonal remodeling of the mossy fiber pathway could alter the functional properties of hippocampal circuitry by altering synaptic connectivity in local circuits within the hilus of the dentate gyrus and by increasing the divergence of the mossy fiber terminal field along the septotemporal axis. J. Comp. Neurol. 390:578–594, 1998. © 1998 Wiley-Liss, Inc.     

The organization and development of the hippocampal mossy fiber system

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat. The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus. During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.     

Lesion-induced synapse reorganization in the hippocampus of cats: sprouting of entorhinal, commissural/associational, and mossy fiber projections after unilateral entorhinal cortex lesions, with comments on the normal organization of these pathways.

This study evaluates whether three forms of sprouting occur in the hippocampus of the cat following unilateral entorhinal cortex (EC) lesions: (1) sprouting of projections from the EC contralateral to the lesion; (2) sprouting of the commissural/associational system; and (3) sprouting of mossy fibers. Tract tracing techniques were used to define the normal organization of the entorhinal cortical projection system, the commissural/associational (C/A) systems, and the mossy fiber projections in normal cats. The same techniques were then used to evaluate whether there were changes in these projections in animals with long-standing unilateral EC lesions. The projections from the entorhinal cortex were evaluated autoradiographically following injections of 3H proline into the entorhinal area. The projections of the C/A system were traced using the Fink-Heimer technique after lesions of the hippocampal commissures, and by using autoradiographic techniques after injections of 3H proline into the hippocampus. The distribution of mossy fibers was evaluated using the Timm's stain. The results reveal that unilateral lesions of the EC in cats lead to the same sorts of sprouting that have been described in rats. There is: (1) an increase in the density of the crossed projection from the surviving EC to the contralateral dentate gyrus that had been deprived of its normal EC inputs; (2) an expansion of the terminal field of the C/A projection system into portions of the molecular layer of the dentate gyrus normally occupied by EC projections; and (3) an increase in supragranular mossy fibers in some animals. The mossy fiber sprouting was especially prominent when the lesions encroached upon the hippocampus. The studies also reveal additional details about the normal organization of hippocampal pathways in cats. The most important points are: (1) there is a crossed projection from the entorhinal cortex to the contralateral dentate gyrus; and (2) there is a complex laminar organization of the commissural and associational terminal fields in the molecular layer of the dentate gyrus that appears to be related to the point of origin of the projections along the septotemporal axis of the hippocampus. This heretofore unrecognized aspect of the laminar organization of C/A terminations has important implications for the temporal competition hypothesis, which has been advanced to account for the development of these afferent systems.     

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

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

New insights into the role of hilar ectopic granule cells in the dentate gyrus based on quantitative anatomic analysis and three-dimensional reconstruction

The dentate gyrus is one of two main areas of the mammalian brain where neurons are born throughout adulthood, a phenomenon called postnatal neurogenesis. Most of the neurons that are generated are granule cells (GCs), the major principal cell type in the dentate gyrus. Some adult-born granule cells develop in ectopic locations, such as the dentate hilus. The generation of hilar ectopic granule cells (HEGCs) is greatly increased in several animal models of epilepsy and has also been demonstrated in surgical specimens from patients with intractable temporal lobe epilepsy (TLE). Herein we review the results of our quantitative neuroanatomic analysis of HEGCs that were filled with Neurobiotin following electrophysiologic characterization in hippocampal slices. The data suggest that two types of HEGCs exist, based on a proximal or distal location of the cell body relative to the granule cell layer, and based on the location of most of the dendrites, in the molecular layer or hilus. Three-dimensional reconstruction revealed that the dendrites of distal HEGCs can extend along the transverse and longitudinal axis of the hippocampus. Analysis of axons demonstrated that HEGCs have projections that contribute to the normal mossy fiber innervation of CA3 as well as the abnormal sprouted fibers in the inner molecular layer of epileptic rodents (mossy fiber sprouting). These data support the idea that HEGCs could function as a "hub" cell in the dentate gyrus and play a critical role in network excitability.     

Mossy fiber sprouting as a potential therapeutic target for epilepsy

Hippocampal mossy fibers, axons of dentate granule cells, converge in the dentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. In the hippocampal formation of temporal lobe epilepsy patients, however, this stereotyped pattern of projection is often collapsed; the mossy fibers branch out of the dentate hilus and abnormally innervate the dentate inner molecular layer, a phenomenon that is termed mossy fiber sprouting. Experimental studies have replicated this sprouting in animal models of temporal lobe epilepsy, including kindling and pharmacological treatment with convulsants. Because these axon collaterals form recurrent excitatory inputs into dendrites of granule cells, the circuit reorganization is assumed to cause epileptiform activity in the hippocampus, whereas some recent studies indicate that the sprouting is not necessarily associated with early-life seizures. Here we review the mechanisms of mossy fiber sprouting and consider its potential contribution to epileptogenesis. Based on recent findings, we propose that the sprouting can be regarded as a result of disruption of the molecular mechanisms underlying the axon guidance. We finally focus on the possibility that prevention of the abnormal sprouting might be a new strategy for medical treatment with temporal lobe epilepsy.     

Epilepsy induced collateral sprouting of hippocampal mossy fibers: Does it induce the development of ectopic synapses with granule cell dendrites?

In the present study, using Golgi and electron microscopy techniques, experimentally induced epilepsy (kindling and kainate treatment) elicited collateral sprouting of mossy fibers in rat hippocampus. Collateral branches invade the hilus, cross the granule cell layer, and distribute throughout the inner third of the molecular layer. These newly developed collaterals may acquire the typical features of mossy fibers including giant fiber varicosities (mousses), although the mean surface of these mousses was thinner in these collaterals than in terminal branches. Granule cell dendrites may develop giant thorny excrescences, suggesting that the targets of these collaterals are granule cells. Giant synaptic boutons appear in the inner third of molecular layer of epileptic rats. These boutons acquire the morphological features of mossy fiber boutons and made multiple synaptic contacts with dendritic spines. The analysis of the profile types suggests that some of the newly developed collateral mossy fibers made hypotrophic synaptic contacts.     

Fluoxetine induces input‐specific hippocampal dendritic spine remodeling along the septotemporal axis in adulthood and middle age

Fluoxetine, a selective serotonin‐reuptake inhibitor (SSRI), is known to induce structural rearrangements and changes in synaptic transmission in hippocampal circuitry. In the adult hippocampus, structural changes include neurogenesis, dendritic, and axonal plasticity of pyramidal and dentate granule neurons, and dedifferentiation of dentate granule neurons. However, much less is known about how chronic fluoxetine affects these processes along the septotemporal axis and during the aging process. Importantly, studies documenting the effects of fluoxetine on density and distribution of spines along different dendritic segments of dentate granule neurons and CA1 pyramidal neurons along the septotemporal axis of hippocampus in adulthood and during aging are conspicuously absent. Here, we use a transgenic mouse line in which mature dentate granule neurons and CA1 pyramidal neurons are genetically labeled with green fluorescent protein (GFP) to investigate the effects of chronic fluoxetine treatment (18 mg/kg/day) on input‐specific spine remodeling and mossy fiber structural plasticity in the dorsal and ventral hippocampus in adulthood and middle age. In addition, we examine levels of adult hippocampal neurogenesis, maturation state of dentate granule neurons, neuronal activity, and glutamic acid decarboxylase‐67 expression in response to chronic fluoxetine in adulthood and middle age. Our studies reveal that while chronic fluoxetine fails to augment adult hippocampal neurogenesis in middle age, the middle‐aged hippocampus retains high sensitivity to changes in the dentate gyrus (DG) such as dematuration, hypoactivation, and increased glutamic acid decarboxylase 67 (GAD67) expression. Interestingly, the middle‐aged hippocampus shows greater sensitivity to fluoxetine‐induced input‐specific synaptic remodeling than the hippocampus in adulthood with the stratum‐oriens of CA1 exhibiting heightened structural plasticity. The input‐specific changes and circuit‐level modifications in middle‐age were associated with modest enhancement in contextual fear memory precision, anxiety‐like behavior and antidepressant‐like behavioral responses. © 2015 Wiley Periodicals, Inc.     

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

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

Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers.

Morphological data from humans with temporal lobe epilepsy and from animal models of epilepsy suggest that seizure-induced damage to dentate hilar neurons causes granule cells to sprout new axon collaterals that innervate other granule cells. This aberrant projection has been suggested to be an anatomical substrate for epileptogenesis. This hypothesis was tested in the present study with intra- and extracellular recordings from granule cells in hippocampal slices removed from rats 1-4 months after kainate treatment. In this animal model, hippocampal cell loss leads to sprouting of mossy fiber axons from the granule cells into the inner molecular layer of the dentate gyrus. Unexpectedly, when slices with mossy fiber sprouting were examined in normal medium, extracellular stimulation of the hilus or perforant path evoked relatively normal responses. However, in the presence of the GABAA-receptor antagonist, bicuculline, low-intensity hilar stimulation evoked delayed bursts of action potentials in about one-quarter of the slices. In one-third of the bicuculline-treated slices with mossy fiber sprouting, spontaneous bursts of synchronous spikes were superimposed on slow negative field potentials. Slices from normal rats or kainate-treated rats without mossy fiber sprouting never showed delayed bursts to weak hilar stimulation or spontaneous bursts in bicuculline. These data suggest that new local excitatory circuits may be suppressed normally, and then emerge functionally when synaptic inhibition is blocked. Therefore, after repeated seizures and excitotoxic damage in the hippocampus, synaptic reorganization of the mossy fibers is consistently associated with normal responses; however, in some preparations, the mossy fibers may form functional recurrent excitatory connections, but synaptic inhibition appears to mask these potentially epileptogenic alterations.     

Growth of hippocampal mossy fibers: a lesion and coculture study of organotypic slice cultures

In hippocampal slice cultures, the mossy fibers from the dentate granule cells project as normally to cells in the dentate hilus (CA4) and the hippocampal CA3 pyramidal cells. After lesions in vivo and intracerebral transplantation, the mossy fibers can alter their normal distribution within CA3 and even contact CA1 pyramidal cells. The present study examined whether similar changes could be induced in the more simple, virtual two-dimensionally organized slice cultures. For this purpose slices of 7-day-old hippocampi were prepared and subjected to one of the two following manipulations: (1) transection of the mossy fiber layer in CA3 or (2) rearrangement of the geometrical relations between the dentate granule cells in their potential targets (CA3 and CA1) by coculturing dentate slices with CA3 or CA1 slices. Two to 8 weeks later the distribution of the mossy fiber system was visualized by histochemical Timm sulphide silver staining of the terminals. The distribution of the mossy fiber system observed in previous studies of ordinary hippocampal slice cultures was confirmed. In addition, mossy fibers were found to cross the cuts through the mossy fiber layer with formation of a reduced number of characteristic Timm-stained terminals in CA3 distal to the lesion. Close proximity and contiguity of the cut surfaces were important for such growth to occur. Significantly fewer mossy fiber terminals were found when separate slices of dentate and CA3 tissue were joined and grown as cocultures. Similar apposition of slices of dentate and CA1 tissue only rarely resulted in the ingrowth of mossy fibers into CA1. The Timm-stained mossy fiber terminals were then of subnormal size. The results show the potentials of the slice culture technique in supplementing lesion and transplant studies in situ. The growth of mossy fibers across a transection of their pathway is thus a new observation, difficult to demonstrate in the brain. The limited growth in the cocultures of aberrant mossy fibers into Ca1 does, on the other hand, emphasize the importance of close structural contact for the formation of nerve connections, and such contact is apparently more easily obtained in the brain. When the growth of the mossy fibers and that of the cholinergic septohippocampal fibers are compared, it is evident that the cholinergic axons grow better both in vitro and in vivo after lesions and transplantation.     

Cellular and connective organization of slice cultures of the rat hippocampus and fascia dentata

This study examined the cellular and connective organization of hippocampal tissue taken from 6-8-day-old rats and cultured by the roller tube technique for 3-6 weeks. In the cultures containing the fascia dentata and the hippocampus proper (CA1, CA3, CA4) the main cell and neuropil layers were organotypically organized when observed in ordinary cell stains. The normal distribution of smaller cell populations of AChE-positive neurons and somatostatin-reactive neurons was demonstrated by histochemical and immunohistochemical methods. Both cell types were mainly confined to str. oriens of CA3 and CA1 and the dentate hilus (CA4). Individual dentate granule cells and hippocampal pyramidal cells were injected with lucifer yellow and HRP, revealing great stability of the dendritic patterns of these cells in the culture condition. The same was found for the axonal branching and termination of HRP-filled mossy fibers arising from an HRP-injected granule cell. The preservation of organotypic afferent patterns in the cultures was also shown by Timm staining of the terminal distribution of the mossy fiber system. Mossy fiber terminals, with characteristic ultrastructural features verified in the electron microscope, were thus found in the hilus (CA4) and along the CA3 pyramidal cell layer onto the CA3-CA1 transition. Depending on the amount of dentate tissue relative to CA3 the terminals could stop before reaching CA1 (small fascia dentata) or take up additional intra and infrapyramidal locations along CA3 (small CA3). In cultures with a gap in the CA3 pyramidal cell layer some mossy fiber terminals were found in contact with the CA3 pyramidal cells beyond the gap. In all cultures there was an aberrant projection of supragranular mossy fibers. This projection is analogous to the one known from lesion and transplant studies to form in the absence of the entorhinal perforant path input to the dentate molecular layer. Also, in accordance with these studies the Timm staining pattern of the outer parts of the dentate molecular layer and the entire molecular layer of the hippocampus was altered corresponding to the spread of afferents normally confined to the inner zone of the dentate and str. radiatum of CA3 and CA1. Possibly as a consequence of the lack of normal targets for projections from CA1, this subfield contained an unusually dense Timm staining suggestive of autoinnervation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholecystokinin-, enkephalin-, and substance P-like immunoreactivity in the dentate area,hippocampus, and subiculum of the domestic pig

The distribution of cholecystokinin-like, enkephalin-like, and substance P-like immunoreactivities is described in the dentate area, hippocampus, and subiculum of the domestic pig (Sus scrofa domesticus) as a baseline for future experimental studies. The distributions in the pig are compared with previous observations in other species. Cholecystokinin-like immunoreactive nerve cell bodies were intensely stained and present in large numbers in all subfields studied. Cholecystokinin-like immunoreactive terminals appeared as stained puncta, whereas fibers were only rarely encounterd. The puncta were mainly seen in the dentate molecular layer and dentate granule cell layer, the pyramidal cell layer of the hippocampal regio inferior, stratum moleculare of the hippocampal regio superior, and in the subiculum. Enkephalin-like immunoreactive nerve cell bodies were faintly stained and generally present in very small numbers, except for some pyramidal cells in the subicular cell layer. Enkephalin-like immunoreactive fibers were few in number, whereas stained puncta appeared with variable densities. Puncta of particularly high densities were found in the dentate molecular layer, whereas they appeared of moderate density in the dentate hilus, stratum moleculare of the hippocampal regio superior, and in the subiculum. Substance P-like immunoreactive nerve cell bodies were few and very faintly stined. They primarily occurred in the dentate hilus, stratum oriens of the hippocampus, and in the subicular cell layer. Stained fibers were few in number, whereas stained puncta were present in abundant numbers corresponding to the mossy fiber projection in the dentate hilus and the layer of mossy fibers of the hippocampal regio inferior, and in moderate numbers in stratum moleculare of the hippocampal regio superior and in the subiculum. For all three neuropeptides there were consistent and very characteristic variations in the distribution of immunoreactivity along the septotemporal axis of the hippocampus. When viewed in a comparative perspective the distribution of enkephalin-like and substance P-like terminals in the domestic pig displayed striking differences from the basic pattern observed in other species. This contrasted with the distribution of cholecystokinin-like neurons and terminals, which resembled more closely these species. © 1993 Wiley-Liss, Inc.     

Axon arbors and synaptic connections of hippocampal mossy cells in the rat in vivo

The axon collateralization patterns and synaptic connections of intracellularly labeled and electrophysiologically identified mossy cells were studied in rat hippocampus. Light microscopic analysis of 11 biocytin-filled cells showed that mossy cell axon arbors extended through an average of 57% of the total septotemporal length of the hippocampus (summated two-dimensional length, not adjusted for tissue shrinkage). Axon collaterals were densest in distant lamellae rather than in lamellae near the soma. Most of the axon was concentrated in the inner one-third of the molecular layer, with the hilus containing an average of only 26% of total axon length and the granule cell layer containing an average of only 7%. Ultrastructural analysis was carried out on three additional intracellularly stained mossy cells, in which axon collaterals and synaptic targets were examined in serial sections of chosen axon segments. In the central and subgranular regions of the hilus, mossy cell axons established a low density of synaptic contacts onto dendritic shafts, neuronal somata, and occasional dendritic spines. Most hilar synapses were made relatively close to the mossy cell somata. At greater distances from the labeled mossy cell (1–2 mm along the septotemporal axis), the axon collaterals ramified predominantly within the inner molecular layer and made a high density of asymmetric synaptic contacts almost exclusively onto dendritic spines. Quantitative measurements indicated that more than 90% of mossy cell synaptic contacts in the ipsilateral hippocampus are onto spines of proximal dendrites of presumed granule cells. These results are consistent with a primary mossy cell role in an excitatory associational network with granule cells of the dentate gyrus. © 1996 Wiley-Liss, Inc.     

The development of the dentate area and the hippocampal mossy fiber projection of the rat

The development of the dentate area and the hippocampal mossy fiber system of the rat has been investigated at the light microscopic level by using fluorescent tracing, Nissl, and Timm's histochemical methods. Although the cytoarchitectonic development of the dentate granular layer is mainly a postnatal phenomenon, the initial events take place before birth. The aggregation and maturation of the cells in the granular layer proceed in a graded fashion from the lateral to the medial and from the superficial to the deep aspects of the layer. The earliest-formed granule cells are probably derived directly from the cells of the ventricular zone. They start to form mossy fibers prenatally, either during the relatively long period of migration to the granular layer or soon after their arrival. However, most of the granule cells are derived from a secondary proliferative center in the hilus. They start to produce mossy fibers postnatally a while after arriving at the granular layer. The total complement of granule cells starts to grow mossy fibers in a sequence that is related to the final position of the cells of origin within the granular layer. This sequence also proceeds in a graded fashion from the lateral to the medial and from the superficial to the deep aspects of the layer. In the beginning the mossy fibers elongate relatively rapidly. Already at birth the Timm-stained mossy fiber zone occupies the anterolateral part of the hilus and the adjacent suprapyramidal parts of the regio inferior. Once the mossy fibers have reached the distal end of the regio inferior they elongate along the longitudinal axis of the hippocampus more slowly. At the same time the Timm-stainability of the mossy fiber zone, which, during the first postnatal week, is weaker toward the regio superior, develops a mature pattern in which the distal part of the zone stains most intensely. Throughout development, fibers from the granule cells that form first are longer and diverge more in the septotemporal dimension than fibers from later-forming granule cells. In contrast to other axonal systems which appear to be sculptured from a diffuse set of connections the results presented here provide evidence that the topographic relationships of the mossy fiber system develop in a stepwise fashion.     

Fetal Hippocampal Cells Grafted to Kainate-Lesioned CA3 Region of Adult Hippocampus Suppress Aberrant Supragranular Sprouting of Host Mossy Fibers

Selective lesion of the rat hippocampus using an intracerebroventricular administration of kainic acid (KA) represents an animal model for studying both lesion recovery and temporal lobe epilepsy. This KA lesion leads initially to loss of CA3 hippocampal neurons, the postsynaptic target of mossy fibers, and later results in aberrant mossy fiber sprouting into the dentate supragranular layer (DSGL). Because of the close association of this aberrant mossy fiber sprouting with an increase in the seizure susceptibility of the dentate gyrus, delayed therapeutic strategies capable of suppressing the sprouting of mossy fibers into the DSGL are of significant importance. We hypothesize that neural grafting can restore the disrupted hippocampal mossy fiber circuitry in this model through the establishment of appropriate mossy fiber projections onto grafted pyramidal neurons and that these appropriate projections will lead to reduced inappropriate sprouting into the DSGL. Large grafts of Embryonic Day 19 hippocampal cells were transplanted into adult hippocampus at 4 days post-KA lesion. Aberrant mossy fiber sprouting was quantified after 3–4 months survival using three different measures of Timm's staining density. Grafts located near the degenerated CA3 cell layer showed dense ingrowth of host mossy fibers compared to grafts elsewhere in the hippocampus. Aberrant mossy fiber sprouting throughout the dentate gyrus was dramatically and specifically reduced in animals with grafts near the degenerated CA3 cell layer compared to “lesion only” animals and those with ectopic grafts away from the CA3 region. These results reveal the capability of appropriately placed fetal hippocampal grafts to restore disrupted hippocampal mossy fiber circuitry by attracting sufficient host mossy fibers to suppress the development of aberrant circuitry in hippocampus. Thus, providing an appropriate postsynaptic target at early postlesion periods significantly facilitates lesion recovery. The graft-induced long-term suppression of aberrant sprouting shown here may provide a new avenue for amelioration of hyperexcitability that occurs following hippocampal lesions.     

Synaptic reorganization in explanted cultures of rat hippocampus

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.