Mossy Cells Research Articles

Endocannabinoid-Mediated Depolarization-Induced Suppression of Inhibition in Hilar Mossy Cells of the Rat Dentate Gyrus

Hilar mossy cells represent a unique population of local circuit neurons in the hippocampus and dentate gyrus. Here we use electrophysiological techniques in acute preparations of hippocampal slices to demonstrate that depolarization of a single hilar mossy cell can produce robust inhibition of local GABAergic afferents. This depolarization-induced suppression of inhibition (DSI) can be observed as a transient reduction in frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) or as a transient reduction in amplitude of evoked IPSCs (eIPSCs). We find that DSI of eIPSCs as observed in hilar mossy cells is enhanced by activation of muscarinic acetylcholine receptors, blocked by chelation of postsynaptic calcium, and critically dependent on retrograde activation of presynaptic cannabinoid type 1 (CB1) receptors. We further report that activation of CB1 receptors on GABAergic afferents to hilar mossy cells (by either endogenous or exogenous agonists) preferentially inhibits calcium-dependent exocytosis and that endocannabinoid-dependent retrograde signaling in this system is subject to tight spatial constraints.

Galanin expressed in the excitatory fibers attenuates synaptic strength and generalized seizures in the piriform cortex of mice

The neuropeptide galanin is considered to be an endogenous antiepileptic agent, presumably acting via inhibition of glutamate release. Previously, we have demonstrated that in mice ectopically overexpressing galanin in cortical and hippocampal neurons, particularly in granule cells and their axons, the mossy fibers, hippocampal kindling epileptogenesis is suppressed and is associated with attenuated frequency facilitation in mossy fiber-CA3 cell synapses. We hypothesized that changes in synaptic transmission might occur also in other excitatory synapses of the galanin overexpressing (GalOE) mouse, contributing to seizure suppression. Lateral olfactory tract (LOT) synapses, formed by axons of olfactory bulb (OB) mitral cells and targeting piriform cortex (PC) pyramidal cells, ectopically express galanin in GalOE mice. Using whole-cell patch-clamp recordings, we found that excitatory synaptic responses recorded in PC pyramidal cells during high frequency stimulation of the LOT were attenuated in GalOE mice as compared to wild-type controls. This effect was mimicked by bath application of galanin or its agonist galnon to wild-type slices, supporting the notion of ectopic galanin action. Since the high frequency activation induced in vitro resembles epileptic seizures in vivo, we asked whether the observed synaptic inhibition would result in altered epileptogenesis when animals were kindled via the same synapses. In male GalOE mice, we found that the latency to convulsions was prolonged, and once animals had experienced the first stage 5 seizure, generalized seizures were less sustainable. These data indicate that the PC is a possible target for epilepsy treatment by ectopically overexpressing galanin to modulate seizure activity.

Selective spread of neurotropic herpesviruses in the rat hippocampus

Transsynaptically spreading viruses are widely used for tracing neuronal circuits in both the central and peripheral nervous systems. However, viruses are capricious tools with selective spreading properties that can produce false-negative results. Using herpes simplex virus type 1 and two pseudorabies virus strains, we aimed at mapping quantitatively neuronal connections in the rat hippocampus. We found that none of the tested viruses infected CA3 pyramidal neurons across synapses following inoculation into the CA1 area. Combined injections of the viruses with the retrograde tracer cholera toxin B (CTB) resulted in CTB, but not virus labeling of CA3 pyramidal neurons. In contrast, other brain regions known to send inputs to the CA1 (the entorhinal cortex, medial septum and diagonal band of Broca, raphe nuclei) were transsynaptically infected. Our results indicate that Schaffer collaterals of CA3 pyramidal cells lack the appropriate cellular machinery for successful neurotropic herpesvirus infection. After injections of viruses into the dentate gyrus/CA3 area, we found labeling in commissurally projecting mossy cells and their afferent granule cells but not in contralateral CA3 pyramidal cells. Using this unique spreading property, we estimated that single mossy cells receive input from a compact cluster of 30-40 granule cells.

Dendritic excrescences seem to characterize hippocampal CA3 pyramidal neurons in humans

Excrescences are unique dendritic postsynaptic structures of the hippocampal formation. Only CA3 pyramidal neurones and hilar mossy cells possess these complex dendritic structures. Dendritic excrescences have so far only been investigated in rabbit, rat and rhesus monkey. Applying a Golgi impregnation method optimized for human brain tissue, we describe the detailed morphology of excrescences of CA3 pyramidal neurons of man. Human thorny excrescences possess a thin and single spine neck and multiple spine heads (4 on average, sometimes more than 10). Human cluster excrescences sit upon the dendrite with a broad stem, and exhibit a "papilloma-like" surface. Some human CA3 pyramidal neurons seem to possess markedly longer spine necks and larger spine heads compared to human neocortical pyramid cells; they were named long-neck spines. Thorny excrescences, cluster excrescences and the newly described long-neck spines can also be found on the dendritic main stem of human CA3 pyramidal neurons.CA2 pyramidal neurons neither possess these long neck spines nor thorny or cluster excrescences. Thus, the unique excrescences of CA3 pyramidal neurones seem to be another criterion for a demarcation between the CA3- and CA2 region of the human hippocampus.

Sex differences in the hilar mossy cells of the guinea-pig before puberty

Numerous sex differences have been detected in the morphology of the dentate and hippocampal neurons and hippocampus-dependent memory functions. The aim of the present study was to ascertain whether the mossy cells, an interneuron population forming a recurrent excitatory circuit with the dentate granule cells, are sexually dimorphic. The brains of juvenile (15–16 days old) and peripubescent (45–46 days old) male and female guinea-pigs were Golgi-Cox stained. Mossy cells were sampled from the hilus in the septal third of the dentate gyrus and their dendritic tree and somata were analyzed. The analysis was separately conducted on mossy cells with soma located in the portions of the hilus that face the upper blade (upper hilus) and lower blade (lower hilus), respectively. The mossy cells in the upper hilus were found to be sexually dimorphic in both juvenile and peripubescent animals. At both ages females had a larger dendritic tree than males. This difference was due to a greater mean branch length and, in peripubescent animals, also to a greater number of branches. In juvenile males, the spines on the proximal dendrites (thorny excrescences) had a greater density than in females. No differences in spine density were present in peripubescent animals. Unlike the mossy cells in the upper hilus, the mossy cells in the lower hilus showed very few sex differences in juvenile animals and no differences in peripubescent animals. The few differences favored females, that had more proximal branches and a greater spine density on the distal dendrites than males. The results show that the mossy cells of the guinea-pig are sexually dimorphic prior to puberty. Extending a previous investigation, the present data provide evidence that sex differences are mainly confined to the dentate region corresponding to the upper blade and upper hilus. The observed segregation of the sexual dimorphism in the upper blade/upper hilus suggests that this region might underlie the sexual dimorphism in hippocampus-dependent memory functions.

Synaptic plasticity at hippocampal mossy fibre synapses

The dentate gyrus provides the main input to the hippocampus. Information reaches the CA3 region through mossy fibre synapses made by dentate granule cell axons. Synaptic plasticity at the mossy fibre-pyramidal cell synapse is unusual for several reasons, including low basal release probability, pronounced frequency facilitation and a lack of N-methyl-D-aspartate receptor involvement in long-term potentiation. In the past few years, some of the mechanisms underlying the peculiar features of mossy fibre synapses have been elucidated. Here we describe recent work from several laboratories on the various forms of synaptic plasticity at hippocampal mossy fibre synapses. We conclude that these contacts have just begun to reveal their many secrets.

Initiation and Propagation of Neuronal Coactivation in the Developing Hippocampus

Correlated neuronal activity is ubiquitous in developing nervous systems, where it may introduce spatiotemporal coherence and contribute to the organization of functional circuits. In this report, we used voltage-sensitive dyes and optical imaging to examine the spatiotemporal pattern of a spontaneous network activity, giant depolarizing potentials (GDPs), in rat hippocampal slices during the first postnatal week. The propagation pattern of the GDP is closely correlated to the anatomical organization of the network. In the hilus, where mossy cells and interneurons are not organized in layers, GDPs propagate at the same velocity in all directions. In CA3 and CA1, the activation is synchronous along the axis of the pyramidal cells' dendritic tree. The velocity of wave propagation is significantly different in three hippocampal subfields: it is slowest in the hilus, faster in CA3, and fastest in CA1. The velocity of horizontal propagation (along the axis of the pyramidal layer) has a large variation from trial to trial, suggesting that the horizontal velocity is determined to some extent by dynamic network factors. Imaging revealed that each GDP event is initiated from a small focus. The location of the initiation focus differs from event to event. All together, our data suggest that GDP is a propagating excitation wave, initiated from a small site, and propagating to the whole hippocampus. The spatiotemporal patterns of the wave in CA3 and CA1 areas show better synchrony along the pyramidal cell dendritic trees and progressive activation along the axis of the pyramidal cell layer.

Borna Disease Virus Replication in Organotypic Hippocampal Slice Cultures from Rats Results in Selective Damage of Dentate Granule Cells

In the hippocampus of Borna disease virus (BDV)-infected newborn rats, dentate granule cells undergo progressive cell death. BDV is noncytolytic, and the pathogenesis of this neurodevelopmental damage in the absence of immunopathology remains unclear. A suitable model system to study early events of the pathology is lacking. We show here that organotypic hippocampal slice cultures from newborn rat pups are a suitable ex vivo model to examine BDV neuropathogenesis. After challenging hippocampal slice cultures with BDV, we observed a progressive loss of calbindin-positive granule cells 21 to 28 days postinfection. This loss was accompanied by reduced numbers of mossy fiber boutons when compared to mock-infected cultures. Similarly, the density of dentate granule cell axons, the mossy fiber axons, appeared to be substantially reduced. In contrast, hilar mossy cells and pyramidal neurons survived, although BDV was detectable in these cells. Despite infection of dentate granule cells 2 weeks postinfection, the axonal projections of these cells and the synaptic connectivity patterns were comparable to those in mock-infected cultures, suggesting that BDV-induced damage of granule cells is a post-maturation event that starts after mossy fiber synapses are formed. In summary, we find that BDV infection of rat organotypic hippocampal slice cultures results in selective neuronal damage similar to that observed with infected newborn rats and is therefore a suitable model to study BDV-induced pathology in the hippocampus.

Hippocampal and visuospatial learning defects in mice with a deletion of frizzled 9, a gene in the Williams syndrome deletion interval

Wnt signaling regulates hippocampal development but little is known about the functions of specific Wnt receptors in this structure. Frizzled 9 is selectively expressed in the hippocampus and is one of about 20 genes typically deleted in Williams syndrome. Since Williams syndrome is associated with severe visuospatial processing defects, we generated a targeted null allele for frizzled 9 to examine its role in hippocampal development. Frizzled 9-null mice had generally normal gross anatomical hippocampal organization but showed large increases in apoptotic cell death in the developing dentate gyrus. This increase in programmed cell death commenced with the onset of dentate gyrus development and persisted into the first postnatal week of life. There was also a perhaps compensatory increase in the number of dividing precursors in the dentate gyrus, which may have been a compensatory response to the increased cell death. These changes in the mutants resulted in a moderate decrease in the number of adult dentate granule cells in null mice and an increase in the number of hilar mossy cells. Heterozygous mice (the same frizzled 9 genotype as Williams syndrome patients) were intermediate between wild type and null mice for all developmental neuronanatomic defects. All mice with a mutant allele had diminished seizure thresholds, and frizzled 9 null mice had severe deficits on tests of visuospatial learning/memory. We conclude that frizzled 9 is a critical determinant of hippocampal development and is very likely to be a contributing factor to the neurodevelopmental and behavioral phenotype of patients with Williams syndrome.

Subchronic treatment with imipramine ameliorates the decreased number in neuropeptide Y-positive cells in the hippocampus of learned helplessness rats

Learned helplessness, but not immobilization stress, decreased the numbers of neuropeptide Y (NPY)-positive cells (interneuron), but not calcitonin gene-related peptide (CGRP)-positive cells (mossy cell), in the hilus of the hippocampus. Subchronic treatment of learned helplessness rats, but not naive rats, with imipramine ameliorated the decrease in the number of NPY-positive cells. Therefore, NPY-positive cells in the hippocampus may contribute to depression.

Spontaneous Release of Neuropeptide Y Tonically Inhibits Recurrent Mossy Fiber Synaptic Transmission in Epileptic Brain

In the pilocarpine model of temporal lobe epilepsy, mossy fibers coexpress the inhibitory transmitter neuropeptide Y (NPY) with glutamate. The effects of endogenous and applied NPY on recurrent mossy fiber synaptic transmission were investigated with the use of whole-cell voltage-clamp and field recordings in rat hippocampal slices. Applied NPY reversibly inhibited synaptic transmission at recurrent mossy fiber synapses on dentate granule cells but not at perforant path or associational-commissural synapses. It also reduced the frequency of miniature EPSCs (mEPSCs) in granule cells from epileptic, but not control, rats and depressed granule cell epileptiform activity dependent on the recurrent mossy fiber pathway. These actions of NPY were mediated by activation of presynaptic Y2 receptors. The Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]argininamide (BIIE0246) not only blocked the effects of NPY but also enhanced recurrent mossy fiber synaptic transmission, the frequency of mEPSCs, and the magnitude of mossy fiber-evoked granule cell epileptiform activity when applied by itself. Several observations supported the selectivity of BIIE0246. These results suggest that even the spontaneous release of NPY (or an active metabolite) from recurrent mossy fibers is sufficient to depress glutamate release from this pathway. Tonic release of NPY accounts at least partially for the low probability of glutamate release from recurrent mossy fiber terminals, impedes the ability of these fibers to synchronize granule cell discharge, and may protect the hippocampus from seizures that involve the entorhinal cortex. This pathway may synchronize granule cell discharge more effectively in human brain than in rat because of its lower expression of NPY.

Long- and short-term plasticity at mossy fiber synapses on mossy cells in the rat dentate gyrus

Mossy cells give rise to the commissural and associational pathway of the dentate gyrus, and receive their major excitatory inputs from the mossy fibers of granule cells. Through these feed-back excitatory connections, mossy cells have been suggested to play important roles in both normal signal processing in learning and memory, as well as in seizure propagation. However, the nature of the activity-dependent modifications of the mossy fiber inputs to mossy hilar cells is not well understood. We studied the long- and short-term plasticity properties of the mossy fiber-mossy cell synapse, using the minimal stimulation technique in slices in whole cell recorded mossy cells retrogradely prelabeled with the fluorescent dye DiO from the contralateral dentate gyrus. Following tetanic stimulation, mossy fiber synapses showed significant NMDA receptor-independent long-term potentiation (LTP), associated with increased excitatory postsynaptic currents (EPSC) amplitude and decreased failure rates. Coefficient of variance and failure rate analyses suggested a presynaptic locus of LTP induction. Mossy fiber synapses on mossy cells also showed activity-dependent short-term modification properties, including both frequency-dependent facilitation (stimuli at higher frequencies evoked larger EPSCs with lower failure rates) and burst facilitation (each EPSC in a burst had a larger amplitude and higher probability of occurrence than the preceding EPSCs within the burst). The data show that mossy fiber-mossy cell synapses exhibit both long- and short-term plasticity phenomena that are generally similar to the mossy fiber synapses on CA3 pyramidal cells.

Mossy cells and different subpopulations of pyramidal neurons are immunoreactive for cocaine- and amphetamine-regulated transcript peptide in the hippocampal formation of non-human primates and tree shrew ( Tupaia belangeri)

Cocaine- and amphetamine-regulated transcript peptide mRNA was discovered in the rat striatum following cocaine and amphetamine administration. Since both psychostimulants elicit memory-related effects, localization of cocaine- and amphetamine-regulated transcript peptide in the hippocampal formation may have functional importance. Previous studies demonstrated different cellular localizations of cocaine- and amphetamine-regulated transcript peptide in humans and in rodents. Mossy cells were cocaine- and amphetamine-regulated transcript-positive in the human dentate gyrus, whereas granule cells contained this peptide in the rat. In the present study, the localization of cocaine- and amphetamine-regulated transcript peptide was examined using immunohistochemistry in the hippocampal formation of the rhesus monkey ( Macaca mulatta), the common marmoset monkey ( Callithrix jacchus) and in the tree shrew ( Tupaia belangeri). In these species principal neurons of the hippocampal formation were cocaine- and amphetamine-regulated transcript-immunoreactive. In both monkeys and tree shrews, mossy cells of the hilus were cocaine- and amphetamine-regulated transcript-positive whereas granule cells of the dentate gyrus were cocaine- and amphetamine-regulated transcript-negative. The dense cocaine- and amphetamine-regulated transcript-immunoreactive axonal plexus of the associational pathway outlined the inner one-third of the dentate molecular layer. In the hippocampus of the tree shrew and marmoset monkey, a subset of CA3 pyramidal cells were cocaine- and amphetamine-regulated transcript-immunoreactive. In the marmoset monkey, cocaine- and amphetamine-regulated transcript labeling was found only in layer V pyramidal cells of the entorhinal cortex, while in the rhesus monkey, pyramidal cells of layers II and III were cocaine- and amphetamine-regulated transcript-immunopositive. Our results show that cocaine- and amphetamine-regulated transcript positive neurons in the dentate gyrus of non-human primates are similar to that of the human. Furthermore, in the hippocampal formation of the tree shrew similar cocaine- and amphetamine-regulated transcript-immunoreactive cell-types were observed as in monkeys, supporting their evolutionary relationship with primates. Mossy cells and granule cells are members of a mutual excitatory intrahippocampal circuitry, therefore cocaine- and amphetamine-regulated transcript-immunoreactivity of these neurons in primates and rodents suggests that psychostimulants cocaine and amphetamine may induce memory-related effects at different points of the same excitatory circuitry in the hippocampal formation.

Sprouting of mossy fibers and presynaptic inhibition by group II metabotropic glutamate receptors in pilocarpine-treated rat hippocampal slice cultures

Mossy fibre sprouting (MFS) is a phenomenon observed in the epileptic hippocampus. We have studied MFS, in 7, 14 and 21 day in vitro (DIV) organotypic slice cultures, or in slice cultures treated with pilocarpine (0.5 mM) or pilocarpine and atropine (0.1 mM or 0.5 mM) for 48–72 h at 5 DIV and tested at 21 DIV. Acute application of pilocarpine directly activated hilar neurons and elicited epileptic-like discharges in CA3 pyramids and mossy cells of 5–8 DIV cultures, without causing substantial cell death, as assessed by lactate dehydrogenase measurements. Timm staining revealed increases in MFS in chronic pilocarpine-treated cultures, which was prevented by prior application of atropine. Extracellular synaptic responses were recorded in the granule cell layer and elicited by antidromic mossy fibre stimulation. The GABA A antagonist 6-imino-3-(4-methoxyphenyl)-1(6 H)-pyridazinebutanoic acid (1μM) induced a greater increase in the coastline bursting index in pilocarpine-treated cultures than in 21 DIV controls. However, there was no significant increase in the frequency of spontaneous or miniature synaptic events recorded in granule cells from pilocarpine-treated cultures. Granule cells were filled with biocytin and morphometric analysis revealed that the length of axon collaterals in the granule and molecular layer was longer in pilocarpine-treated cultures than in 21 DIV controls. Dual recordings between granule cells and between granule and hilar neurons showed that pilocarpine-treated cultures had a larger proportion of monosynaptic and polysynaptic connections. The group II metabotropic glutamate receptor (mGluR) agonist LY354740 (0.5 μM) suppressed excitatory but not inhibitory monosynaptic currents. LY354740 also inhibited antidromically evoked action currents in granule cells from pilocarpine- and to a lesser extent in pilocarpine and atropine-treated cultures, suggesting that group II mGluRs can reside along the axon and suppress action potential invasion. We provide direct evidence for the development of functional MFS and suggest a novel, axonal mechanism by which presynaptic group II mGluRs can inhibit selected synapses.

Stress suppresses and learning induces plasticity in CA3 of rat hippocampus: A three-dimensional ultrastructural study of thorny excrescences and their postsynaptic densities

Chronic stress and spatial training have been proposed to affect hippocampal structure and function in opposite ways. Previous morphological studies that addressed structural changes after chronic restraint stress and spatial training were based on two-dimensional morphometry which does not allow a complete morphometric characterisation of synaptic features. Here, for the first time in such studies, we examined these issues by using three-dimensional (3-D) reconstructions of electron microscope images taken from thorny excrescences of hippocampal CA3 pyramidal cells. Ultrastructural alterations in postsynaptic densities (PSDs) of thorny excrescences receiving input from mossy fibre boutons were also determined, as were changes in numbers of multivesicular bodies (endosome-like structures) within thorny excrescences and dendrites. Quantitative 3-D data demonstrated retraction of thorny excrescences after chronic restraint stress which was reversed after water maze training, whilst water maze training alone increased thorny excrescence volume and number of thorns per thorny excrescence. PSD surface area was unaffected by restraint stress but water maze training increased both number and area of PSDs per thorny excrescence. In restrained rats that were water maze trained PSD volume and surface area increased significantly. The proportion of perforated PSDs almost doubled after water maze training and restraint stress. Numbers of endosome-like structures in thorny excrescences decreased after restraint stress and increased after water maze training. These findings demonstrate that circuits involving contacts between mossy fibre terminals and CA3 pyramidal cells at stratum lucidum level are affected conversely by water maze training and chronic stress, confirming the remarkable plasticity of CA3 dendrites. They provide a clear illustration of the structural modifications that occur after life experiences noted for their different impact on hippocampal function.

Phenotype of cerebellar glutamatergic neurons is altered in stargazer mutant mice lacking brain‐derived neurotrophic factor mRNA expression

Brain-derived neurotrophic factor (BDNF) influences neuronal survival, differentiation, and maturation. More recently, its role in synapse formation and plasticity has also emerged. In the cerebellum of the spontaneous recessive mutant mouse stargazer (stg) there is a specific and pronounced deficit in BDNF mRNA expression. BDNF protein levels in the cerebellum as a whole are reduced by 70%, while in the granule cells (GCs) there is a selective and near total reduction in BDNF mRNA expression. Recently, we published data demonstrating that inhibitory neurons in the cerebella of stgs have significantly reduced levels (approximately 50%) of gamma-aminobutyric acid (GABA) and fewer, smaller inhibitory synapses compared to wildtype (WT) controls. Our current investigations indicate that the stargazer mutation has an even more pronounced effect on the phenotype of glutamatergic neurons in the cerebellum. There is a profound decrease in the levels of glutamate-immunoreactivity (up to 77%) in stg compared to WT controls. The distribution profile of presynaptic vesicles is also markedly different: stgs have proportionally fewer docked vesicles and fewer vesicles located adjacent to the active zone ready to dock than WTs. Furthermore, the thickness of the postsynaptic density (PSD) at mossy fiber-granule cell (MF-GC) and parallel fiber-Purkinje cell (PF-PC) synapses is severely reduced (up to 33% less than WT controls). The number and length of excitatory synapses, however, appear to be relatively unchanged. It is possible that at least some of theses changes in phenotype are directly attributable to the lack of BDNF in the cerebellum of the stg mutant.

Functional Consequences of Hilar Mossy Cell Loss in Temporal Lobe Epilepsy: Proepileptic or Antiepileptic?

Functional Consequences of Hilar Mossy Cell Loss in Temporal Lobe Epilepsy: Proepileptic or Antiepileptic?

Nor-1 regulates hippocampal axon guidance, pyramidal cell survival, and seizure susceptibility.

The nuclear receptor transcription factor, nor-1, is expressed during mammalian development predominantly in the nervous system and is induced in a cell-specific manner in nonneuronal cells in response to a variety of extracellular stimuli. To elucidate the essential developmental functions of this transcription factor, we have analyzed the consequences of its elimination on central nervous system development in mice. Here we show that null mutant mice lacking nor-1 respond with increased limbic seizure activity to the excitotoxic glutamate receptor agonist kainic acid. We demonstrate that these abnormalities are associated with defective postnatal hippocampal development exemplified by abnormal axonal guidance of dentate gyrus granule and mossy cells, disorganization of the pyramidal cell layer, and early postnatal death of pyramidal neurons in the CA1 field of the hippocampus. Our data indicate that nor-1 plays a critical role in neuronal survival and axonal guidance in the developing murine hippocampus and that hippocampal dysgenesis in nor-1-/- mice may be an underlying cause of seizure susceptibility.

NMDA receptor GluRepsilon/NR2 subunits are essential for postsynaptic localization and protein stability of GluRzeta1/NR1 subunit.

In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2A and GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-C2') cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRzeta1-C2 and GluRzeta1-C2'. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 +/- 3.3% for GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-C1) was, although low in cerebellar content, also reduced to 12.7 +/- 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.

Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures.

Axonal sprouting like that of the mossy fibers is commonly associated with temporal lobe epilepsy, but its significance remains uncertain. To investigate the functional consequences of sprouting of mossy fibers and alternative pathways, kainic acid (KA) was used to induce robust mossy fiber sprouting in hippocampal slice cultures. Physiological comparisons documented many similarities in granule cell responses between KA- and vehicle-treated cultures, including: seizures, epileptiform bursts, and spontaneous excitatory postsynaptic currents (sEPSCs) >600 pA. GABAergic control and contribution of glutamatergic synaptic transmission were similar. Analyses of neurobiotin-filled CA1 pyramidal cells revealed robust axonal sprouting in both vehicle- and KA-treated cultures, which was significantly greater in KA-treated cultures. Hilar stimulation evoked an antidromic population spike followed by variable numbers of postsynaptic potentials (PSPs) and population spikes in both vehicle- and KA-treated cultures. Despite robust mossy fiber sprouting, knife cuts separating CA1 from dentate gyrus virtually abolished EPSPs evoked by hilar stimulation in KA-treated but not vehicle-treated cultures, suggesting a pivotal role of functional afferents from CA1 to dentate gyrus in KA-treated cultures. Together, these findings demonstrate striking hyperexcitability of dentate granule cells in long-term hippocampal slice cultures after treatment with either vehicle or KA. The contribution to hilar-evoked hyperexcitability of granule cells by the unexpected axonal projection from CA1 to dentate in KA-treated cultures reinforces the idea that axonal sprouting may contribute to pathologic hyperexcitability of granule cells.