Intracellular recording and labeling of mossy cells and proximal CA3 pyramidal cells in macaque monkeys

Little is known about the morphological characteristics and intracellular electrophysiological properties of neurons in the primate hippocampus and dentate gyrus. We have therefore begun a program of studies using intracellular recording and biocytin labeling in hippocampal slices from macaque monkeys. In the current study, we investigated mossy cells and proximal CA3 pyramidal cells. As in rats, macaque mossy cells display fundamentally different traits than proximal CA3 pyramidal cells. Interestingly, macaque mossy cells and CA3 pyramidal neurons display some morphological differences from those in rats. Macaque monkey mossy cells extend more dendrites into the molecular layer of the dentate gyrus, have more elaborate thorny excrescences on their proximal dendrites, and project more axon collaterals into the CA3 region. In macaques, three types of proximal CA3 pyramidal cells are found: classical pyramidal cells, neurons with their dendrites confined to the CA3 pyramidal cell layer, and a previously undescribed cell type, the "dentate" CA3 pyramidal cell, whose apical dendrites extend into and ramify within the hilus, granule cell layer, and molecular layer of the dentate gyrus. The basic electrophysiological properties of mossy cells and proximal CA3 cells are similar to those reported for the rodent. Mossy cells have a higher frequency of large amplitude spontaneous depolarizing postsynaptic potentials, and proximal CA3 pyramidal cells are more likely to discharge bursts of action potentials. Although mossy cells and CA3 pyramidal cells in macaque monkeys display many morphological and electrophysiological features described in rodents, these findings highlight significant species differences, with more heterogeneity and the potential for richer interconnections in the primate hippocampus.     

Commissurally projecting inhibitory interneurons of the rat hippocampal dentate gyrus: a colocalization study of neuronal markers and the retrograde tracer Fluoro-gold.

Improved methods for detecting neuronal markers and the retrograde tracer Fluoro-Gold (FG) were used to identify commissurally projecting neurons of the rat hippocampus. In addition to the dentate hilar mossy cells and CA3 pyramidal cells shown previously to transport retrograde tracers after injection into the dorsal hippocampus, FG-positive interneurons of the dentate granule cell layer and hilus were detected in numbers greater than previously reported. FG labeling of interneurons was variable among animals, but was as high as 96% of hilar somatostatin-positive interneurons, 84% of parvalbumin-positive cells of the granule cell layer and hilus combined, and 33% of hilar calretinin-positive cells. By comparison, interneurons of the dentate molecular layer and all hippocampal subregions were conspicuously FG-negative. Whereas hilar mossy cells and CA3 pyramidal cells were FG-labeled throughout the longitudinal axis, FG-positive interneurons exhibited a relatively homotopic distribution. "Control" injections of FG into the neocortex, septum, and ventral hippocampus demonstrated that the homotopic labeling of dentate interneurons was injection site-specific, and that the CA1-CA3 interneurons unlabeled by contralateral hippocampal FG injection were nonetheless able to transport FG from the septum. These data suggest a hippocampal organizing principle according to which virtually all commissurally projecting hippocampal neurons share the property of being monosynaptic targets of dentate granule cells. Because granule cells innervate their exclusively ipsilateral target cells in a highly lamellar pattern, these results suggest that focal granule cell excitation may result in commissural inhibition of the corresponding "twin" granule cell lamella, thereby lateralizing and amplifying the influence of the initiating discharge.     

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.     

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.     

Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.     

Extensive target cell loss during development results in mossy fibers in the regio superior (CA1) of the rat hippocampal formation

The axons of dentate granule cells (mossy fibers) have been reported to appear in the regio superior (CA1) of the rat hippocampal formation following destruction of the pyramidal cells in the regio inferior (CA3). We undertook the present experiments to confirm this finding and to determine the requirements for this dramatic neuronal rearrangement. We found that extensive (greater than 80%) loss of CA3 cells, as well as the presence of surviving CA1 neurons within a narrow period of development (postnatal days 3-5) is necessary, however apparently not sufficient, for the appearance of CA1 mossy fibers. That the absence of normal target cells during a restricted period of mossy fiber development will lead to their association with novel targets suggests that much of the specificity of this developing connection depends on the presence of normal targets during a critical period.     

Mossy cell dendritic structure quantified and compared with other hippocampal neurons labeled in rats in vivo

Mossy cells are likely to contribute to normal hippocampal function and to the pathogenesis of neurologic disorders that involve the hippocampus, including epilepsy. Mossy cells are the least well-characterized excitatory neurons in the hippocampus. Their somatic and dendritic morphology has been described qualitatively but not quantitatively. In the present study rat mossy cells were labeled intracellularly with biocytin in vivo. Somatic and dendritic structure was reconstructed three-dimensionally. For comparison, granule cells, CA3 pyramidal cells, and CA1 pyramidal cells were labeled and analyzed using the same approach. Among the four types of hippocampal neurons, granule cells had the smallest somata, fewest primary dendrites and dendritic branches, and shortest total dendritic length. CA1 pyramidal cells had the most dendritic branches and longest total dendritic length. Mossy cells and CA3 pyramidal cells both had large somata and similar total dendritic lengths. However, mossy cell dendrites branched less than CA3 pyramidal cells, especially close to the soma. These findings suggest that mossy cells have dendritic features that are not identical to any other type of hippocampal neuron. Therefore, electrotonic properties that depend on soma-dendritic structure are likely to be distinct in mossy cells compared to other neurons.     

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.     

Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity

Two neuronal calcium-binding proteins, calbindin-D28k (CaBP) and parvalbumin (PV), were localized in the normal rat hippocampus by using immunocytochemical methods to determine 1) their location and 2) whether a correlation exists between the presence of these two calcium-binding proteins and the selective vulnerability of different hippocampal neuronal populations to experimental seizure activity. CaBP-like immunoreactivity (CaBP-LI) is present in all dentate granule cells and some, but not all, CA1 and CA2 pyramidal cells. Some CA1 pyramidal cells lack CaBP-LI, and those that do are lightly stained compared to the dentate granule cells. CA3 pyramidal cells appear to contain neither CaBP- nor PV-LI, and no granule or pyramidal cells exhibit PV-LI. CaBP-LI is present in distinct populations of dentate and hippocampal interneurons but absent from others. In area dentata, CaBP-LI is present in a small number of interneurons of the molecular and granule cell layers and in a small population of presumed basket cells in or below the granule cell layer. Conversely, more presumed dentate basket cells exhibit PV-LI than CaBP-LI. In the hilus of area dentata, few cells are CaBP- or PV-immunoreactive. The hilar somatostatin/neuropeptide Y (NPY)-immunoreactive cells and hilar mossy cells, two distinct and large populations, lack CaBP- and PV-LI. In the CA3 region, CaBP-LI is present in a relatively small number of interneurons in each stratum. PV-immunoreactive interneurons in area CA3 are more numerous. In area CA1, CaBP-LI is present in many interneurons in strata radiatum and lacunosum-moleculare. Some, but relatively fewer, CaBP-positive interneurons are present in strata pyramidale and oriens. Conversely, PV-immunoreactive interneurons are numerous in strata pyramidale and oriens but rare in strata radiatum and lacunosum-moleculare. Staining with the particulate chromagen benzidine hydrochloride revealed a previously undescribed dense band of CaBP-LI in the inner dentate molecular layer, a lamina enriched with kainate-displaceable glutamate-binding sites and innervated by the apparently excitatory ipsilateral associational/commissural (IAC) pathway that originates in the CaBP-negative hilar mossy cells. Bilateral electrical stimulation of the perforant path was performed in order to destroy the hilar mossy cells and to determine if this band of CaBP-LI is normally present within the mossy cell terminals. Perforant path stimulation that destroyed hilar mossy cells throughout the dorsal portions of both hippocampi did not abolish the dense CaBP-like immunoreactivity in the inner molecular layer.     

Selective destruction of mossy fibers and granule cells with preservation of the GABAergic network in the inferior region of the rat hippocampus after colchicine treatment

Lesions induced by colchicine injection into the rat hippocampus were investigated by means of electron microscopy and GABA immunocytochemistry. Granule cells were nearly completely destroyed 3 days after colchicine injection; since the necrosis of their axonal endings was delayed, an anterograde degeneration of the mossy fibers had probably taken place. The selectivity of the lesions was not limited to granule cells, for some pyramidal neurons in CA1 pyramidal layer were damaged. It was, however, striking to observe that throughout the hippocampal structure GABAergic neurons were spared from the effects of colchicine. For instance, GABAergic neurons were found in the vicinity of the completely destroyed granule cell layer. GABAergic neurons and terminals were also present in the CA3 region where the GABA-containing terminals formed a dense network of synapses with somata and dendrites of pyramidal cells. It was interesting to note that, consistent with previous studies, the GABAergic neurons in CA3 are innervated by mossy fibers. We conclude that after colchicine treatment the destruction of the granule cells was not associated with a lesion of the GABAergic network. This selective lesion provides a useful model with which to study the properties of CA3 neurons deprived of their major excitatory input but with an intact inhibitory network.     

Postnatal development of CA3 pyramidal neurons and their afferents in the Ammon's horn of rhesus monkeys

Previous studies described the postnatal development of CA3 pyramidal neurons and their afferents in the rat. However, the postnatal development of the primate hippocampus was not previously studied. Thus, pyramidal neurons of the CA3 area of the monkey hippocampus were analyzed postnatally in the present study. At birth, a few thorny excrescences, the complex spines postsynaptic to mossy fibers, were found on the proximal segments of both apical and basal dendrites, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number until the third month. A continuous increase in the number of spines per unit length along the distal dendrites was observed during the first 12 months. The ultrastructural features of somata and dendrites of pyramidal cells in newborn monkeys were similar to those of adults. The analysis of the afferents to the CA3 pyramidal neurons was limited to the development of mossy fibers, the axons of granule cells, and myelinated axons in the alveus, stratum oriens, and stratum lacunosum-moleculare. At birth, most mossy fiber terminals were densely packed with synaptic vesicles and formed mainly axospinous synapses with CA3 pyramidal cells. By 1 month of age, the number of mitochondria and embedded spines increased to mature amounts. In the first postnatal month, degenerating axons and axon terminals were frequently observed in the mossy fiber bundles in stratum lucidum. The proportion of myelinated axons increased simultaneously in all three examined layers. At birth most axons were unmyelinated, whereas at 7 months of age the proportion of myelinated axons was similar to that found in adults. The present study indicates that most pyramidal neurons of the CA3 region in monkeys are in an advanced stage of development at the time of birth. Thus, mossy fibers from granule cells in the dentate gyrus have established mature-looking synapses, and the thorny excrescences of pyramidal cells that are postsynaptic to mossy fibers are also adult-like. Nevertheless, several of the adult features, such as the spine density of distal dendrites of pyramidal neurons and the myelination of afferent axons, develop during an extended period of time in the first year. The significance of this early anatomical maturation in a brain region involved in memory function is consistent with recent behavioral data that show a rapid postnatal maturation of limbic-dependent recognition memory in rhesus monkeys. © 1995 Wiley-Liss, Inc.     

Changes in mint1, a novel synaptic protein, after transient global ischemia in mouse hippocampus.

Mints (munc18-interacting proteins) are novel multimodular adapter proteins in membrane transport and organization. Mint1, a neuronal isoform, is involved in synaptic vesicle exocytosis. Its potential effects on development of ischemic damage to neurons have not yet been evaluated. The authors examined changes in mint1 and other synaptic proteins by immunohistochemistry after transient global ischemia in mouse hippocampus. In sham-ischemic mice, immunoreactivity for mint1 was rich in fibers projecting from the entorhinal cortex to the hippocampus and in the mossy fibers linking the granule cells of the dentate gyrus to CA3 pyramidal neurons. Munc18-1, a binding partner of mint1, was distributed uniformly throughout the hippocampus, and synaptophysin 2, a synaptic vesicle protein, was localized mainly in mossy fibers. After transient global ischemia, mint1 immunoreactivity in mossy fibers was dramatically decreased at 1 day of reperfusion but actually showed enhancement at 3 days. However, munc18-1 and synaptophysin 2 were substantially expressed in the same region throughout the reperfusion period. These findings suggest that mint1 participates in neuronal transmission along the excitatory pathway linking the entorhinal cortex to CA3 in the hippocampus. Because mint1 was transiently decreased in the mossy fiber projection after ischemia, functional impairment of neuronal transmission in the projection from the dentate gyrus to CA3 pyramidal neurons might be involved in delayed neuronal death.     

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.     

Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study

Specific antibodies against gamma aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) were used to study the organization of the GABAergic system in the rat hippocampal formation. Both the number of GABA-like-immunoreactive (Li) somata and neuropil density were assessed in semithin sections. Cell counts revealed that approximately 11% of the hippocampal neuronal population showed GABA-Li within the various planes of section. Each layer in the hippocampal formation had a characteristic organization of GABA-Li elements. In Ammon's horn, 80-95% of the neuronal somata within the apical and basal dendritic regions were GABA-Li positive. Within the pyramidal cell layer 5-8% of the cells were GABA-Li in the CA1 to CA3 subfields of Ammon's horn and only 3% were GABA-Li within that portion of the pyramidal cell layer that inserts into the hilus. Only slight differences in the density of the GABA-Li neuropil were observed within the CA1-CA3 dendritic regions. Restricted to the stratum lucidum was a dense band of GABA-Li label. Counts of immunoreactive grains localized on the perimeter of pyramidal (CA1-CA3) and granule somata revealed more terminal boutons on the CA3 cells than on CA1 and granule neuronal somata. A topographical distribution of GABA-Li somata and neuropil was found in the fascia dentata: There the label particularly concerned its suprapyramidal and rostrolateral portions. Approximately 40% of neurons in the molecular layer, 60% in the polymorph layer, and 18% within the hilar region were GABA-Li. Within the granule cell layer only 2% of the neurons were GABA-Li positive. Distinct differences in the density of the GABA-Li neuropil were present in the molecular, pericellular granular, and hilar regions of the fascia dentata. While the morphology of GABA-Li neuronal somata varied according to their hippocampal layer, the most heterogeneous cell types were found in the regio inferior of the hippocampus. There we have identified neurons that are reminiscent of the inferior region interneuron described in Golgi material by Amaral and Woodward (Brain Res. 124:225-236, '77). Moreover, particularly in the sagittal plane, we have identified oval, triangular, and round cells and that have processes oriented in a parallel arrangement, appearing to be aligned along the granule cell mossy fibers.     

Evidence for neuronal interactions by electrical field effects in the CA3 and dentate regions of rat hippocampal slices

During population spikes in slices of rat hippocampus, transmembrane differential recordings (intracellular minus extracellular) revealed electrical field-effect depolarizations in CA3 pyramidal and dentate granule neurons. The field-effect depolarizations were not seen in single-ended recordings, and were consistently shown to increase neuronal excitability. Therefore, as previously shown for the CA1 area, large population spikes from CA3 pyramidal and dentate granule cells are associated with transmembrane depolarizations that increase neuronal excitability.     

Stimulation-induced status epilepticus: role of the hippocampal mossy fibers in the seizures and associated neuropathology

A study of seizure activity and neuronal cell death produced by intracerebroventricular kainic acid had suggested that seizures conveyed by the hippocampal mossy fibers are more damaging to CA3 pyramidal cells than seizures conveyed by other pathways. To test this idea, the effects of a unilateral mossy fiber lesion were determined on seizure activity and neuronal degeneration provoked by repetitive electrical stimulation of the hippocampal fimbria in unanesthetized rats. Fimbrial stimulation resulted in self-sustained status epilepticus accompanied by neuronal degeneration in several brain regions, including area CA3 of the hippocampal formation. A unilateral mossy fiber lesion more readily attenuated the electrographic and behavioral seizures provoked by fimbrial stimulation than those provoked by kainic acid. If status epilepticus developed in the presence of a mossy fiber lesion, denervated CA3 pyramidal cells were still destroyed, although similar lesions protect these neurons from kainic acid-induced status epilepticus. Thus the two models of status epilepticus employ somewhat different seizure circuitries and neurodegenerative mechanisms. Seizures which involve the mossy fiber projection are not necessarily more damaging to CA3 pyramidal cells than seizures which do not.     

Roles of afadin in the formation of the cellular architecture of the mouse hippocampus and dentate gyrus

The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.     

Reorganization of CA3 area of the mouse hippocampus after pilocarpine induced temporal lobe epilepsy with special reference to the CA3-septum pathway

We showed that when CA3 pyramidal neurons in the caudal 80% of the dorsal hippocampus had almost disappeared completely, the efferent pathway of CA3 was rarely detectable. We used the mouse pilocarpine model of temporal lobe epilepsy (TLE), and injected iontophoretically the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. In the afferent pathway, the number of neurons projecting to CA3 from medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei increased significantly. In the hippocampus, where CA3 pyramidal neurons were partially lost, calbindin, calretinin, parvalbumin immunopositive back-projection neurons from CA1-CA3 area were observed. Sprouting of Schaffer collaterals with increased number of large boutons in both sides of CA1 area, particularly in the stratum pyramidale, was found. When CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus have almost disappeared completely, surviving CA3 neurons in the rostral 20% of the dorsal hippocampus may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons or to dorsal part of the lateral septum. We concluded that reorganization of CA3 area with its downstream or upstream nuclei may be involved in the occurrence of epilepsy.     

Amygdala kindling develops without mossy fiber sprouting and hippocampal neuronal degeneration in rats

Repeated electrical stimulation of limbic structures has been reported to produce the kindling effect together with morphological changes in the hippocampus such as mossy fiber sprouting and/or neuronal loss. However, to argue against a causal role of these neuropathological changes in the development of kindling-associated seizures, we examined mossy fiber sprouting in amygdala (AM)-kindled rats using Timm histochemical staining, and evaluated the hippocampal neuronal degeneration in AM-kindled rats by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labelling (TUNEL). Amygdala kindling was established by 10.3 +/- 0.7 electrical stimulations, and no increase in Timm granules (neuronal sprouting) was observed up to the time of acquisition of a fully kindled state. However, the density and distribution of Timm granules increased significantly in the dentate gyrus compared with unkindled rats after 29 after-discharges or more than 10 kindled convulsions. In addition, no significant increase in TUNEL-positive cells was found in the hilar polymorphic neurons or in CA3 pyramidal neurons of the kindled rats that had fewer than 29 after-discharges. However, a significant increase of TUNEL-positive cells was found in the granule cell layer in the dentate gyrus of the stimulated side after 18 after-discharges or 10 kindled convulsions. Our result show that AM kindling develops without evidence of mossy fiber sprouting, and that mossy fiber sprouting may appear after repeated kindled convulsions, following death of the granule cells in the dentate gyrus.