Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1

Reelin is a positional signal for the lamination of the dentate gyrus. In the reeler mutant lacking Reelin, granule cells are scattered all over the dentate gyrus. We have recently shown that the reeler phenotype of the dentate gyrus can be rescued in vitro by coculturing reeler hippocampal slices with slices from wild-type hippocampus. Here we studied whether Reelin from other brain regions can similarly induce this rescue effect and whether it is mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR). We found that coculturing reeler hippocampal slices with slices from wild-type olfactory bulb, cerebellum, and neocortex rescued the reeler phenotype as seen before with hippocampal slices, provided that the Reelin-synthesizing cells of these regions were placed near the marginal zone of the reeler hippocampal slice. However, coculturing wild-type hippocampal slices with hippocampal slices from mutants deficient in ApoER2 and VLDLR did not rescue the reeler-like phenotype in these cultures. Similarly, no rescue of the reeler-like phenotype was observed in slices from mutants lacking Disabled 1 (Dab1), an adapter protein downstream of Reelin receptors. Conversely, reeler hippocampal slices were rescued by coculturing them with slices from Dab1(-/-) mutants or ApoER2(-/-)/VLDLR(-/-) mice. These findings show that Reelin from other brain regions can substitute for the loss of hippocampal Reelin and that rescue of the reeler phenotype observed in our coculture studies is mediated via lipoprotein receptors for Reelin and Dab1.     

Malformation of the radial glial scaffold in the dentate gyrus of reeler mice,scrambler mice,and ApoER2/VLDLR-deficient mice

We studied the postnatal development of the radial glial scaffold in the dentate gyrus of reeler mice, lacking the extracellular matrix protein Reelin, in scrambler mice, deficient in the intracellular adaptor protein disabled1 (Dab1), which is required for the transmission of the Reelin signal into the cell, and in mutant mice lacking the Reelin receptors apolipoprotein receptor 2 (ApoER2) and/or the very low density lipoprotein receptor (VLDLR), known to transmit the Reelin signal via Dab1. By immunolabeling for the glial fibrillary acidic protein (GFAP), we show that a regular dentate radial glial scaffold fails to form in mutants deficient of Reelin, Dab1, and VLDLR and ApoER2. Mutant mice lacking only one of the Reelin receptors, VLDLR or ApoER2, display a gradual expression of the radial glial defects seen in mutants that lack both receptors. Our results suggest that Reelin signaling via ApoER2, VLDLR, and Dab1 is required for the formation of a regular radial glial scaffold in the dentate gyrus.     

Development of Commissural Connections in the Hippocampus of Reeler Mice: Evidence of an Inhibitory Influence of Cajal–Retzius Cells

Reelin is a large, extracellular matrix protein involved in neuronal migration and axonal growth. To analyze the contribution of Reelin to the development of the commissural projection in the hippocampus, we analyzed the ontogeny of this projection in the reeler mutant mouse. Injections of the lipophilic tracer DiI revealed many commissural fibers in the hippocampus of both reeler and control mice at P1-P2. At P5, at P12, and in the adult, the topography of commissural connections was normal in the CA1 region of reeler mice, with axons innervating the stratum radiatum and stratum oriens. In contrast, in the CA3/CA2 region, commissural fibers abnormally innervated the stratum lacunosum-moleculare and, in the dentate gyrus, some fibers were observed in the outer molecular layer. Next, we monitored the distribution of Cajal-Retzius cells in the hippocampus of reeler mutant mice and noted that the stratum lacunosum-moleculare of the CA3/CA2 region was largely devoid of Cajal-Retzius (CR) cells. Taken together, the above results indicate that in the absence of CR cells in the CA3/CA2, commissural axons abnormally grow to the stratum lacunosum-moleculare. To test this hypothesis a series of coculture experiments was performed in collagen gels, in which the CA3 axonal growth was monitored when confronted to the marginal zone. These experiments showed that the marginal zone containing CR cells exerts short-range inhibitory influences for commissural axonal growth.     

Balance between neurogenesis and gliogenesis in the adult hippocampus: role for reelin

The extracellular matrix protein reelin is essential for the proper radial migration of cortical neurons. In reeler mice lacking reelin, there is a malformation of the radial glial scaffold required for granule cell migration. Immunostaining for glial fibrillary acidic protein (GFAP) reveals abundant radial glial cells with long fibers traversing the granular layer in the wild type, but almost exclusively astrocytes in the reeler mutant. With the concept that radial glial cells are precursors of neurons, we hypothesized that the balance between neurogenesis and gliogenesis is altered in the reeler mutant. To this end, adult reeler mutants and their wild-type littermates were injected with bromodeoxyuridine (BrdU), a marker of newly generated cells. When compared to wild-type animals, we found a reduction in the number of BrdU-labeled cells in the adult reeler dentate gyrus. Moreover, whereas there was a dramatic decrease in the number of newly generated granule cells identified by double labeling for BrdU and NeuN, the number of BrdU-labeled, GFAP-positive astrocytes had increased. Decreased neurogenesis in the adult reeler dentate gyrus was confirmed by immunostaining for doublecortin, a marker of newly generated neurons. These results indicate that adult neurogenesis is altered in the reeler dentate gyrus and that newly generated cells preferentially differentiate into astrocytes.     

Connections of the hippocampal formation in humans: I. The mossy fiber pathway

The hippocampal formation has been one of the most extensively studied cortical regions in rats, yet little is known about the anatomical connections of the hippocampus in primates, especially humans. With the use of an antibody against the calcium-binding protein, calbindin-D28K, in normal autopsy tissue and the neuronal tracers biocytin or biotinylated dextrans in in vitro slice preparations from tissue removed during surgery for intractable epilepsy, we examined the human hippocampal mossy fiber pathway. The injections of biocytin into the dentate granule cell layer labeled neurons in a Golgi-like manner, revealing the presence of basal dendrites on about 30% of the granule cells. The granule cell axons, the mossy fibers, initially formed a diffuse plexus of fibers in the polymorphic layer before organizing into fiber fascicles in the hilar pyramidal region. These fiber fascicles were much more prominent rostrally than caudally. Within the hilus and proximal portions of the extrahilar CA3 field, the mossy fibers ran through the pyramidal cell layer, and while near the transition to field CA2, the fibers turned superficially and crossed the pyramidal layer to run in the stratum lucidum. All of these features, seen following injections of tracer into hippocampal slices from the brains of epileptics, were confirmed by calbindin-staining of mossy fibers in normal brains. Biocytin-labeled mossy fiber axons revealed two characteristic types of enlargements: small varicosities and larger expansions. The expansions were found throughout the neuropil and were highly irregular, diaminobenzidine-dense profiles that had pleiomorphic modes of attachment to the parent axon. Electron microscopic images of these biocytin labeled expansions revealed that they were large synaptic boutons bearing asymmetric synapses. This study indicates that the human mossy fiber pathway shows some minor deviations from the rodent brain but little difference from monkeys. We argue that these changes mirror a phylogenetic growth of the CA3 pyramidal neurons (subfield CA3c) into the hilus rather than an evolutionary change of the mossy fiber pathway. This growth of subfield CA3c and the increase in mossy fibers running through the pyramidal layer (and a presumed accompanying increase in proximal basal dendritic contacts) may reflect a growing role of the projection from the dentate granule cells to subfield CA3c and from there to field CA1 in the primate hippocampus. J. Comp. Neurol. 385:325–351, 1997. © 1997 Wiley-Liss, Inc.     

Localization of ApoER2, VLDLR and Dab1 in radial glia: groundwork for a new model of reelin action during cortical development

The reelin signaling pathway regulates laminar positioning of radially migrating neurons during cortical development. It has been suggested that reelin secreted by Cajal-Retzius cells in the marginal zone could provide either a stop or an attractant signal for migratory neurons expressing reelin receptors, but the proposed models fail to explain recent experimental findings. Here we provide evidence that the reelin receptor machinery, including the lipoprotein receptors ApoER2 and VLDLR along with the cytoplasmic adaptor protein Dab1, is located in radial glia precursors whose processes span the entire cortical wall from the ventricular zone to the pial surface. Moreover, in reeler mice, defective in reelin, decreased levels of Dab1 in the ventricular zone correspond to an accumulation of the protein in radial end-feet beneath the pia matter. Our results support that neural stem cells receive a functional reelin signal. They are also consistent with a working model of reelin action, according to which reelin signaling on the newborn neuron-inherited radial process regulates perikaryal translocation and positioning.     

Different Primary Target Cells Are Important for Fiber Lamination in the Fascia Dentata: A Lesson from Reeler Mutant Mice

The factors determining the lamina-specific termination of entorhinal and commissural afferents to the fascia dentata are poorly understood. Recently it was shown that early generated Cajal-Retzius (CR) cells in the outer molecular layer and reelin, synthesized by CR cells, play a role in the lamina-specific termination of entorhinal fibers which form transient synapses with CR cells before establishing their definite contacts with granule cell dendrites (J. A. del Rio et al., 1997, Nature 385, 70-74). By using anterograde tracing with Phaseolus vulgaris leukoagglutinin we show that the normal, sharply delineated entorhinal projection to the outer molecular layer is retained in reeler mutant mice lacking reelin. This coincides with the regular presence of CR cells, the primary, transient target cells of entorhinal fibers. In contrast, the commissural fibers were found to terminate in an abnormal broad, not clearly defined area. This widespread projection coincides with the distribution of granule cells which in the mutant do not form a dense cell layer but are scattered all over the hilus due to a migration defect. Unlike the entorhinal fibers, the commissural fibers arrive in their target layer late in development, when granule cell dendrites are already there. We hypothesize from these results that the presence of the adequate postsynaptic element at the time of fiber ingrowth, CR cells for the early ingrowing entorhinal fibers and granule cells for the late-arriving commissural fibers, is crucial for the normal formation of these layer-specific projections.     

A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus

The axon collaterals of dentate granule cells have been analyzed with the aid of a computerized microscope, following intracellular injections of horseradish peroxidase in hippocampal slice preparations. The axon of each granule cell gives rise to approximately seven primary collaterals; these collaterals usually divide into secondary and tertiary branches, which form an extensive plexus within the hilar region of the dentate gyrus. Individual axon collaterals vary greatly in length, but most have been found to be between 100 and 300 microns long. On average, the summed lengths of the collaterals (exclusive of the parent mossy fiber) are approximately 2,300 microns. Except for an occasional collateral that is given off by a mossy fiber in the proximal part of field CA3 of the hippocampus, the collaterals of the granule cell axons are confined to the hilar region; they are rarely seen in the granule cell layer itself and have never been observed in the molecular layer. In the longitudinal dimension of the dentate gyrus, most of the collaterals are contained within a zone about 400 microns wide. The distribution of the collaterals within the hilar region is correlated with the location of the granule cell body. Those that arise from cells near the tip of the suprapyramidal blade tend to be confined to the region above field CA3; those from cells nearer the crest and from the infrapyramidal blade ramify widely throughout the hilus. Two types of varicosities are present on the collaterals. Numerous small (approximately 2 microns), round varicosities are distributed unevenly along the collaterals; in electron micrographs these varicosities can be seen to make asymmetric synaptic contacts with dendritic shafts. On average, each granule cell collateral plexus has about 160 of these varicosities. The second type of varicosity is irregular in shape and ranges from 2 to 4 microns in diameter; there is usually only one such varicosity per collateral. In all respects except size, these varicosities resemble the expansions found on the parent mossy fibers. Mossy fiber trajectories in the proximal part of field CA3 were studied after extracellular injections of HRP into localized regions of the granule cell layer. Granule cells at different locations around the blade send their mossy fibers to different depths within the pyramidal cell layer in the proximal part of field CA3. However, further distally, mossy fibers from all parts of the granule cell layer contribute to the suprapyramidal bundle that occupies the stratum lucidum.     

Reelin receptors in developing laminated brain structures of mouse and human

Reelin is an extracellular matrix protein secreted by a variety of cell types throughout the developing brain. The target cells for reelin express the cytoplasmic adapter protein Dab1, which binds to the reelin receptors VLDLR and ApoER2. In the present work, we have studied the localization of both receptors in developing mouse and human cortex, olfactory bulb and cerebellum. In mouse, some Cajal-Retzius cells express reelin and VLDLR; in humans, all the components of the signalling pathway (Reelin, Dab1, VLDLR and ApoER2) are present in subsets of Cajal-Retzius cells. In the mouse cortical plate, VLDLR and ApoER2 are present from E15 to postnatal stages; in human cortical plate they are most prominent at approximately 20 gestational weeks. In mice, cerebellar Purkinje cells only express VLDLR whereas in humans they express both VLDLR and ApoER2. Mitral cells of the mouse olfactory bulb are ApoER2-positive and VLDLR-negative. In sum, the receptor expression patterns are similar in the human and mouse cortical plate but differ in Cajal-Retzius and Purkinje cells, which in humans express additional components of the reelin-Dab1 pathway.     

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.     

Abnormal positioning of granule cells alters afferent fiber distribution in the mouse fascia dentata: morphologic evidence from reeler, apolipoprotein E receptor 2-, and very low density lipoprotein receptor knockout mice

The fascia dentata of the hippocampal formation is characterized by the nonoverlapping and lamina-specific termination of afferent fibers: entorhinal fibers terminate in the outer molecular layer and commissural/associational fibers terminate in the inner molecular layer. It has been proposed that this fiber lamination depends on the presence of the correct postsynaptic partner at the time of fiber ingrowth during development. Pioneer neurons that guide afferent fibers to their correct layers as well as signals located on granule cells have both been implicated. To study the role of granule cells for the lamina-specific ingrowth of afferents, the cyto- and fiberarchitecture of three mouse mutants (very low density lipoprotein receptor knockout mouse, apolipoprotein E receptor 2 knockout mouse, and reeler mouse) that show different degrees of granule cell migration defects were analyzed. Anterograde tracing with Phaseolus vulgaris-leucoagglutinin was used to visualize the afferent fiber systems, and immunohistochemistry was used to determine the position of their putative target cells. In controls, granule cells are packed in a single layer. This laminar organization is mildly altered in very low density lipoprotein receptor knockout mice, moderately disturbed in apolipoprotein E receptor 2 knockout mice, and severely disrupted in reeler mice. These changes in granule cell distribution are mirrored by the distribution of commissural fibers. In contrast, changes in granule cell distribution do not severely affect the laminar termination of entorhinal fibers. These data provide further evidence for a role of granule cells in the laminar termination of commissural/associational afferents to the fascia dentata.     

Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis.

A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these "granule-like" neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry.     

Binding of purified Reelin to ApoER2 and VLDLR mediates tyrosine phosphorylation of Disabled-1

Reelin, Disabled-1 (Dab1), apolipoprotein E receptor 2 (ApoER2), and very low density lipoprotein receptor (VLDLR) participate in a signaling pathway required for layer formation during mammalian brain development. Binding of Reelin to ApoER2 and VLDLR induces a rapid increase in tyrosine phosphorylation of Dab1, an adaptor protein that associates with the cytoplasmic domain of the receptors. However, Reelin has also been proposed to signal through integrin and protocadherin. Here we compare the roles of ApoER2 and VLDLR in Reelin signaling. We used layer-specific markers to identify the final positions of early- and late-born neurons in the cortices of mice lacking ApoER2, VLDLR, or both ApoER2 and VLDLR. Subtle alterations were observed in mice lacking VLDLR, whereas more severe abnormalities were detected in the absence of ApoER2, and major disruptions were obvious in mice lacking both receptors. Purified Reelin associated more readily with ApoER2 than with VLDLR and no synergy was observed in the presence of both receptors. Consistent with the binding data, the level of Reelin-induced Dab1 phosphorylation was more severely reduced in neurons lacking ApoER2 than in neurons lacking VLDLR. However, similarly low levels of Dab1 tyrosine phosphorylation were observed in ApoER2(-/-) and VLDLR(-/-) mice in vivo. Finally, there was a complete absence of Reelin-induced tyrosine phosphorylation of Dab1 in cortical neurons from mice lacking both ApoER2 and VLDLR. These findings demonstrate that ApoER2 and VLDLR are essential for Reelin signaling and that no other receptor molecules can compensate for their role in mediating tyrosine phosphorylation of Dab1.     

Electrophysiological characteristics and morphological properties of dentate granule--and CA3 pyramidal cells in slices cut from neonatally irradiated rats

Neonatal irradiation reduces the dentate granule cells by 60-80%, and consequently the mossy fiber projection toward the CA3 and hilar areas decreases. The number of hilar cells diminishes. Thorny excrescences on the dendrites of the CA3 pyramidal cells get smaller both in number (from 20-30 per neuron in normal to 1-6 per neuron after irradiation) and in size. In spite of these morphological changes functional efficacy of the mossy-fiber projection to CA3 pyramidal cells remains sufficient to generate monosynaptic action potentials when stimulated electrically. Inhibitory circuits activated by mossy fiber volleys seem to be unaffected by irradiation. Main biophysical properties of CA3 pyramidal and surviving granule cells remain within the normal range. Further work should determine if efficacy of the mossy fiber projection increases to compensate for the substantial decrease of presynaptic input, or the power of transmission far exceeds the level needed to fire postsynaptic cells in normal rats.     

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.     

Hilar mossy cells share developmental influences with dentate granule neurons

Mossy cells are the major class of excitatory neurons in the dentate hilus. Although mossy cells are involved in a range of physiological and pathological conditions, very little is known about their ontogeny. To gain insight into this issue, we first determined the developmental stage at which mossy cells can be reliably identified with the molecular markers calretinin and GluR2/3 and found that hilar mossy cells were first identifiable around the end of the 1st postnatal week. Birthdating studies combined with staining for these markers revealed that the appearance of mossy cells coincided with the first wave of dentate granule cell production during mid-gestation. Since mossy cells are born as the first granule cells are produced and it is believed that mossy cells originate from the neuroepithelium adjacent to the dentate progenitor zone, we examined to what extent the development of mossy cells is controlled by the same molecular pathways as that of granule cells. To do this, we analyzed the production of mossy cells in Lef1 and NeuroD mutant animals, in which granule cell production is disrupted during precursor proliferation or neuronal differentiation, respectively. The production of mossy cells was almost entirely lost in both mutants. Collectively, these data suggests that hilar mossy cells, unlike CA subfield pyramidal cells, are influenced by many of the same developmental cues as dentate granule cells.     

Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2‐specific markers Purkinje cell protein 4 (PCP4) and regulator of G‐Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin‐expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE‐associated pathological restructuring. © 2015 Wiley Periodicals, Inc.