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Prolonged dentate granule cell discharges produce hippocampal injury and chronic epilepsy in rats. In preparing to study this epileptogenic process in genetically altered mice, we determined whether the background strain used to generate most genetically altered mice, the C57BL/6 mouse, is vulnerable to stimulation-induced seizure-induced injury. This was necessary because C57BL/6 mice are reportedly resistant to the neurotoxic effects of kainate-induced seizures, which we hypothesized to be related to strain differences in kainate's effects, rather than genetic differences in intrinsic neuronal vulnerability. Bilateral perforant pathway stimulation-induced granule cell discharge for 4 hours under urethane anesthesia produced degeneration of glutamate receptor subunit 2 (GluR2)-positive hilar mossy cells and peptide-containing interneurons in both FVB/N (kainate-vulnerable) and C57BL/6 (kainate-resistant) mice, indicating no strain differences in neuronal vulnerability to seizure activity. Granule cell discharge for 2 hours in C57BL/6 mice destroyed most GluR2-positive dentate hilar mossy cells, but not peptide-containing hilar interneurons, indicating that mossy cells are the neurons most vulnerable to this insult. Stimulation for 24 hours caused extensive hippocampal neuron loss and injury to the septum and entorhinal cortex, but no other detectable damage. Mice stimulated for 24 hours developed hippocampal sclerosis, granule cell mossy fiber sprouting, and chronic epilepsy, but not the granule cell layer hypertrophy (granule cell dispersion) produced by intrahippocampal kainate. These results demonstrate that perforant pathway stimulation in mice reliably reproduces the defining features of human mesial temporal lobe epilepsy with hippocampal sclerosis. Experimental studies in transgenic or knockout mice are feasible if electrical stimulation is used to produce controlled epileptogenic insults.

Progesterone's effects on hippocampus-dependent behavior and synaptic connectivity maybe mediated through the progestin receptor (PR). Although estrogen induces PR mRNA and cytosolic PR in the hippocampus, nuclear PR immunoreactivity is undetectable by light microscopy, suggesting that PR is present at extranuclear sites. To determine whether this is the case, we used immunoelectron microscopy to examine PR distribution in the hippocampal formation of proestrus rats. Ultrastructural analysis revealed that PR labeling is present in extranuclear profiles throughout the CA1 and CA3 regions and dentate gyrus, and, in contrast to light microscopic findings, in nuclei of a few pyramidal and subgranular zone cells. Most neuronal PR labeling is extranuclear and is divided between pre- and postsynaptic compartments; approximately 30% of labeled profiles were axon terminals and 30% were dendrites and dendritic spines. In most laminae, except in CA3 stratum lucidum, about 15% of PR-immunoreactive profiles were unmyelinated axons. In stratum lucidum, where the mossy fiber axons course, more than 50% of PR-labeled profiles were axonal. The remaining 25% of PR-labeled profiles were glia, some resembling astrocytes. PR labeling is strongly dependent on estrogen priming, insofar as few PR-labeled profiles were detected in ovariectomized, oil-replaced females. Synapses formed by PR-labeled terminals were predominantly asymmetric, consistent with a role for progesterone in directly regulating excitatory transmission. These findings suggest that some of progesterone's actions in the hippocampal formation may be mediated by direct and rapid actions on extranuclear PRs and that PRs are well positioned to regulate progesterone-induced changes at synapses.

Nectin and afadin constitute a novel intercellular adhesion system that organizes adherens junctions in cooperation with the cadherin-catenin system in epithelial cells. Nectin is a Ca(2+)-independent immunoglobulin-like adhesion molecule and afadin is an actin filament (F-actin)-binding protein that connects nectin to the actin cytoskeleton. At the puncta adhaerentia junctions (PAs) between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, the nectin-afadin system also colocalizes with the cadherin-catenin system and has a role in the formation of synapses. ZO-1 is another F-actin-binding protein that localizes at tight junctions (TJs) and connects claudin to the actin cytoskeleton in epithelial cells. The nectin-afadin system is able to recruit ZO-1 to the nectin-based cell-cell adhesion sites in nonepithelial cells that have no TJs. In the present study, we investigated the localization of ZO-1 in the mouse hippocampus. Immunofluorescence and immunoelectron microscopy revealed that ZO-1 also localized at the PAs between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, as described for afadin. ZO-1 colocalized with afadin during the development of synaptic junctions and PAs. Microbeads coated with the extracellular fragment of nectin, which interacts with cellular nectin, recruited both afadin and ZO-1 to the bead-cell contact sites in cultured rat hippocampal neurons. These results indicate that ZO-1 colocalizes with nectin and afadin at the PAs and that the nectin-afadin system is involved in the localization of ZO-1.

Factors that regulate the formation, spatial patterning, and maturation of CNS synapses are poorly understood. We used organotypic hippocampal slice cultures derived from developing (P5-P7) rat to test whether synaptic activity regulates the development and organization of postsynaptic structures at mossy fiber (MF) giant synapses. Antibodies to a prominent postsynaptic density (PSD) scaffold protein, PSD95, identified large (>1 microm) and irregularly shaped PSD assemblies that codistributed with synapsin-I or metabotropic glutamate receptor 7b (mGluR7b) -immunolabeled MF terminals in area CA3. To investigate the spatial organization of synaptic PSDs on individual pyramidal cells, neurons in slice cultures were transfected with a vector encoding a GFP-PSD95 fusion protein. Confocal three-dimensional reconstructions revealed clusters of PSDs along proximal dendrites of transfected pyramidal neurons in area CA3, but not in CA1. Clusters averaged 7.6 microm in length (range, 2.2-29 microm) and contained up to 35 individual PSDs (mean, 8.3). PSD clusters failed to form when slices were cultured without MFs, indicating that MFs induce cluster assembly. Chronic blockade of N-methyl-D-apartate- and AMPA/kainate-type glutamate receptors did not disrupt MF targeting or de novo formation of PSD clusters with a normal distribution on target cells. Additionally, glutamate receptor blockers did not alter the ultrastructural development of MF giant synapses containing multiple puncta adherens-like junctions and asymmetric synaptic junctions at dendritic shaft and spine domains, respectively. The results indicate that MF axons can induce the assembly and clustering of PSD95-containing postsynaptic complexes, displaying a normal subcellular and tissue distribution, by mechanisms that are independent of ionotropic glutamate receptor activation.

The present investigation examined the structural integrity of the aged hippocampus by using computer-aided morphometry to quantify the volume of principal hippocampal circuits in young, mature adult, and aged Long-Evans rats. A key feature of the experimental design was that the status of hippocampal-dependent learning and memory was documented prior to histologic evaluation. The following regions, which were visualized by using Timm staining, were included in the analysis: 1) outer portions of the dentate gyrus molecular layer (OML) innervated by the lateral entorhinal cortex, 2) middle portions of the molecular layer (MML) that receive input from the medial entorhinal cortex, 3) the commissural/associational zone (IML) immediately adjacent to the granule cell layer, and 4) the hilus and mossy fiber projection to the CA3 pyramidal cell field (MF). To identify morphometric changes that emerge during the same segment of the life span as age-related learning impairment, analysis of the volumetric results focused on comparisons between the mature adult group and the aged group. Among the individual regions that were analyzed, age-related decreases in total volume were restricted to the MML. This effect, however, occurred against a background of other, subtle changes that, together, reflected substantial reorganization in the normal balance of hippocampal circuitry. Age-related decreases in the proportion of the molecular layer (ML) that comprises the MML were accompanied by a corresponding increase in relative IML volume. The ratio between the volumes of the MML and the MF also displayed significant age-related decline. Overall, aging affected septal levels of the hippocampus disproportionately, and, with the exception of MML/MF volume ratio, the temporal hippocampus was spared. Finally, the status of spatial learning among the aged animals correlated selectively with decreases in the MML/ML and MML/MF ratios. These results demonstrate that the effects of aging are regionally selective and circuit specific, and they suggest that connectional reorganization may contribute to age-related decline in the computational functions of the hippocampus. J. Comp. Neurol. 403:459‐470, 1999. r 1999 Wiley-Liss, Inc. Indexing terms: entorhinal cortex; histological techniques; maze learning; stereology

The goal of the present study was to determine whether alumina gel injections into temporal lobe structures cause complex partial seizures (CPS) and pathological changes observed in human temporal lobe epilepsy. Rhesus monkeys with alumina gel injections in the amygdala, perirhinal and entorhinal cortices, or Ammon's horn and dentate gyrus all initially displayed focal pathological electroencephalographic (EEG) slowing limited to the site of injection. After clinical seizures developed, they also displayed widespread pathological EEG slowing over both hemispheres, interictal and ictal epileptiform EEG abnormalities limited to the mesial-inferior temporal lobe on the side of injection, and different degrees of spread to other ipsilateral and contralateral structures. Noninjected control and nonepileptic monkeys with injections into the middle and inferior temporal gyri displayed no hippocampal neuronal loss or mossy fiber sprouting. When alumina gel was injected into the amygdala, CPS began within 3-6 weeks and degeneration of neurons and gliosis occurred in the perirhinal cortex or the hippocampus, with consequent sprouting of mossy fibers in the dentate gyrus. Dispersion of the granule cell layer was also observed. Other monkeys with alumina gel in the perirhinal and entorhinal cortices developed CPS within 2-3 weeks after the injections and displayed mossy fiber sprouting only after 4 weeks after the injections. Alumina gel in Ammon's horn and the dentate gyrus also induced CPS, but mossy fiber sprouting was limited to sites immediately adjacent to the injection, probably because none survived more than 4 weeks after the injections. This nonhuman primate model of CPS displayed similar anatomical, behavioral, and EEG features as observed in human temporal lobe epilepsy and provides opportunities to analyze the chronological sequence of epileptogenesis and to test potential therapies.

Thyroid hormone deficiency has been reported to interfere with synaptogenesis, particularly in those regions of the brain where the neurons display a late and protracted histogenesis, although the extent of the synaptic alterations remains unknown. To provide detailed quantitative data on the effects of hypothyroidism upon synapses, a link of the hippocampal circuitry was selected: the contact between mossy fibers and dendritic excrescences of CA3 pyramidal cells (MF-CA3 synapses). Groups of six male and six female rats aged 30 and 180 days were analyzed separately after being treated as follows: (1) hypothyroid from day 0 until day 30 (30 day old hypothyroid group); (2) hypothyroid from day 0 until day 180 (180 day old hypothyroid group); (3) hypothyroid until day 30 and thenceforth maintained euthyroid (recovery group); and (4) and (5) 30 and 180 day old control groups, respectively. Timm staining, Golgi impregnation, and electron microscopy were employed to estimate the volume of the mossy fiber system, the number and size of mossy fiber boutons, and the number and related features of MF-CA3 synapses. The volume of the mossy fiber system and the number of synaptic boutons were reduced in all experimental groups. The total number of synapses was decreased in 30 day old hypothyroid rats, but did not differ among 180 day old animals. Postsynaptic densities were shorter in hypothyroid and recovery groups than in controls, although the reduction was not as marked in recovery rats as it was in hypothyroid animals. Structural alterations were noted in the pre- and postsynaptic compartments of MF-CA3 synapses of both 180 day old hypothyroid and recovery rats. These changes can be regarded as mechanisms of reorganization as they underlie the compensation for the hypothyroid-induced numerical reduction of synapses observed in 30 day old animals and enable a complete catch-up of their total number. However, synaptic reorganization was not fully achieved, as revealed by the reduction in the size of the synaptic sites in hypothyroid and recovery animals. Finally, we demonstrate that hypothyroidism did not interfere with the sex-related differences of MF-CA3 synapses described in normal rats.

Estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta), and progestin receptor (PR) immunoreactivities are localized to extranuclear sites in the rat hippocampal formation. Because rats and mice respond differently to estradiol treatment at a cellular level, the present study examined the distribution of ovarian hormone receptors in the dorsal hippocampal formation of mice. For this, antibodies to ERalpha, ERbeta, and PR were localized by light and electron immunomicroscopy in male and female mice across the estrous cycle. Light microscopic examination of the mouse hippocampal formation showed sparse nuclear ERalpha and PR immunoreactivity (-ir) most prominently in the CA1 region and diffuse ERbeta-ir primarily in the CA1 pyramidal cell layer as well as in a few interneurons. Ultrastructural analysis additionally revealed discrete extranuclear ERalpha-, ERbeta-, and PR-ir in neuronal and glial profiles throughout the hippocampal formation. Although extranuclear profiles were detected in all animal groups examined, the amount and types of profiles varied with sex and estrous cycle phase. ERalpha-ir was highest in diestrus females, particularly in dendritic spines, axons, and glia. Similarly, ERbeta-ir was highest in estrus and diestrus females, mainly in dendritic spines and glia. Conversely, PR-ir was highest during proestrus, mostly in axons. Except for very low levels of extranuclear ERbeta-ir in mossy fiber terminals in mice, the labeling patterns in the mice for all three antibodies were similar to the ultrastructural labeling found previously in rats, suggesting that regulation of these receptors is well conserved across the two species.

Projections of glutamatergic somatosensory and auditory fibers to the cochlear nucleus (CN) are mostly nonoverlapping: projections from the spinal trigeminal nucleus (Sp5) terminate primarily in the granule cell domains (GCD) of CN, whereas type I auditory nerve fibers (ANFs) project to the magnocellular areas of the VCN (VCNm) and deep layers of Dorsal CN (DCN). Vesicular glutamate transporters (VGLUTs), which selectively package glutamate into synaptic vesicles, have different isoforms associated with distinct subtypes of excitatory glutamatergic neurons. Here we examined the distributions of VGLUT1 and VGLU2 expression in the CN and their colocalization with Sp5 and ANF terminals following injections of anterograde tracers into Sp5 and the cochlea in the guinea pig. The CN regions that showed the most intense expression of VGLUT1 and VGLUT2 were largely nonoverlapping and were consistent with ANF and Sp5 projections, respectively: VGLUT1 was highly expressed in VCNm and the molecular layer of the DCN, whereas VGLUT2 was expressed predominantly in the GCD. Half (47% +/- 3%) of the Sp5 mossy fiber endings colabeled with VGLUT2, but few (2.5% +/- 1%) colabeled with VGLUT1. In contrast, ANFs colabeled predominantly with VGLUT1. The pathway-specific expression of VGLUT isoforms in the CN may be associated with the intrinsic synaptic properties that are unique to each sensory pathway.

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP+ of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP+ cells were observed migrating into the DG. Subsets of these EGFP+ cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP+ cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP+ cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP+ cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP+/RIP+ oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis. J. Comp. Neurol. 497:833–845, 2006. © 2006 Wiley-Liss, Inc.

Synaptogyrins comprise a family of tyrosine-phosphorylated proteins with two neuronal (synaptogyrins 1 and 3) and one ubiquitous (cellugyrin) isoform. Previous studies have indicated that synaptogyrins are involved in the regulation of neurotransmitter release. Synaptogyrin 1 is a synaptic vesicle protein; cellugyrin, by contrast, is absent from synaptic vesicles. In an effort to further characterize the synaptogyrin family, we studied the distribution of the synaptogyrin 3 protein in the nervous system. Subcellular fractionation and immunoprecipitation of synaptic vesicles from mouse brain showed that synaptogyrin 3 is associated with synaptic vesicles and that synaptogyrins 1 and 3 can reside on the same synaptic vesicle. Immunofluorescent staining of cultured hippocampal neurons confirmed the synaptic localization of synaptogyrin 3. Analysis of the relative distributions of synaptogyrins 1 and 3 in mouse brain revealed a more restricted expression pattern for synaptogyrin 3 compared to the ubiquitous distribution of synaptogyrin 1. Strong synaptogyrin 3 labeling was observed in the mossy fiber region of the hippocampus, substantia nigra pars reticulata, pallidum, and deep cerebellar nuclei. By comparison, the striatum and reticular and ventral posterolateral thalamic nuclei, which all showed synaptogyrin 1 labeling, contained significantly less synaptogyrin 3. Finally, we used in situ hybridization experiments to correlate synaptogyrin 3 mRNA in cell bodies with synaptogyrin 3 protein at synapses. Altogether, our data indicate that neuronal synaptogyrins are differentially expressed protein isoforms that may represent functionally distinct populations of synapses and/or synaptic vesicles.

The postnatal development of excitatory amino acid receptor types including kainate, N-methyl-D-aspartate (NMDA), and -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was assessed in the hippocampus, entorhinal cortex, and adjacent neocortex in normal and prenatally protein malnourished rats ages 15, 30, 90, and 220 postnatal days by quantitative autoradiography. Tritiated ligands used to measure binding site density were 3 [H]kainate, 3 [H]MK-801, and 3 [H]AMPA, respectively. Kainate receptors showed statistically significant increases in binding density in stratum lucidum of CA3 (hippocampal mossy fiber zone) in 90and 220-day-old malnourished rats compared with age- and sex-matched controls but not in 15or 30-day-old malnourished rats. Compared with previous anatomic studies, these results are mostly in agreement with a significantly decreased hippocampal mossy fiber plexus in 15-, 90-, and 220-day-old rats but not in 30-day-old rats. These results suggested that the increased density of postsynaptic kainate receptors located mainly on proximal apical dendrites of CA3 pyramidal cells may be compensatory to decreased glutamate release due to the reduction in mossy fiber plexus. In contrast, the density of putative NMDA and AMPA receptors quantified in prenatally malnourished rats was comparable to the density quantified in age- and sex-matched control rats, as were all three receptor types in entorhinal cortex and adjacent neocortex. Thus, the selectivity of the compensation of 3 [H]kainate-labeled mossy fiber plexus in adult but not in early postnatal developing malnourished rats may help ensure continued breeding and survival of the species under otherwise adverse environmental conditions. J. Comp. Neurol. 456:350‐360, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: ligand binding; autoradiography; glutamatergic; undernutrition; mossy fibers (stratum lucidum of CA3)

We have recently revealed that large multipolar neurons, presumed mossy cells in the hamster dentate gyrus (DG), were calretinin (CR)-immunoreactive (IR) at the ventral level, although these neurons were CR-negative at the dorsal level. In the present study, we confirmed this identification with several methods and analyzed structural features of hamster mossy cells in detail. Golgi impregnationi and intracellular Lucifer yellow labeling studies revealed that mossy cells in the hamster dentate hilus had extraordinarily prominent thorny excrescences on their somata as well as on their proximal dendrites. Mossy cells exhibited dorsoventral differences in their structural features; proximal dendrites of single mossy cells were fewer, and thorny excrescences were larger and more complicated at the dorsal level than at the ventral level. Electron microscopic serial section three-dimensional reconstructions revealed that somatic thorny excrescences consisted of large and complicated spines, which received numerous asymmetrical synapses from mossy fiber terminals. In addition, our confocal laser scanning microscopic observations also revealed many glutamic acid decarboxylase-immunoreactive punctae abutting the mossy cell somata and dendrites. Our present and previous observations revealed the structural features of hamster mossy cells and their differences along the dorsoventral axis and further indicated that mossy cells were prominently different in their chemical and morphological features among species.

We have recently found a novel cell–cell adhesion system at cadherin-based adherens junctions. This system consists of at least two components: nectin, an immunoglobulin-like cell adhesion molecule with Ca2+-independent homophilic binding activity, and l-afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. In the present study, we investigated immunocytochemically the localization of l-afadin in the mouse hippocampus. At the light microscopic level, l-afadin immunoreactivity was demonstrated as flattened disks in the stratum lucidum of the CA3 area. By immunoelectron microscopy, signals for l-afadin were highly concentrated in a symmetrical manner at the puncta adhaerentia-like junctions between the mossy fiber terminals and the dendritic trunks of pyramidal cells. We furthermore immunostained the hippocampus with antibodies recognizing both l-afadin and s-afadin, a small splicing variant of l-afadin that is identical to AF-6. Immunoreactivity for l- and s-afadins was demonstrated not only as the flattened disks similar to that for l-afadin, but also as numerous fine dots widely distributed in all synaptic layers of the CA1 and CA3 areas. The latter finding may correspond with the recent report by Buchert et al. (1999, J. Cell. Biol. 144:361–371), who found that s-afadin (AF-6) and/or l-afadin was localized at the postsynaptic membranes of asymmetric synaptic junctions. Our present results indicate that l- and s-afadins are differentially distributed in the hippocampus and suggest that l-afadin localized at the puncta adhaerentia-like junctions in the mossy fiber terminals may regulate the structural and functional organization of these complex synaptic structures. J. Comp. Neurol. 424:297–306, 2000. © 2000 Wiley-Liss, Inc.

Chandelier cells are specialized cortical GABAergic neurons that establish synaptic contacts exclusively with the axon initial segments of principal neurons. They are found in all regions of the hippocampal formation. Here we describe their morphological features in the hilus and in regions CA1 and CA3 by using Golgi/electron microscopy. Attempts were also made to identify the target neurons of chandelier cells in the hilus and entorhinal cortex. Golgi-impregnated chandelier cells display a complex axonal arbor in CA1, with many collaterals forming strings of boutons. The axon plexuses of such cells are less developed in CA3, whereas those in the hilus cover the entire region, although single collaterals are rather simple, with only a few boutons. The dendrites of chandelier cells in CA1 and CA3 have an orientation similar to that of pyramidal cell dendrites and are thus likely to be activated by the same afferent fiber systems. The hilar chandelier cells do not give rise to dendrites invading the molecular layer. Thus, these cells may not receive a dense input from the entorhinal cortex but may be driven by the abundant mossy fiber collaterals in the hilar region. In the CA1 and CA3 regions, the axons of chandelier cells contact the axon initial segments of pyramidal cells. In the hilar region, gold-toned boutons were found to impinge on the initial segments of neurons displaying characteristics of mossy cells. This notion was substantiated by electron microscopic analysis of mossy cells identified by intracellular injection of Lucifer yellow. Those cells regularly showed numerous symmetric synapses on their axon initial segments. Entorhinohippocampal projection cells, identified by injection of horseradish peroxidase into the hippocampus, were found to be preferential targets of chandelier cells in the entorhinal cortex. Our data point to regional variations in chandelier cell morphology and connectivity and indicate that chandelier cells are a principal component of inhibitory mechanisms in all stations of the main excitatory pathway of the hippocampal formation.

Opioid peptides, including leu-enkephalin (LE), are important neuromodulators in the hippocampal formation where they may play a role in learning and memory as well as epileptogenesis. We examined the cellular substrates that underlie the function of LE in each lamina of the rat hippocampal formation by immunocytochemistry at the electron microscopic level in single section analysis. LE-like immunoreactivity (LE-LI) was primarily associated with large dense-core vesicles (80-100 nm), usually found in axons and axon terminals, but was also observed in perikarya and occasionally in dendrites. The morphology and synaptic associations of LE-LI-containing terminals were strikingly distinct in each region of the hippocampal formation. In the molecular layer of the dentate gyrus, terminals with LE-LI were typically small (0.6 microns) and formed primarily asymmetric (excitatory type) synapses on single dendritic spines, which is consistent with the presence of LE in the lateral perforant path. In the hilus of the dentate gyrus, two types of LE-containing terminals were present: (1) small round terminals that were heterogeneous in size (0.4-1 microns) and in type of contact formed and (2) larger (3-5 microns) terminals exhibiting the characteristic morphology of mossy fiber boutons that formed asymmetric synapses on spines. This variation in morphology and the type of contact suggests LE may have a heterogeneous influence on diverse hilar interneurons. In the CA3 region of the hippocampus, LE-LI was localized to large mossy fiber boutons (3-7 microns) that formed multiple asymmetric synapses on complex spiny dendritic processes and often formed puncta adherentia with the shafts of large CA3 pyramidal cell dendrites, indicating that this peptide may be directly released onto pyramidal cells. At the border of stratum radiatum and lacunosum moleculare in the CA1 region of the hippocampus, LE-labeled terminals averaged 0.8 microns in diameter and often formed symmetric (inhibitory type) synapses on dendritic shafts, which is consistent with a role in disinhibition. In conclusion, these heterogeneous cellular interactions indicate that LE has diverse functional roles and mechanisms of action within each lamina of the hippocampal formation and may directly and indirectly modulate hippocampal cell activity.

There is increasing evidence that the various types of hippocampal nonpyramidal neurons control the principal cells in different ways. In the present study a type of spiny nonpyramidal cell in stratum lucidum of rat hippocampal region CA3 was studied by Golgi impregnation. Three Golgi-impregnated and gold-toned neurons of this type were further analyzed by electron microscopy and postembedding immunocytochemistry. The dendrites of these bipolar neurons seemed to be restricted to stratum lucidum and ran parallel with the mossy fibers that terminate in this layer. A characteristic feature of this neuron is the presence of long, thin spines on both cell body and dendrites. Although these dendrites were exposed to a large number of mossy fibers, no thorny excrescences were formed which are characteristic postsynaptic elements of CA3 pyramidal neurons for synaptic contact with the mossy fibers. Semithin sections of Golgi-impregnated and gold-toned stratum lucidum cells displayed immunoreactivity of the cell body region for glutamate but not for GABA. A fine-structural analysis of gold-toned sections revealed a large cell body with numerous cytoplasmic organelles and an indented nucleus. Numerous asymmetric synapses were found on dendritic shafts as well as on the long, thin somatic and dendritic spines. Usually, several presynaptic boutons contacted a single spine. The majority of these asymmetric spine synapses were probably of mossy fiber origin, although no giant mossy fiber synapses were formed. The long spines were contacted by much smaller en passant synapses of preterminal axons. In contrast, giant mossy fiber boutons were found presynaptic to dendritic shafts and cell bodies of these cells. Our morphological analysis of a glutamate-immunoreactive, GABA-negative type of nonpyramidal neuron that receives convergent mossy fiber input suggests that the impulse flow within the "trisynaptic pathway" is more complex than previously assumed.

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.

This paper is the third in a series to quantify differences in the composition of subcellular organelles and three-dimensional structure of dendritic spines that could contribute to their specific biological properties. Proximal apical dendritic spines of the CA3 pyramidal cells receiving synaptic input from mossy fiber (MF) boutons in the adult rat hippocampus were evaluated in three sets of serial electron micrographs. These CA3 spines are unusual in that they have from 1 to 16 branches emerging from a single dendritic origin. The branched spines usually contain subcellular organelles that are rarely found in adult spines of other brain regions including ribosomes, multivesicular bodies (MVB), mitochondria, and microtubules. MVBs occur most often in the spine heads that also contain smooth endoplasmic reticulum, and ribosomes occur most often in spines that have spinules, which are small nonsynaptic protuberances emerging from the spine head. Most of the branched spines are surrounded by a single MF bouton, which establishes synapses with multiple spine heads. The postsynaptic densities (PSDs) occupy about 10-15% of the spine head membrane, a value that is consistent with spines from other brain regions, with spines of different geometries, and with immature spines. Individual MF boutons usually synapse with several different branched spines, all of which originate from the same parent dendrite. Larger branched spines and MF boutons are more likely to synapse with multiple MF boutons and spines, respectively, than smaller spines and boutons. Complete three-dimensional reconstructions of representative spines with 1, 6, or 12 heads were measured to obtain the volumes, total surface areas, and PSD surface areas. Overall, these dimensions were larger for the complete branched spines than for unbranched or branched spines in other brain regions. However, individual branches were of comparable size to the large mushroom spines in hippocampal area CA1 and in the visual cortex, though the CA3 branches were more irregular in shape. The diameters of each spine branch were measured along the cytoplasmic path from the PSD to the origin with the dendrite, and the lengths of branch segments over which the diameters remained approximately uniform were computed for subsequent use in biophysical models. No constrictions in the segments of the branched spines were thin enough to reduce charge transfer along their lengths.(ABSTRACT TRUNCATED AT 400 WORDS)

A semiquantitative, electron microscopic immunocytochemical procedure based on the use of colloidal gold particles as markers was employed to analyze the subcellular distribution of glutamate and glutamine, a major glutamate precursor, in a subpopulation of spinocerebellar mossy fiber terminals. These terminals were identified by anterograde transport of a horseradish peroxidase-wheat germ agglutinin conjugate, injected in the thoracic spinal cord. Gold particles signalling glutamate-like immunoreactivity were enriched over clusters of synaptic vesicles relative to organelle-free cytoplasmic matrix, and there was a strong positive correlation between gold particle and synaptic vesicle densities (correlation coefficient 0.94). Gold particles indicating glutamine-ike immunoreactivity showed a much weaker correlation with vesicle density (correlation coefficient 0.36) and were about equally concentrated over cytoplasmic matrix as over clusters of synaptic vesicles. Compared with the mossy fibers, the putative GABAergic Golgi cell terminals exhibited a lower level of glutamate-like immunoreactivity, which was very weakly correlated with the vesicle density (correlation coefficient 0.27). The level of glutamine-like immunoreactivity in the Golgi cell terminals was similar to that in mossy fibers, but much lower than that in glial cells. The anterogradely labelled mossy fiber terminals were not enriched in immunoreactivities for aspartate or GABA. These results suggest that the level and subcellular distribution of glutamate in presumed glutamatergic terminals differs from that in terminals in which glutamate only serves metabolic or precursor roles, and that these differences can be exploited in immunocytochemical studies aimed at identifying glutamate-using neurons. In contrast, glutamine immunocytochemistry does not seem to be generally useful in this regard.

The commissural connections of the area dentata were investigated with the Fink-Heimer silver impregnation method and the commissural terminals in the hilus of the fascia dentata further studied by electron microscopy of anterograde degeneration. The commissural endings in the molecular layer were found to terminate as previously reported by others. In addition it was shown that the hilus also receives a significant commissural input. The commissural afferents to both the molecular layer and the hilus terminate along the full rostro-caudal extent of the area dentata, but with varying densities. The degeneration in the molecular layer is maximal dorso-rostrally and declines in the caudo-ventral direction, whereas the degeneration in the hilus varies inversely. The commissural terminals in the hilus make asymmetrical contacts with dendritic spines and to a lesser extent with dendritic stems. Dark, but otherwise apparently normal terminals with the features of mossy fiber boutons, were encountered in low numbers in both decommissurated and control animals. The commissural projection to the dentate area originates in the opposite hilus and possibly the adjacent part of CA3 (CA3c). Fibers from middle dorso-basal levels of the hilus to the opposite molecular layer are distributed more rostrally than ventrally relative to the level of the source of the fibers.

Terminal boutons of mossy fiber collaterals in the fastigial nucleus originating from the nucleus reticularis tegmenti pontis, the bulbar reticular formation, and the medial vestibular nucleus were studied with high-resolution autoradiography in order to examine their ultrastructural features and synaptic relations. Labeled mossy fiber boutons ranged in size from 0.5 to 5 micron in diameter, and they all contained clear and spherical vesicles in an electron-lucent matrix, mitochondria, and some fine tubular elements. These boutons form asymmetric synapses with dendritic profiles of different sizes. No evidence was found for mossy fiber termination on the soma of fastigial neurons. Two types of mossy fiber terminals were distinguished on the basis of the aggregation of synaptic vesicles: one type with clustered vesicles and one type with densely packed vesicles, occurring in equal number from all sources. Furthermore, the applicability of the congruity hypothesis is confirmed for the general identification of terminals.

Much of the recent data on cells, synapses, and other structures in the dentate gyrus and hippocampus as a function of age are packing density or volume fraction data. In order to estimate total numbers, volumes, or surface areas of cells, synapses, vessels, etc., as a function of age, the total volumes of the subregions of the dentate gyrus and hippocampus must be known. The volumes of these subregions, visualized with the Timm stain, have been determined in 24 F344 rats from 4 to 37 months of age. Volumes of the various structures showed age-related increases which were statistically significant for the perforant path zone of the dentate gyrus molecular layer, as well as the total molecular layer, the hilus, and regio inferior and total mossy fiber systems. If the 4-month age group is eliminated from consideration, only the ratio of the volume of the mossy fiber zones to the volume of the perforant path zones of the dentate molecular layer increases significantly with age. Our general finding of lack of volumetric reorganization of the subdivisions of the hippocampal region between 12 and 37 months suggests that studies of the packing densities of structures in most of these zones may be considered comparable across ages, assuming comparability of sampling regions.

The neuronal generation patterns of hippocampal pyramids were analyzed in two inbred strains of house mice (SM/J and BALB/cJ) using tritiated thymidine radioautography. These two strains of mice possess markedly different patterns of mossy fiber synapses upon pyramidal neurons within a specific segment of regio inferior. The results of this study show that this same segment of regio inferior also displays markedly different patterns of pyramidal neuron generation in the two strains. Specifically, the pyramids of this segment of regio inferior are generated according to the typical [inside-out] sequence in SM/J mice, but this pattern is reversed into an [outside-in] sequence in BALB/cJ mice. The remaining segment of regio inferior, and all of regio superior, is formed in an inside-out sequence in both strains. These results strongly suggest that altered temporal patterns of pyramidal neuron generation may play a major role in determining mossy fiber connectivity patterns.

Intraventricular injections of kainic acid were used to create a model of selective cell death in order to study the fate of afferent projections that are deprived of their postsynaptic targets. This treatment rapidly destroyed hipocampal CA3 pyramidal cells, but not those neurons that give rise to their mossy fiber and entorhinal afferents. Light microscopic studies with the Timm's sulfide silver stain indicated that half or more of the mossy fiber boutons in area CA3b were lost within the first 1–3 days after kainic acid administration. This finding was confirmed by electron microscopy. Electron-dense, usually vacuolated mossy fiber boutons accounted for about 10–20% of the total population present at a 4-hour survival time, but were not encountered in control rats nor at survival times longer than 1 day. Other mossy fiber boutons remained electron lucent, but enlarged became more rounded in shape, and suffered and apparent loss of synaptic vesicles. It is suggested that degeneration of some mossy fiber boutons and resorption of others into the axon may have accounted for the precipitous decline in their number. The dendritic excrescences contacted by these boutons were nearly all undergoing electron-dense degeneration 4 hours after kainic acid administration. In rats that survived 6–8 weeks mossy fiber boutons remained somewhat scarce, individual boutons appeared relatively small, and only one-third the normal percentage were observed to be engaged in more than one synaptic contact within a single cross section. A qualitative electron microscopic study of the entorhinal projection to area CA3 suggested a response to kainic acid treatment similar to that of the mossy fiber projection, except that no entorhinal boutons were seen to become electron dense. These findings suggest that presynaptic fibers in the mature hippocampus adjust the size of their terminal arborizations and number of synaptic contacts to accommodate a reduction in the target cell population. The rapid loss of mossy fiber boutons may be attributable to an unsual fragility of these structures when they are deprived of the mechanical support normally provided by the pyramidal cell. Finally, the ability of kainic acid administration to alter the number and distribution of presynaptic elements must be taken into account whenever this toxin is used to make selective lesions of postsynaptic cells.

The boutons of the hippocampal mossy fibers stain particularly well with Timm's sulfide silver reaction for zinc and other metals. In the present study anterograde degeneration was produced in the mossy fibers in the rat by placing lesions in the fascia dentata, after which the Timm reaction was checked at different postoperative intervals. No unequivocal changes were seen as early as five hours after operation. Barely discernible blanching of the affected parts of the mossy fiber layer was observed after seven hours, a significant reduction of stainability was present after ten hours, and a nearly complete loss after 24 hours. The latter situation persisted for the rest of the period studied, viz., up to eight days after operation. Topographical coincidence of reduced Timm staining with areas of anterograde degeneration was verified by impregnating alternating sections of the sulfide treated tissue according to the reduced silver method of Fink and Heimer. These sections moreover confirmed that the affected boutons retain their structural identity for at least several days after the disappearance of the stainability with Timm's method. These findings are compatible with the concept that the zinc may be associated with some rapidly metabolized substance directly or indirectly involved in synaptic transmission.

Udgivelsesdato: 1984-Jul-10 A quantitative and qualitative description has been made of the components of the Timm-stained hippocampus of the European hedgehog. While the laminar organization and the relative size of the major subdivisions of the hippocampus (i.e., area dentata, Ammon's horn, and subiculum) are similar to those of the albino laboratory rat, the relative proportions and the staining characteristics of some of the components of the subdivisions are different. The differences are particularly evident in Ammon's horn where regions are poorly differentiated along the dentatosubicular axis and the mossy fiber zone is relatively extensive. The description characterizes a hippocampal form that can be used as a basal reference in comparative studies of the mammalian hippocampus.

The rat hippocampus and fascia dentata contain neurons and terminal fields that react immunohistochemically with antisera raised against the peptides somatostatin, cholecystokinin (CCK), and enkephalin. Neurons with overlapping distribution synthesize acetylcholinesterase (AChE) as shown histochemically after systemic administration of diisopropylfluorophosphate (DFP). We used these techniques to examine the organization of peptidergic neurons and fibers and AChE-positve neurons in transplants of late embryonic and early postnatal rat hippocampi inserted into the brain of immature and adult rats. The astroglial organization was examined using an antibody raised against the astroglial marker, glial fibrillary acidic protein (GFAP). Eighty percent of the transplants were recovered. Within the transplants the peptidergic neurons, and the AChE-producing neurons retained their characteristic distributions. An organotypical astroglial pattern was also retained with some indication of a higher than normal density of cells and processes. The transplant neuropil displayed characteristic patterns of terminal field-like staining with the enkephalin and CCK antibodies. An enkephalin-like reactivity characteristic of the large mossy fiber terminals was found in the normal mossy fiber zones. A similar staining, but of smaller particle size, was found in and above the granule cell layer. It corresponded to the aberrant, supragranular mossy fiber projection, known to form after denervation of the dentate molecular layer. Also the CCK-like reactivity of the transplant neuropil was distributed in laminar patterns characteristic of the different subfields. Deviations from the normal patterns again followed known principles for lesion-induced aberrant nerve growth. Normalization of the enkephalin and CCK staining patterns in the dentate molecular layer was observed in transplants with intimate contact with the host dentate molecular layer or entorhinal cortex, but only in immature recipients, and not in every case. The staining was typically of the enkephalin-reacting lateral perforant path and the CCK-reacting medial perforant path and distributed in normal, mutually exclusive laminae in the dentate molecular layer. An invasion of the transplant dentate molecular layer by heterologous, host enkephalin- and CCK-like reacting afferents in transplants located in the host basal ganglia was limited at most. Outgrowth of CCK and enkephalin reacting transplant fibers into the host brains was not observed in this study. We conclude (1) that peptidergic and AChE-producing hippocampal and dentate neurons can survive intracerebral transplantation and that an organotypic, astroglial pattern of the tissue was retained after transplantation, (2) that the enkephalin- and CCK-reacting afferents have a growth capacity similar to most other hippocampal afferents and participate in the intrinsic connective reorganizations like these afferents, and (3) that enkephalin- and CCK-reacting host afferents, presumably of entorhinal origin, can innervate the transplant dentate molecular layer in a homotypic, laminar fashion, in immature recipients with close contact between the transplant and the host dentate or entorhinal area.

The Ammonic system of inbred mouse strains was examined with light and electron microscopic methods in order to determine whether genetically-associated variations occur in the distribution of dentate granule cell synapses (mossy fiber boutons) upon the dendrites of hippocampal pyramidal neurons in Regio Inferior. The results of this investigation showed that both qualitative and quantitative variations of the mossy fiber system occur among the strains examined. The most striking variation was seen in strain BALB/cJ. These mice are unique in that their mossy fibers do not form an appreciable infrapyramidal synaptic field, but form an extensive intrapyramidal synaptic field instead. In marked contrast, SM/J mice exhibit an extensive development of infrapyramidal mossy fiber boutons while their intrapyramidal mossy fiber synaptic field, when present at all, is limited to a very small portion of Regio Inferior adjacent to the hilus of Area Dentata. Since the mouse strains used in this study do not exhibit neurological defects (i.e., they are not neurological mutants identified through the presence of gross behavioral or external anatomical abnormalities), the described variations may be thought of as representing genotype-dependent differences in the connectivity of the Ammonic system which might occur within a “normal,” heterogeneous population of mice, Genetic, developmental and functional implications of the observed variations in the distribution of mossy fiber boutons upon Regio inferior pyramids are discussed.

Mossy fiber-deafferentated rats (20) were permitted to survive from 34 to to 120 days and subsequently examined following Golgi-Cox preparation or after processing for electron microscopy. The primary response to mossy fiber deafferentation was transneuronal degeneration of the granule cell system. Morphological evidence is provided that suggests that the mossy fiber varicosity plays an important role in the fragmentation and removal of the granule cell digitiform dendrite. Computer-assisted image analysis of Golgi-impregnated Purkinje cells indicated significant losses in both smooth branch and spiny branchlet numbers following loss of the mossy fiber input. Ultrastructural examination revealed that a secondary transneuronal degeneration occurred within the dendritic arborization of both Purkinje cells and molecular layer interneurons. Although an overall reduction in the number of dendritic spines occurred along the terminal branchlets following mossy fiber deafferentation, several of the existing spines underwent marked changes in length, with some elongating to more than twice their size. By increasing the length of their spines, denervated Purkinje cells may acquire new synaptic contacts.