Mossy Cells in the Dorsal and Ventral Dentate Gyrus Differ in Their Patterns of Axonal Projections.

Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function.SIGNIFICANCE STATEMENT Mossy cells (MCs), a major cell type in the hilus of the dentate gyrus (DG), are unique in providing extensive longitudinal and commissural projections throughout the DG. Although it has been assumed that all MCs have similar patterns of termination in the inner molecular layer of the DG, we discovered that the axonal projections of dorsal and ventral MCs differ. While ventral MC projections exhibit the classical pattern, with dense innervation in the inner molecular layer, dorsal MCs have a more diffuse distribution and expand into the middle molecular layer where they overlap and interact with innervation from the perforant path. These distinct locations and patterns of axonal projections suggest that dorsal and ventral MCs may have different functional roles.

Early Aberrant Growth of Mossy Fibers after Status Epilepticus in the Immature Rat Brain.

Axonal sprouting is recognized to be an important mean of repair after neurologic injury. Some characteristic aftermaths of pilocarpine-induced status epilepticus (SE) in the immature rat are nerve cell loss and rearrangement of neuronal fibers. SE induced cell degeneration exclusively in the hippocampal CA1 subfield. Development of neuronal death becomes evident within hours after SE, following a delayed time course ranging from 6 to 48 h post-SE. An incidental finding is that pilocarpine induces within 48 h an aberrant growth of hippocampal mossy fibers in the hippocampus, especially in the infrapyramidal region of the CA3-subfield. We found a strong infrapyramidal band of mossy fibers along the entire stratum oriens of the CA3-region. No mossy fibers sprouting into the inner molecular layer of the dentate gyrus, or CA1 sprouting into the stratum moleculare of CA1 were noted. Signs of aberrant connectivity were found in six of the 10 pilocarpine-treated animals. This study provides the demonstration that pilocarpine within 48 h consistently results in the formation of ectopic hippocampal mossy fibers in a 2-week-old pup. This indicates a high degree of axonal reorganization in the hippocampus. It remains controversial whether such reorganization is the cause or consequence of chronic seizures. We assume that these additional infrapyramidal mossy fibers may influence the way in which granule cells drive pyramidal cells in CA3.

Npas4 Is a Critical Regulator of Learning-Induced Plasticity at Mossy Fiber-CA3 Synapses during Contextual Memory Formation.

Synaptic connections between hippocampal mossy fibers (MFs) and CA3 pyramidal neurons are essential for contextual memory encoding, but the molecular mechanisms regulating MF-CA3 synapses during memory formation and the exact nature of this regulation are poorly understood. Here we report that the activity-dependent transcription factor Npas4 selectively regulates the structure and strength of MF-CA3 synapses by restricting the number of their functional synaptic contacts without affecting the other synaptic inputs onto CA3 pyramidal neurons. Using an activity-dependent reporter, we identified CA3 pyramidal cells that were activated by contextual learning and found that MF inputs on these cells were selectively strengthened. Deletion of Npas4 prevented both contextual memory formation and this learning-induced synaptic modification. We further show that Npas4 regulates MF-CA3 synapses by controlling the expression of the polo-like kinase Plk2. Thus, Npas4 is a critical regulator of experience-dependent, structural, and functional plasticity at MF-CA3 synapses during contextual memory formation.

Quantitative analysis of zinc in rat hippocampal mossy fibers by nuclear microscopy.

Zinc (Zn) is involved in regulating mental and motor functions of the brain. Previous approaches have determined Zn content in the brain using semi-quantitative histological methods. We present here an alternative approach to map and quantify Zn levels in the synapses from mossy fibers to CA3 region of the hippocampus. Based on the use of nuclear microscopy, which is a combination of imaging and analysis techniques encompassing scanning transmission ion microscopy (STIM), Rutherford backscattering spectrometry (RBS), and particle induced X-ray emission (PIXE), it enables quantitative elemental mapping down to the parts per million (µg/g dry weight) levels of zinc in rat hippocampal mossy fibers. Our results indicate a laminar-specific Zn concentration of 240±9µM in wet weight level (135±5µg/g dry weight) in the stratum lucidum (SL) compared to 144±6µM in wet weight level (81±3µg/g dry weight) in the stratum pyramidale (SP) and 78±10µM in wet weight level (44±5µg/g dry weight) in the stratum oriens (SO) of the hippocampus. The mossy fibers terminals in CA3 are mainly located in the SL. Hence the Zn concentration is suggested to be within this axonal presynaptic terminal system.

Endothelin receptors A and B are expressed in distinct cellular compartments of rat hippocampus following global ischemia: an immunocytochemical study.

OBJECTIVES: The syntheses of endothelin receptors A and B were previously shown to be upregulated in rat dorsal hippocampus after traumatic brain injury. Here we characterize endothelin receptor A and endothelin receptor B cellular distribution in hippocampus after permanent global brain ischemia and their possible association to nerve cell injury. METHODS: Twenty-minute global ischemia was induced using the Pulsinelli's four-vessel occlusion in conjunction with systemic hypovolemia in male rats. Endothelin receptor A and endothelin receptor B immunoreactivities from sham-operated and ischemic rats were assessed qualitatively in dentate gyrus, Cornu Ammonis, and hilus regions of the hippocampus. Quantitative immunoreactivity measurements were also obtained by optical densitometry. RESULTS: In sham-operated control hippocampus, endothelin receptor A immunoreactivity was absent in nerve cell bodies but strongly expressed in the mossy fiber pathway (axons of dentate gyrus granule cells). After ischemia endothelin receptor A immunoreactivity in the same regions was reduced by 40-50% from control. In contrast, endothelin receptor B immunoreactivity in control hippocampus was widely distributed in pyramidal neurons, granule cells and glial cells, this immunoreactivity increasing by approximately 25-30% after ischemia. DISCUSSION: Endothelin receptor A's marked decrease in mossy fibers after ischemia may contribute to glutamate release from mossy fiber terminals, thus enhancing excitotoxic effects on their Cornu Ammonis synaptic targets. Additionally, endothelin receptor B increased expression in neurons and glia could be related to a more generalized activation of survival mechanisms involving elements of the neurovascular unit.

The GABAB1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses.

GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed that GABA(B(1a,2)) receptors are more abundant than GABA(B(1b,2)) receptors at glutamatergic terminals. Accordingly, it was found that GABA(B(1a,2)) receptors are more efficient than GABA(B(1b,2)) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA(B(1a,2)) and GABA(B(1b,2)) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA(B1a) than GABA(B1b) protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations >or=1 microm, both GABA(B(1a,2)) and GABA(B(1b,2)) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA(B(1a,2)) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA(B(1a,2)) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA(B(1a,2)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance.

Selective apoptosis induction in the hippocampal mossy fiber pathway by exposure to CT105, the C-terminal fragment of Alzheimer's amyloid precursor protein.

Beta-amyloid protein (Abeta), a proteolytic byproduct of Alzheimer's amyloid precursor protein (APP), has been shown to play a central role in the development of Alzheimer's disease (AD). In addition, recent studies strongly suggest that other byproducts of proteolysis, such as C-terminal fragments of APP (APP-CTF), are also critically involved in the AD pathology. To explore this possibility, we investigated the histopathological changes induced by repeated low-dose intrahippocampal injection of a recombinant 105 amino acid C-terminal fragment of APP (CT105). First, we carried out a behavioral analysis by using the three-panel runway task, and found that the working memory was significantly impaired by CT105 exposure. Then, via propidium iodide staining, we encountered a number of cells exhibiting fragmented or shrank nuclei in the mossy fiber pathway (stratum lucidum and dentate hilus) in CT105-treated rats. These cells were positive for single-stranded DNA (ssDNA), an apoptosis-specific marker, and thus were considered to be apoptotic. Some of the ssDNA-positive cells were also positive for somatostatin. But neither ionized calcium-binding adapter molecule 1 (Iba1) nor S100beta occurred in ssDNA-positive cells. These findings suggest that CT105 induces apoptotic changes in cells of neuronal origin. Quantitative analysis showed that the densities of ssDNA-positive cells in the mossy fiber pathway were significantly higher in CT105-treated rats than in control animals. The present results suggest that CT105 causes dysfunction in the hippocampal mossy fiber system, and also provide some key to understand the relationship between APP-CTF and glutamatergic synaptic dysregulation in AD.

Synaptic release of zinc from brain slices: factors governing release, imaging, and accurate calculation of concentration.

Cerebrocortical neurons that store and release zinc synaptically are widely recognized as critical in maintenance of cortical excitability and in certain forms of brain injury and disease. Through the last 20 years, this synaptic release has been observed directly or indirectly and reported in more than a score of publications from over a dozen laboratories in eight countries. However, the concentration of zinc released synaptically has not been established with final certainty. In the present work we have considered six aspects of the methods for studying release that can affect the magnitude of zinc release, the imaging of the release, and the calculated concentration of released zinc. We present original data on four of the issues and review published data on two others. We show that common errors can cause up to a 3000-fold underestimation of the concentration of released zinc. The results should help bring consistency to the study of synaptic release of zinc.

Localization of brain-derived neurotrophic factor to distinct terminals of mossy fiber axons implies regulation of both excitation and feedforward inhibition of CA3 pyramidal cells.

Hippocampal dentate granule cells directly excite and indirectly inhibit CA3 pyramidal cells via distinct presynaptic terminal specializations of their mossy fiber axons. This mossy fiber pathway contains the highest concentration of brain-derived neurotrophic factor (BDNF) in the CNS, yet whether BDNF is positioned to regulate the excitatory and/or inhibitory pathways is unknown. To localize BDNF, confocal microscopy of green fluorescent protein transgenic mice was combined with BDNF immunohistochemistry. Approximately half of presynaptic granule cell-CA3 pyramidal cell contacts were found to contain BDNF. Moreover, enhanced neuronal activity virtually doubled the percentage of BDNF-immunoreactive terminals contacting CA3 pyramidal cells. To our surprise, BDNF was also found in mossy fiber terminals contacting inhibitory neurons. These studies demonstrate that mossy fiber BDNF is poised to regulate both direct excitatory and indirect feedforward inhibitory inputs to CA3 pyramdal cells and reveal that seizure activity increases the pool of BDNF-expressing granule cell presynaptic terminals contacting CA3 pyramidal cells.

Involvement of highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive granule cells in the amygdaloid-kindling-induced sprouting of a hippocampal mossy fiber trajectory.

The mossy fiber system in the hippocampus of amygdaloid-kindled rats was examined by using highly polysialylated neural cell adhesion molecule (PSA-NCAM) as a marker for immunohistochemical detection of immature dentate granule cells and mossy fibers in combination with bromodeoxyuridine (BrdU) labeling of newly generated granule cells. Statistically significant increases in BrdU-labeled cells and PSA-NCAM-positive cells occurred in the dentate gyrus following kindling. The increase in PSA-NCAM-immunoreactive neurites was confined to the entire stratum lucidum of CA3. Immunoelectron-microscopic examination also revealed that PSA-NCAM-positive immature synaptic terminals of the sprouting mossy fibers increased in the stratum lucidum of CA3 in the kindled rats. The increase in the numbers of PSA-NCAM-positive granule cells correlated well with the increase in the immunopositive neurites and synaptic terminals on the mossy fiber trajectory. The increase in these PSA-NCAM-immunopositive structures is thought to reflect the enhancement of sprouting and synaptogenesis of mossy fibers by a subset of granule cells newly generated during amygdaloid-kindling and suggests that the reorganization of the mossy fiber system on the normal trajectory at least in part contributes to the acquisition and maintenance of an epileptogenic state.

Plasticity of GABA(B) receptor-mediated heterosynaptic interactions at mossy fibers after status epilepticus.

Several neurotransmitters, including GABA acting at presynaptic GABA(B) receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABA(B) receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABA(B) receptor agonist baclofen. Autoradiography revealed a reduction in GABA(B) receptor binding in the stratum lucidum after status epilepticus. Failure of GABA(B) receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus.

Mossy fiber pathfinding in multilayer organotypic cultures of rat hippocampal slices.

1. Using a novel technique of organotypic cultures, in which two hippocampal slices were cocultured in a bilayer style, we found that the mossy fibers arising from the dentate gyrus grafted onto another dentate tissue grew along the CA3 stratum lucidum of the host hippocampal slice. The same transplantation of a CA1 microslice failed to form a network with the host hippocampus. 2. Thus the type of grafted neurons is important to determine whether they can form an appropriate network after transplantation.

Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus.

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.

Gene expression associated with in vivo induction of early phase-long-term potentiation (LTP) in the hippocampal mossy fiber-Cornus Ammonis (CA)3 pathway.

Affymetrix microarray technology was used to characterize whole-hippocampus gene expression associated with in vivo N-methyl-D-aspartate (NMDA)-R-independent long-term potentiation (LTP) in the mossy fiber (MF)-Cornus Ammonis (CA)3 pathway of adult male F344 rats. Acute MF responses were evoked by stimulation of the MF bundle and recorded in stratum lucidum of CA3. Following recording of baseline responses at 0.05 Hz, animals received either CPP (NMDA-R antagonist, 10 mg/kg) or naloxone (opioid-R antagonist, 10 mg/kg). LTP was induced by two 100 Hz 1-sec trains at the intensity sufficient to evoke 50% of the maximal response. Responses were collected for an additional hour. In controls, MF responses were collected at 0.05 Hz for 1 hr, but 100 Hz trains were not delivered. Hippocampi were harvested prior to total RNA isolation. Fragmented cRNA was hybridized to a rat U34 neurobiology array. F344 rats exhibited characteristic LTP in the presence of CPP and LTP blockade in the presence of naloxone. As a result, genes associated with both NMDA-independent LTP and naloxone-induced blockade were identified. These include genes involved in transmitter transport, intracellular messengers, growth factors and ion channels. Up-regulated include NMDA-R2D, neuropeptide Y (NPY), proenkephalin, BDNF and NGFR. Down-regulated genes include IGF-1 and GABA-B.

Nectin-dependent localization of synaptic scaffolding molecule (S-SCAM) at the puncta adherentia junctions formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the mouse hippocampus.

BACKGROUND: Two types of intercellular junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAs), are observed at the synapses between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the hippocampus. SJs are associated with active zones and postsynaptic densities (PSDs) where neurotransmission occurs, whereas PAs are not associated with either of them. We have found that the nectin-afadin unit as well as the N-cadherin-catenin unit localizes at the PAs and that both the units cooperatively organize the PAs. Nectins are Ca2+-independent Ig-like cell-cell adhesion molecules and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Synaptic scaffolding molecule (S-SCAM) is a neural scaffolding protein which interacts with many proteins including neuroligin, NMDA receptors, neural plakophilin-related armadillo-repeat protein/delta-catenin, a GDP/GTP exchange protein for Rap1 small G protein (PDZ-Rap-GEP), and beta-catenin. S-SCAM has been suggested to be a component of PSDs, but its precise localization at the synapses remains unknown. RESULTS: S-SCAM was not concentrated at the PSDs but highly concentrated and co-localized with nectins at both the sides of the PAs formed between the mossy fibre terminals and the dendrites of pyramidal cells in the CA3 area of the adult mouse hippocampus. S-SCAM co-localized with nectin-1 at the primitive synapses where the SJs and the PAs were not morphologically differentiated, and they co-localized during the maturation of the SJs and the PAs. Nectin-1 had a potency to recruit S-SCAM to the nectin-1-based cell-cell adhesion sites formed in cadherin-deficient L cells as a model system. This recruitment was dependent on the C-terminal PDZ domain-binding motif of nectin-1 which is necessary for the binding of afadin, suggesting that nectins recruit S-SCAM through afadin. Consistently, S-SCAM was co-immunoprecipitated with afadin by the anti-S-SCAM antibody from the mouse brain, but S-SCAM did not directly bind afadin. CONCLUSION: These results indicate that S-SCAM localizes at the PAs in the CA3 area of the hippocampus in a nectin-dependent manner and suggest that S-SCAM serves as a scaffolding molecule at the PAs after maturation of the synapses and at the SJs during the maturation.

Activation of presynaptic P2X7-like receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase.

P2X(7) receptor subunits form homomeric ATP-gated, calcium-permeable cation channels. In this study, we used Western blots and immunocytochemistry to demonstrate that P2X(7) receptors are abundant on presynaptic terminals of mossy fiber synapses in the rat hippocampus. P2X(7)-immunoreactive protein was detected using a specific P2X(7) antibody in Western blots of protein isolated from whole hippocampus and from a subcellular fraction containing mossy fiber synaptosomes. P2X(7) immunoreactivity was colocalized with syntaxin 1A/B-immunoreactivity in mossy fiber terminals in the dentate hilus and stratum lucidum of CA3. Extracellular and whole-cell voltage-clamp recordings in CA3 revealed that bath application of the potent P2X(7) agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP) caused a long-lasting inhibition of neurotransmission at mossy fiber-CA3 synapses. Consistent with a presynaptic action at mossy fiber synapses, Bz-ATP had no significant effect on neurotransmission at associational-commissural synapses in CA3 but increased paired-pulse facilitation during depression of mossy fiber evoked currents. In addition, Bz-ATP had no postsynaptic effect on holding current or conductance of CA3 neurons. Bz-ATP-induced mossy fiber synaptic depression was blocked by the P2X(7) antagonist oxidized ATP but not by the P2X(1-3,5,6) antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or the P2Y antagonist reactive blue 2. Finally, an antagonist of p38 MAP kinase activation [4-(4-fluorophenyl)2-(4-methylsulfinylphenyl)5-(4-pyridyl)imidazole] but not extracellular signal-regulated kinase 1/2 MAP kinase (2'-amino-3'-methoxyflavone) blocked the synaptic depression mediated by Bz-ATP, suggesting that this presynaptic inhibition was mediated by activation of p38 MAP kinase. The results of the present study demonstrate that activation of presynaptic P2X(7) receptors depresses mossy fiber-CA3 synaptic transmission through activation of p38 MAP kinase.

Timing of cognitive deficits following neonatal seizures: relationship to histological changes in the hippocampus.

Neonatal seizures are frequently associated with cognitive impairment and reduced seizure threshold. Previous studies in our laboratory have demonstrated that rats with recurrent neonatal seizures have impaired learning, lower seizure thresholds, and sprouting of mossy fibers in CA3 and the supragranular region of the dentate gyrus in the hippocampus when studied as adults. The goal of this study was to determine the age of onset of cognitive dysfunction and alterations in seizure susceptibility in rats subjected to recurrent neonatal seizures and the relation of this cognitive impairment to mossy fiber sprouting and expression of glutamate receptors. Starting at postnatal day (P) 0, rats were exposed to 45 flurothyl-induced seizures over a 9-day period of time. Visual-spatial learning in the water maze and seizure susceptibility were assessed in subsets of the rats at P20 or P35. Brains were evaluated for cell loss, mossy fiber distribution, and AMPA (GluR1) and NMDA (NMDAR1) subreceptor expression at these same time points. Rats with neonatal seizures showed significant impairment in the performance of the water maze and increased seizure susceptibility at both P20 and P35. Sprouting of mossy fibers into the CA3 and supragranular region of the dentate gyrus was seen at both P20 and P35. GluR1 expression was increased in CA3 at P20 and NMDAR1 was increased in expression in CA3 and the supragranular region of the dentate gyrus at P35. Our findings indicate that altered seizure susceptibility and cognitive impairment occurs prior to weaning following a series of neonatal seizures. Furthermore, these alterations in cognition and seizure susceptibility are paralleled by sprouting of mossy fibers and increased expression of glutamate receptors. To be effective, our results suggest that strategies to alter the adverse outcome following neonatal seizures will have to be initiated during, or shortly following, the seizures.

Immunoelectron microscopic localization of the neural recognition molecules L1, NCAM, and its isoform NCAM180, the NCAM-associated polysialic acid, beta1 integrin and the extracellular matrix molecule tenascin-R in synapses of the adult rat hippocampus.

We have investigated the possibility that morphologically different excitatory glutamatergic synapses of the "trisynaptic circuit" in the adult rodent hippocampus, which display different types of long-term potentiation (LTP), may express the immunoglobulin superfamily recognition molecules L1 and NCAM, the extracellular matrix molecule tenascin-R, and the extracellular matrix receptor constituent beta1 integrin in a differential manner. The neural cell adhesion molecules L1, NCAM (all three major isoforms), NCAM180 (the largest major isoform with the longest cytoplasmic domain), beta1 integrin, polysialic acid (PSA) associated with NCAM, and tenascin-R were localized by pre-embedding immunostaining procedures in the CA3/CA4 region (mossy fiber synapses) and in the dentate gyrus (spine synapses) of the adult rat hippocampus. Synaptic membranes of mossy fiber synapses where LTP is expressed presynaptically did not show detectable levels of immunoreactivity for any of the molecules/epitopes studied. L1, NCAM, and PSA, but not NCAM180 or beta1 integrin, were detectable on axonal membranes of fasciculating mossy fibers. In contrast to mossy fiber synapses, spine synapses in the outer third of the molecular layer of the dentate gyrus, which display postsynaptic expression mechanisms of LTP, were both immunopositive and immunonegative for NCAM, NCAM180, beta1 integrin, and PSA. Those spine synapses postsynaptically immunoreactive for NCAM or PSA also showed immunoreactivity on their presynaptic membranes. NCAM180 was not detectable presynaptically in spine synapses. L1 could not be found in spine synapses either pre- or postsynaptically. Also, the extracellular matrix molecule tenascin-R was not detectable in synaptic clefts of all synapses tested, but was amply present between fasciculating axons, axon-astrocyte contact areas, and astrocytic gap junctions. Differences in expression of the membrane-bound adhesion molecules at both types of synapses may reflect the different mechanisms for induction and/or maintenance of synaptic plasticity.

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

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

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique.

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca(2+) channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca(2+) channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca(2+) channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca(2+) channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.