Cholinergic innervation of mossy cells in the rat fascia dentata

Hilar mossy cells are the main cells of origin of the commissural/associational projection to the inner molecular layer of the rat fascia dentata. In order to analyze the cholinergic innervation of hilar mossy cells, a light and electron microscopic double‐labeling technique was used. Immunolabeling for calcitonin gene‐related peptide (CGRP) was employed to identify mossy cells and immunocytochemistry for choline acetyltransferase (ChAT) was used to label cholinergic septohippocampal fibers. Cholinergic boutons were abundant around mossy cell somata and on their proximal dendrites. Electron microscopy confirmed that many of these boutons formed synapses with the CGRP‐positive mossy cells. These data demonstrate a direct innervation of hilar mossy cells by cholinergic septohippocampal afferents. This connectivity could contribute to the electrophysiological behavior of mossy cells during theta oscillations. Hippocampus 1999;9:314–320. © 1999 Wiley‐Liss, Inc.     

Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of ‘excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.     

Lesion-induced mossy fibers to the molecular layer of the rat fascia dentata: identification of postsynaptic granule cells by the Golgi-EM technique

The axons of the dentate granule cells, the hippocampal mossy fibers, sprout "backward" into the dentate molecular layer when this is heavily denervated. Using the combined Golgi-electron microscopy (EM) technique we now demonstrate that these aberrant supragranular mossy fibers at least in part terminate on granule cell dendrites. Sprouting of mossy fibers into the dentate molecular layer was induced in adult rats by simultaneous surgical removal of the commissural and entorhinal afferents to the fascia dentata. After at least 7 weeks survival, the presence of mossy fiber terminals in the inner part of the dentate molecular layer was demonstrated by light microscopy. In the electron microscope the mossy fiber terminals were identified by their unique structural characteristics, namely, the unusually large size of the terminals, the dense packing of clear synaptic vesicles with a few dense core vesicles intermingled, the presence of asymmetric synaptic contacts with spines and desmosome-like contacts with dendritic shafts, and the continuity with a thin unmyelinated preterminal axon. Golgi-stained granule cells were first identified in the light microscope, and then, after deimpregnation, the same cells were examined in the electron microscope. In ultrathin, serial sections lesion-induced mossy fiber terminals were found in synaptic contact with spines on proximal dendritic segments of such identified Golgi-impregnated granule cells. From this we conclude that the aberrant, supragranular mossy fibers can innervate dendrites of the parent cell group, the dentate granule cells. The results, moreover, provide an example of reactive synaptogenesis where both the sprouted afferents and its postsynaptic element have been identified.     

Entorhinal fibers form synaptic contacts on parvalbumin-immunoreactive neurons in the rat fascia dentata

The entorhinal cortex gives rise to a dense projection to the outer two-thirds of the dentate molecular layer. The main target neurons are the granule cells. This study demonstrates that entorhinal fibers labeled by anterograde degeneration also terminate on parvalbumin-containing non-granule cells in the rat fascia dentata. Since the calcium-binding protein parvalbumin was recently found coexistent with gamma-aminobutyric acid in inhibitory hippocampal neurons, the described connection provides evidence for a role of entorhinal fibers in feed-forward inhibition of the granule cells.     

Synaptogenesis in the postnatal rat fascia dentata is influenced by 5-HT1a receptor activation.

Neonatal serotonin (5-HT) depletion or 5-HT1a receptor blockade results in a significant and permanent reduction in the number of dentate granule cell dendritic spines. The purpose of this study was to determine whether the loss of spines was accompanied by a reduction in molecular layer synaptic profile density. Rat pups were treated with parachloroamphetamine (PCA), 5,7-DHT or the 5-HT1a receptor antagonist NAN-190. The synaptic profile density (profiles/100 microm2) of the dentate molecular layer was estimated on P14, P21 and P60. Molecular layer synaptic profile density the was significantly reduced in each treatment group on P14 and P21. By P60, the NAN-190 and PCA groups had reached control values, but the 5,7-DHT group remained significantly lower than control. The most dramatic changes were observed among synapses terminating on dendritic spines. Numbers of profiles forming simple spine contacts were significantly reduced by all treatments at P14 and P21, but returned to normal by P60 in the PCA and NAN-190 groups. Simple spine synapses in the 5,7-DHT group remained significantly below control, but numbers of complex spine synapses were higher than either control or the other treatment groups at each age. These results indicate that the loss of dendritic spines observed following 5-HT depletion or 5-HT1a antagonist treatment is accompanied by a decrease in synaptic profile density. This effect appears to be a retardation of synaptogenesis since recovery occurs once 5-HT1a receptor activity resumes. Data from the 5,7-DHT group shows that complex spine synapse formation may represent an effort to attain some degree of functional compensation when synaptogenesis is slowed.     

Expansion and retraction of hippocampal mossy fibers during postweaning development: strain-specific effects of NMDA receptor blockade

We have recently discovered differences in the distribution of the mossy fiber terminal field (MFTF) between adult Long-Evans rats (LER) and Wistar rats(WR): the suprapyramidal MFTF extends into distal stratum oriens (dSO) in LER, but is nearly absent in WR (Holahan et al.,2006, Hippocampus 16:560-570). To our knowledge, there is no developmental evidence that sheds light on how this strain-dependent MFTF innervation in the adult is achieved. Accordingly, the present study examined the time course of MFTF development from postnatal days 0 to 40 and the effect of NMDA-receptor antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) on this developmental organization. In both LER and WR, a MFTF projection to dSO was observed between P18 and P21. By P24, the dSO projection in WR was no longer detectable whereas in LER, the dSO projection seen on P21 remained. We suggest that in WR a retraction of the MFTF projection from dSO to stratum lucidum between P21 and P24 leads to its adult pattern. In WR, CPP administration enhanced the dSO projection, possibly by blocking the retraction process. In LER, CPP administration reduced the dSO projection. Thus, in each strain, NMDA receptor blockade effectively reversed the developmental course of MFTF pattern of innervation. The present results lend strong support to the view that NMDA receptor regulation of input-dependent processes during development is of critical importance in promoting the motility and target selection of presynaptic MF axons. This regulation extends later into development than had previously been thought.     

Interaction with CA4-derived fibers accounts for distribution of septohippocampal fibers in rat fascia dentata after entorhinal lesion

When the perforant path innervation of the rat fascia dentata is removed, cholinergic septohippocampal fibers proliferate and partially replace it. However, the septohippocampal fibers reinnervate only a portion of the perforant path terminal zone; CA4-derived fibers grow into the remaining portion. We tested the possibility that septohippocampal fibers would fill the entire perforant path terminal zone; if CA4-derived fibers were not present. For this purpose, perforant path and CA4-derived fibers were selectively destroyed, either simultaneously or sequentially. If the two lesions were made simultaneously or if the CA4 projection was first destroyed with kainic acid, septohippocampal fibers indeed proliferated in the entire perforant path terminal zone. When perforant path fibers were destroyed first, however, subsequent destruction of the CA4 projection had no further effect on the growth of septohippocampal fibers. They continued to occupy only a portion of the denervated perforant path terminal zone. These data, together with previous results, suggest that, after a perforant path lesion, growing CA4-derived fibers literally push septohippocampal fibers before them, thus limiting the region within which the latter can proliferate. Similarly, repulsion by the residual CA4-derived fibers may account for the limited ability of septohippocampal fibers to grow into a zone denervated by partial destruction of the CA4 projection.     

Mossy cells of the rat fascia dentata are glutamate-immunoreactive

The mossy cells represent a prominent cell type of the hilar region. Whereas the morphology of these neurons, their synaptic connections, and physiological characteristics have been described in some detail, information about their neurotransmitter is still lacking. Using immunocytochemistry in combination with Golgi impregnation, the authors demonstrate that identified mossy cells are GABA-immunonegative but stain for glutamate. These results do not prove that these cells use glutamate as a transmitter, since glutamate is a ubiquitous metabolite. However, together with the lack of GABA staining and a recent report on asymmetric spine synapses formed by identified mossy cell axons, the present results support an excitatory nature of these neurons.     

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

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

Asymmetrical reductions of hippocampal NMDAR1 glutamate receptor mRNA in the psychoses

The psychotomimetic properties of NMDA glutamate receptor antagonists suggest there may be disease related changes of this receptor in schizophrenia. Using in situ hybridisation histochemistry (ISHH), we measured mRNA for the obligatory NMDAR1 subunit of the NMDA glutamate receptor in post-mortem samples of hippocampus from schizophrenics, depressives, bipolar patients and normal controls. A significant main effect of diagnosis was observed in the dentate gyrus (ANOVA, p = 0.004) and a trend in the CA3 region (ANOVA, p = 0.06), with all psychiatric groups having reduced NMDAR1 mRNA levels compared to normal controls. In contrast to the affectively ill groups, the reductions in schizophrenics were more pronounced in the left side compared to the right. Expression of poly A mRNA also showed left-sided losses in the dentate gyrus in schizophrenia but reductions in NMDAR1 remained significant when expressed as a ratio of poly A. The findings confirm a recent report of reduced hippocampal NMDAR1 mRNA in schizophrenia. However, our new evidence suggests that this is a feature of both affective and schizophrenic disorders and that schizophrenia is distinguished from the others by left-sided reductions in hippocampal NMDAR1 gene expression.     

Changes in hippocampal GABAA/cBZR density during limbic epileptogenesis: Relationship to cell loss and mossy fibre sprouting

Reduced GABA_A/central benzodiazepine receptor (GABA_A/cBZR) density, mossy fibre sprouting (MFS) and hippocampal cell loss are well described pathological features of human temporal lobe epilepsy (TLE), and animal models thereof. However, the temporal relationship of their development, and their roles in the emergence of the epilepsy, are uncertain. This was investigated in the kainic acid (KA)-induced post-status epilepticus (SE) model of TLE. Male Wistar rats (7 weeks, n = 53) were randomised into control and KA groups. At 24 h, 2, 4 or 6 weeks sham and KA post-SE animals were euthanised, brains extracted and GABA_A/cBZR density, neuronal loss and MFS measured in hippocampal sub-regions. GABA_A/cBZR density (B_max) was measured by saturation-binding analysis using [³H]-flumazenil. At 24 h post-SE GABA_A/cBZR density was increased in almost all hippocampal subregions, but was decreased at the later time points with the exception of the dentate gyrus. There was significant neuronal loss in the CA3 SPc region (?24±9.3%, p<0.05) at 24h, which remained stable at the later time points associated with an elevated GABA_A/cBZR density per surviving neuron at 24h post-SE (+56.4%; p<0.05) which returned to control levels by 6 weeks post-SE. MFS in the dentate gyrus progressively increased over the 6 weeks following SE (+ 70.6% at 6 weeks), at which time there was a significant inverse relationship with GABA_A/cBZR binding (r² = 0.87; p = 0.02). The temporal evolution of GABA_A/cBZR density changes post-KA-induced SE, and the relationship with decreases in hippocampal pyramidal cell numbers and MFS, may point to a key role for these changes in the pathogenesis of acquired limbic epileptogenesis.     

Spreading depression-like hypoxic depolarization in CA1 and fascia dentata of hippocampal slices: relationship to selective vulnerability

Hippocampal tissue slices were made hypoxic for 4-10 min and then reoxygenated for 60-120 min. Postsynaptic evoked potentials were recorded and extracellular DC potential was monitored continuously in stratum (st.) pyramidale of CA1 and st. granulosum of fascia dentata (FD). In some preparations extracellular potassium ([K+]o) and calcium ([Ca2+]o) were also recorded in both regions. Postsynaptic responses disappeared sooner during hypoxia and were less likely to recover upon reoxygenation in CA1 than in FD. The CA1 region exhibited a spreading depression (SD)-like response to hypoxia more often than did FD. When both regions showed SD-like depolarization, voltage shift and elevation of [K+]o were of greater magnitude and shorter latency in CA1. The probability of posthypoxic recovery of synaptic transmission was inversely related to the time spent in the SD-like state in both CA1 and FD. We conclude that the selective vulnerability of CA1 neurons to hypoxic and ischemic damage may be due, at least in part, to the region's propensity to undergo prolonged and severe SD-like depolarization.     

Distribution of hippocampal mossy fibers in the rat. An experimental study with silver impregnation methods

The paper deals with the distribution of the axons that arise in the fascia dentata and go to the hippocampus proper. lesions were placed in the fascia dentata and degenerating mossy fibers were mapped by silver impregnation, mostly according to the method of Fink and Heimer ('67). The fascia dentata and the layer of mossy fibers in the rat form a curved and twisted structure extending from the level of the posterior aspect of the septum to the temporal tip. The horizontal serial sections used for this study were, therefore, in part interpreted by means of three-dimensional reconstructions. Mossy fibers do not enter the fimbria at any point. Degenerating fibers can be traced to the limit between regio superior (CA 1) and regio inferior. This dentato-hippocampal connection displays a pattern of precise localization, each level of fascia dentata projecting onto a restricted level of regio inferior. The bands of degenerated mossy fibers produced by lesions in the fascia dentata were largely oriented in the transverse direction (slightly in a temporal direction) and were somewhat wider distally than at the origin from the hilus. Very narrow bands were seen in a few animals with particularly small lesions.     

Plasticity and metaplasticity of adult rat hippocampal mossy fibers induced by neurotrophin‐3

Changes in synaptic efficacy and morphology are considered as the downstream mechanisms of consolidation of memories and other adaptive behaviors. In the last decade, neurotrophin‐3 (NT‐3) has emerged as one potent mediator of synaptic plasticity. In the adult brain, expression of NT‐3 is largely confined to the hippocampal dentate gyrus (DG). Our previous studies show that application of high‐frequency stimulation (HFS) sufficient to elicit long‐term potentiation (LTP) at the DG‐CA3 pathway as well as acute intrahippocampal microinfusion of brain‐derived neurotrophin factor produce mossy fiber (MF) structural reorganization. Here, we show that intrahippocampal microinfusion of NT‐3 induces a long‐lasting potentiation of synaptic efficacy in the DG‐CA3 projection accompanied by an MF structural reorganization of adult rats in vivo. It is considered that the capacity of synapses to express plastic changes is itself subject to variation depending on previous experience; taking into consideration the effects of NT‐3 on MF synaptic plasticity, we thus used intrahippocampal microinfusion of NT‐3 to analyse its effects on functional and structural plasticity induced by subsequent MF‐HFS sufficient to induce LTP in adult rats, in vivo. Our results show that NT‐3 modifies the ability of the MF pathway to present subsequent LTP by HFS, and modifies the structural reorganization pattern. The modifications in synaptic efficacy and morphology elicited by NT‐3 at the MF‐CA3 pathway were blocked by the presence of a Trk receptor inhibitor (K252a). These findings support the idea that NT‐3 actions modify subsequent synaptic plasticity, a homeostatic mechanism thought to be essential for maintaining synapses in the adult mammalian brain.     

T-type Ca2+ channel activity increases in rat hippocampal CA1 region during kindling epileptogenesis

Epileptogenesis is a dynamical process that involves synaptic plasticity changes such as synaptic reorganization of excitatory and inhibitory systems and axonal sprouting in the hippocampus, which is one of the most studied epileptogenic regions in the brain. However, the early events that trigger these changes are not understood well. We investigated short-term and long-term synaptic plasticity parameters and T-type Ca²⁺ channel activity changes in the early phase of a rat kindling model. Chronic pentylenetetrazole (PTZ) application was used in order to induce the kindling process in rats. The recordings were obtained from hippocampal slices in the CA1 region at 25th day of PTZ application. Tetraethylammonium was used in order to induce long-term potentiation and T-type Ca²⁺ channel activity was assessed in the presence of mibefradil. We found that tetraethylammonium-induced long-term potentiation was not prevented by mibefradil in the kindling group in contrast to control group. We also found an increase in paired-pulse ratios in the PTZ-applied group. Our findings indicate an increase in the “T-type Ca²⁺ channel component of LTP” in the kindling group, which may be an early mechanism in epileptogenesis.     

Afferent and efferent synaptic connections of somatostatin-immunoreactive neurons in the rat fascia dentata

The aim of this study was to determine whether somatostatin (SS)-immunoreactive neurons of the rat fascia dentata are involved in specific excitatory circuitries that may result in their selective damage in models of epilepsy. Synaptic connections of SS-immunoreactive neurons were determined at the electron microscopic level by using normal and colchicine pretreated rats. Vibratome sections prepared from both fascia dentata of control animals and from rats that had received an ipsilateral lesion of the entorhinal cortex 30-36 hours before sacrifice were immunostained for SS by using a monoclonal antibody (SS8). Correlated light and electron microscopic analysis demonstrated that many SS-immunoreactive neurons in the hilus send dendritic processes into the outer molecular layer of the fascia dentata, and dendrites of the same neurons occupy broad areas in the dentate hilar area. The majority of SS-immunoreactive axon terminals form symmetric synapses with the granule cell dendrites in the outer molecular layer and also innervate deep hilar neurons. Via their dendrites in the outer molecular layer, the SS-immunoreactive neurons receive synaptic inputs from perforant pathway axons which were identified by their anterograde degeneration following entorhinal lesions. The axons from the entorhinal cortex are the first segment of the main hippocampal excitatory loop. The hilar dendrites of the same SS-immunoreactive cells establish synapses with the mossy axon collaterals which represent the second member in this excitatory neuronal chain. These observations suggest that SS-immunoreactive neurons in the dentate hilar area may be driven directly by their perforant path synapses and via the granule cells which are known to receive a dense innervation from the entorhinal cortex. These observations demonstrate that SS-immunoreactive neurons in the hilar region are integrated in the main excitatory impulse flow of the hippocampal formation.     

Mixed electrical-chemical transmission between hippocampal mossy fibers and pyramidal cells

Morphological and electrophysiological studies have shown that granule cell axons, the mossy fibers (MFs), establish gap junctions and therefore electrical communication among them. That granule cells express gap junctional proteins in their axons suggests the possibility that their terminals also express them. If this were to be the case, mixed electrical-chemical communication could be supported, as MF terminals normally use glutamate for fast communication with their target cells. Here we present electrophysiological studies in the rat and modeling studies consistent with this hypothesis. We show that MF activation produced fast spikelets followed by excitatory postsynaptic potentials in pyramidal cells (PCs), which, unlike the spikelets, underwent frequency potentiation and were strongly depressed by activation of metabotropic glutamate receptors, as expected from transmission of MF origin. The spikelets, which persisted during blockade of chemical transmission, were potentiated by dopamine and suppressed by the gap junction blocker carbenoxolone. The various waveforms evoked by MF stimulation were replicated in a multi-compartment model of a PC by brief current-pulse injections into the proximal apical dendritic compartment, where MFs are known to contact PCs. Mixed electrical and glutamatergic communication between granule cells and some PCs in CA3 may ensure the activation of sets of PCs, bypassing the strong action of concurrent feed-forward inhibition that granule cells activate. Importantly, MF-to-PC electrical coupling may allow bidirectional, possibly graded, communication that can be faster than chemical synapses and subject to different forms of modulation.     

Effects of alcohol exposure during different periods of development: changes in hippocampal mossy fibers

Exposure to 10-12 g/kg/day of alcohol either during days 1-10 or 11-21 of gestation had no detectable effect on hippocampal mossy fiber development. Exposing artificially reared rat pups to 7.0-7.5 g/kg/day of alcohol during days 1-10 postpartum dramatically altered the organization of the Timm-stained mossy fiber terminal field when the animals were examined as adults, suggesting that alcohol exposure during a period equivalent to the human third trimester is more deleterious to brain development than exposure during periods equivalent to either the first or second trimesters.     

Timm's sulfide silver reaction for zinc during experimental anterograde degeneration of hippocampal mossy fibers

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.