Potential roles for hyaluronan and CD44 in kainic acid-induced mossy fiber sprouting in organotypic hippocampal slice cultures

The most well-documented synaptic rearrangement associated with temporal lobe epilepsy is mossy fiber sprouting (MFS). MFS is a pronounced expansion of granule cell mossy fiber axons into the inner dentate molecular layer. The recurrent excitatory network formed by MFS is hypothesized to play a critical role in epileptogenesis, which is the transformation of the normal brain into one that is prone to recurrent spontaneous seizures. While many studies have focused on the functional consequences of MFS, relatively few have investigated the molecular mechanisms underlying the increased propensity of mossy fibers to invade the inner molecular layer. We hypothesized that changes in two components of the extracellular matrix, hyaluronan and its primary receptor, CD44, contribute to MFS. Hyaluronan contributes to laminar-specificity in the hippocampus and increases in hyaluronan and CD44 are associated with temporal lobe epilepsy. We tested our hypothesis in an in vitro model of MFS using a combination of histological and biochemical approaches. Application of kainic acid (KA) to organotypic hippocampal slice cultures induced robust MFS into the inner dentate molecular layer compared with vehicle-treated controls. Degradation of hyaluronan with hyaluronidase significantly reduced but did not eliminate KA-induced MFS, suggesting that hyaluronan played a permissive role in MFS, but that loss of hyaluronan signaling alone was not sufficient to block mossy fiber reorganization. Comparison of CD44 expression with MFS revealed that when CD44 expression in the molecular layers was high, MFS was minimal and when CD44 expression/function was reduced following KA treatment or with function blocking antibodies, MFS was increased. The time course of KA-induced reductions in CD44 expression was identical to the temporal progression of KA-induced MFS reported previously in hippocampal slice cultures, suggesting that reduced CD44 expression may help promote MFS. Understanding the molecular mechanisms underlying MFS may lead to therapeutic interventions that limit epileptogenesis.     

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.     

Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures

Sprouting of the mossy fiber axons of the dentate granule cells is a structural neuronal plasticity found in the mature brain of epileptic humans and experimental animals. Mossy fiber sprouting typically arises in experimental animals after repeated seizures and may contribute to the hyperexcitability of the epileptic brain. Investigation of the molecular triggers and spatial cues involved in mossy fiber sprouting has been hampered by the lack of an optimal in vitro model for studying this rearrangement. For an in vitro model to be feasible, the circuitry and receptors involved in convulsant-induced mossy fiber sprouting would have to be localized near the granule cells, rather than being dependent on long-range brain interconnections. However, it is not known whether this is the case. We report here that that application of the convulsant, kainic acid, to organotypic hippocampal explant cultures induces seizures, neuronal cell death, and subsequent dramatic mossy fiber sprouting with a similar laminar preference and time-course to that seen in intact animals. Prolonged (48 h) but not transient (4 h) kainic acid treatment caused regionally selective neuronal cell death. Cultures treated with kainic acid for a prolonged period displayed a time- and dose-dependent increase in supragranular Timm staining reflective of increased mossy fiber innervation to this area. Direct visualization of mossy fiber axons with neurobiotin-labeling revealed that mossy fibers in kainic acid-treated cultures exhibited a dramatic increase in supragranular axonal branch points and synaptic boutons. The cellular and molecular determinants required for kainic acid-induced cell death and subsequent mossy fiber reorganization thus appear to be intrinsic to the hippocampal slice preparation, and are preserved in culture. Given the ease with which functional inhibitors or pharmacological agents may be utilized in this system, slice cultures may provide a powerful model in which to study the molecular components involved in triggering mossy fiber outgrowth and underlying its laminar specificity. Elucidation of these molecular pathways will likely have both specific utility in clarifying the functional consequences of mossy fiber sprouting, as well as general utility in understanding of synaptic reorganization in the mature central nervous system.     

Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures

Apolipoprotein E isoforms may have differential effects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease.We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secretion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this effect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the effects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/cAMP pathway. Isoproterenol (50 nM, a beta-adrenoceptor agonist) enhanced, while clonidine (250 nM, an alpha2-adrenoceptor agonist) decreased, secreted apolipoprotein E. We also analysed the effects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 microM, an alpha1-adrenoceptor agonist) and serotonin (2.5 microM) enhanced, whereas carbachol (10 microM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The effects of these non-selective receptor agonists were modest, probably due to effects on different signalling pathways. Arterenol also potentiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite effects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general effect on protein synthesis.We conclude that astrocyte apolipoprotein E production can be regulated by factors that affect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder.     

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(6H)-pyridazinebutanoic acid (1 microM) 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 microM) 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.     

Glycine-induced neurotoxicity in organotypic hippocampal slice cultures

The role of the neutral amino acid glycine in excitotoxic neuronal injury is unclear. Glycine coactivates glutamate N-methyl-D-aspartate (NMDA) receptors by binding to a distinct recognition site on the NR1 subunit. Purely excitatory glycine receptors composed of NR1 and NR3/NR4 NMDA receptor subunits have recently been described, raising the possibility of excitotoxic effects mediated by glycine alone. We have previously shown that exposure to high concentrations of glycine causes extensive neurotoxicity in organotypic hippocampal slice cultures by activation of NMDA receptors. In the present study, we investigated further properties of in vitro glycine-mediated toxicity. Agonists of the glycine recognition site of NMDA receptors (D-serine and D-alanine) did not have any toxic effect in hippocampal cultures, whereas competitive blockade of the glycine site by 7-chlorokynurenic acid was neuroprotective. Stimulation (taurine, -alanine) or inhibition (strychnine) of the inhibitory strychnine-sensitive glycine receptors did not produce any neurotoxicity. The toxic effects of high-dose glycine were comparable in extent to those produced by the excitatory amino acid glutamate in our model. When combined with sublethal hypoxia/hypoglycemia, the threshold of glycine toxicity was decreased to less than 1 mM, which corresponds to the range of concentrations of excitatory amino acids measured during in vivo cerebral ischemia. Taken together, these results further support the assumption of an active role of glycine in excitotoxic neuronal injury.     

Cytotoxicity of tributyltin in rat hippocampal slice cultures

The neurotoxic effects of tributyltin (TBT), an endocrine-disrupting chemical, were evaluated in organotypic slice cultures of immature rat hippocampus. Confocal microscopy study with propidium iodide showed that TBT induced severe neuronal death in a concentration- and time-dependent manner with CA3 > CA1 > dentate gyrus ranking of vulnerability of the hippocampal subfields. Dead or damaged neurons exhibited chromatin condensation, which is one of the morphological characteristics of apoptosis, as revealed by acridine orange staining. TBT neurotoxicity was alleviated by application of free radical scavengers or antioxidants, such as catalase, superoxide dismutase, Trolox and alpha-tocopherol but not by ascorbic acid or N-acetyl-L-cysteine, which suggests an involvement of free radicals, particularly reactive oxygen species. Neurons displayed a long-lasting increase in intracellular Ca2+ concentrations after TBT treatment. Although neither N-methyl-D-aspartate (NMDA) receptor inhibitors nor voltage-sensitive Ca2+ channel blockers protected hippocampal neurons against TBT neurotoxicity, non-NMDA receptor antagonist completely prevented TBT-induced neurodegeneration. These data suggest that TBT provokes apoptosis-like neuronal cell death, which might be mediated by intracellular Ca2+ elevation and free radical generation via non-NMDA receptor activation.     

Hippocampal mossy fiber sprouting induced by forced and voluntary physical exercise

Alterations in the function and organization of synapses have been proposed to induce learning and memory. Previous studies have demonstrated that mossy fiber induced by overtraining in a spatial learning task can be related with spatial long-term memory formation. In this work we analyzed whether physical exercise could induce mossy fiber sprouting by using a zinc-detecting histologic technique (Timm). Rats were submitted to 3 and 5 days of forced or voluntary exercise. Rat brains were processed for Timm's staining to analyze mossy fiber projection at 7, 12 and 30 days after the last physical exercise session. A significant increase of mossy fiber terminals in the CA3 stratum oriens region was observed after 5 days of forced or voluntary exercise. Interestingly, the pattern of Timm's staining in CA3 mossy fibers was significantly altered when analyzed 12 days after exercise but not at 7 days post-exercise. In contrast, animals trained for only 3 days did not show increments of mossy fiber terminals in the stratum oriens. Altogether, these results demonstrate that sustained or programmed exercise can alter mossy fiber sprouting. Further Investigations are necessary to determine whether mossy fiber sprouting induced by exercise is also involved in learning and memory processes.     

Role of mossy fiber sprouting and mossy cell loss in hyperexcitability: a network model of the dentate gyrus incorporating cell types and axonal topography

Mossy cell loss and mossy fiber sprouting are two characteristic consequences of repeated seizures and head trauma. However, their precise contributions to the hyperexcitable state are not well understood. Because it is difficult, and frequently impossible, to independently examine using experimental techniques whether it is the loss of mossy cells or the sprouting of mossy fibers that leads to dentate hyperexcitability, we built a biophysically realistic and anatomically representative computational model of the dentate gyrus to examine this question. The 527-cell model, containing granule, mossy, basket, and hilar cells with axonal projections to the perforant-path termination zone, showed that even weak mossy fiber sprouting (10-15% of the strong sprouting observed in the pilocarpine model of epilepsy) resulted in the spread of seizure-like activity to the adjacent model hippocampal laminae after focal stimulation of the perforant path. The simulations also indicated that the spatially restricted, lamellar distribution of the sprouted mossy fiber contacts reported in in vivo studies was an important factor in sustaining seizure-like activity in the network. In contrast to the robust hyperexcitability-inducing effects of mossy fiber sprouting, removal of mossy cells resulted in decreased granule cell responses to perforant-path activation in agreement with recent experimental data. These results indicate the crucial role of mossy fiber sprouting even in situations where there is only relatively weak mossy fiber sprouting as is the case after moderate concussive experimental head injury.     

Beta amyloid is neurotoxic in hippocampal slice cultures

We examined the neurotoxicity of the 40 amino acid fragment of beta amyloid peptide (Aβ1–40) in cultured hippocampal slices. When injected into area CA3, Aβ1–40 produced widespread neuronal damage. Injection of the reverse sequence peptide, Aβ40-1, or vehicle alone produced little damage. The distribution Aβ1–40 was highly correlated with the area of neuronal damage. Thioflavine S and electron microscopic analysis confirmed that injected Aβ1–40 formed 7–9 nm AD type amyloid fibrils in the cultures. Aβ1–40 also altered the number of GFAP immunoreactive astrocytes and ED-1 immunoreactive microglia/macrophages within and around the Aβ1–40 deposit. The observed neurotoxicity of Aβ1–40 in hippocampal slice cultures provides evidence that this peptide may be responsible for the neurodegeneration observed in AD.     

Ultrastructure of mossy fiber endings in in vitro hippocampal slices

Summary 0.2 to 0.4 mm thick slices of guinea pig hippocampus were studied morphologically after varying periods of incubation at 36 ° C in Krebs-Ringer solution. Prior to fixation, the slices were tested for the presence of synaptically driven discharges of CA 3 neurons following mossy fiber (mf) stimulation because tissue preservation was satisfactory only in slices in which electrical responses were obtained. The fine structure of the mf layer in slices was compared with the ultrastructure of this region in hippocampal tissue fixed by transcardial perfusion or immersion of the tissue in the fixative.In the central part of the slices many intact neuronal structures of the mf layer could be seen even after 4 h of incubation. In the outer parts of the slices, neurons were swollen and vacuolated. These alterations were not observed in hippocampal tissue fixed by transcardial perfusion or by immersion. In all parts of the slices dark neurons and processes were found. Since dark neurons were also numerous in tissue blocks immersed in the fixative but were rare in perfused material, these changes were obviously caused by damage to unfixed tissue and fixation by immersion.     

Hippocampal mossy fiber sprouting is not impaired in brain-derived neurotrophic factor-deficient mice

In human temporal lobe epilepsy, a loss of hilar neurons followed by the sprouting of recurrent mossy fiber collaterals and the reinnervation of free synaptic sites on granule cell dendrites are discussed as possible mechanisms underlying hippocampal hyperexcitability. Dentate granule cells have been shown to upregulate brain-derived neurotrophic factor (BDNF) as well as TrkB, the high-affinity receptor for BDNF, in response to limbic seizures. This raised the possibility that BDNF is an important factor in hippocampal mossy fiber sprouting. Here we have used slice cultures of hippocampus, in which mossy fibers sprout and form a supragranular plexus in response to granule cell deafferentation, and have compared cultures from early postnatal BDNF-deficient mice and wild-type mice. We demonstrate that there is sprouting of supragranular mossy fibers in cultured slices from both BDNF knock-out and wild-type mice. We conclude that BDNF is not an essential factor for mossy fiber sprouting. However, our data do not exclude a role for BDNF in mossy fiber sprouting in wild-type mice, as compensatory mechanisms might have become effective in the mutant. Received: 6 January 1998 / Accepted: 23 February 1998     

Effect of beta amyloid peptides on neurons in hippocampal slice cultures.

Several investigators have described the neurotrophic and neurotoxic effects of beta amyloid peptide fragments on dissociated hippocampal neurons in culture. In these prior studies, the peptides were added to dissociated cultures between day 0 and day 4 in vitro, before hippocampal neurons are fully mature. We have analyzed the neurotrophic and neurotoxic effects of beta amyloid fragments beta 1-28, beta 25-35 and beta 1-40 on hippocampal slice cultures, whose physiology and morphology resembles the intact hippocampus. Addition of beta 1-28 or beta 25-35 to the growth medium did not produce significant changes in dendritic length or number of branches. Nerve growth factor, previously reported to enhance the neurotoxic effects of beta 1-40 on dissociated hippocampal neurons in culture, did not significantly enhance the neurotrophic effects of beta 1-28. To achieve high local concentrations of peptides and to avoid potential access problems in the cultures, we injected beta 1-28, beta 25-35, and beta 1-40 directly into the cultures. Amyloid-mediated neurotoxicity was not observed for beta 1-28 or beta 25-35, but beta 1-40 appeared to produce neurodegeneration around the site of injection.     

Pertussis toxin suppresses long-term potentiation of hippocampal mossy fiber synapses

The long-term potentiation (LTP) was studied using rat hippocampal slices in vitro. LTP in mossy fiber-CA3 pyramidal cell synapses was markedly suppressed in slices prepared from rats which had previously received intraventricular injection of pertussis toxin (PTX), compared with the bovine serum albumin-injected controls, suggesting the involvement of G-proteins in the mechanism of LTP in mossy fiber synapses. In contrast, LTP in Schaffer/commissural-CA1 pyramidal synapses was not affected by PTX pretreatment.     

AMPA receptor alterations precede mossy fiber sprouting in young children with temporal lobe epilepsy

Following neurological injury early in life numerous events, including excitotoxicity, neural degeneration, gliosis, neosynaptogenesis, and circuitry reorganization, may alone or in concert contribute to hyperexcitability and recurrent seizures in temporal lobe epilepsy. Our studies provide new evidence regarding the temporal sequence of key elements of hippocampal reorganization, mossy fiber sprouting and glutamate receptor subunit up-regulation, in a subset of young temporal lobe epileptic patients. Without evidence of mossy fiber sprouting, the youngest age group (3-10 years old) of mesial temporal lobe epileptic patients demonstrated enhanced glutamate receptor subunit profiles, suggesting that the dendritic change precedes axonal sprouting. However, sclerotic hippocampal specimens from epileptic patients ages 12-15 years old had the characteristic features of glutamate receptor up-regulation and mossy fiber sprouting first identified in the adult, indicating that reconstructed circuits appear early in the course of the disease. Non-sclerotic hippocampal specimens from lesion associated temporal lobe epileptic patients of all age groups showed minimal cell loss, sparse staining of glutamate receptor subunits in the dentate gyrus, and little or no mossy fiber sprouting. These compelling findings suggest a progressive sequence of events in the reorganization of the dentate gyrus of sclerotic hippocampal specimens. We suggest that cell loss and up-regulation of glutamate receptor subunits appear early in temporal lobe epilepsy and contribute to the synaptic plasticity that may facilitate the subsequent sprouting of mossy fiber collaterals which compound an already precipitous state of decline. The combination of pre-synaptic and post-synaptic changes serves as a potential substrate for hyperexcitability.     

Morphological and functional consequences of chronic epilepsy in rat hippocampal slice cultures

We have developed an in vitro model of chronic epilepsy in order to study the consequences of prolonged periods of epileptic activity. After applying the convulsants bicuculline and/or picrotoxin to mature rat hippocampal slice cultures for 3 days, large numbers of swollen and vacuolated cells were observed throughout all hippocampal subfields. The number of dendritic spines of pyramidal cells was massively reduced. These changes were similar to those observed previously in post-mortem studies of hippocampal tissue from human epilepsy patients. Intracellular recordings from CA3 pyramidal cells revealed that spontaneous synaptic activity was greatly reduced in treated cultures. -Aminobutyric acid-mediated inhibition was apparently not affected by sustained convulsant activity, although synaptic excitation was markedly depressed. Acute re-application of bicuculline to treated cultures elicited, upon stimulation of the mossy fibre tract, a typical interictal burst lasting several hundred milliseconds, with a wave form similar to those occurring in untreated cultures, but of a shorter duration. In contrast, ictal bursts (lasting tens of seconds), which always occur spontaneously in control cultures during initial perfusion of bicuculline, were not observed in treated cultures. These pathological changes were reversible when treated cultures were returned to normal medium for 1 week. The surviving cells had a healthy morphology and a normal complement of dendritic spines. Spontaneous synaptic activity was normal, and ictal bursts occurred spontaneously upon perfusion of bicuculline. The findings suggest that the morphological and functional changes are a consequence, rather than a direct cause of epilepsy.     

Muscarinic depression of synaptic transmission at the hippocampal mossy fiber synapse

1. The action of muscarine was studied in the CA3 region of the rat hippocampal slice with single-electrode voltage-clamp techniques. 2. Bath application of 1 or 10 microM muscarine produced an increase in the input resistance of these cells and reduced the slow afterhyperpolarization (sAHP) response. Changes in input resistance were more pronounced around the resting potential of the cell (-50 to -60 mV), but in many cells an effect was also seen at -80 mV. These effects were absent when cesium chloride-containing microelectrodes were used. 3. At 1 microM, muscarine had little effect on synaptic transmission, causing a 0 +/- 7% (mean +/- SE, n = 19) change in excitatory postsynaptic potential (EPSP) and decreasing the excitatory postsynaptic current (EPSC) by 11 +/- 6% (n = 14); neither change was statistically significant. 4. In contrast, 10 microM muscarine produced a reliable depression of both the EPSP and EPSC. This effect was independent of the electrolyte used: with KCl the EPSP was depressed 23 +/- 4% (n = 5) and the EPSC 35 +/- 5% (n = 4); for CsCl the EPSP was depressed 23 +/- 10% (n = 7) and the EPSC 34 +/- 5% (n = 7). 5. Muscarine did not alter the reversal potential of the synaptic current but merely produced a decrease in slope conductance (37 +/- 5%, n = 6). 6. Muscarine did not significantly alter the shape of the EPSC waveform. This was assessed by comparing the 10-90% rise time and the half decay time of the current before and after muscarine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Loss of dynorphin-mediated inhibition of voltage-dependent Ca2+ currents in hippocampal granule cells isolated from epilepsy patients is associated with mossy fiber sprouting

The endogenous kappa receptor selective opioid peptide dynorphin has been shown to inhibit glutamate receptor-mediated neurotransmission and voltage-dependent Ca2+ channels. It is thought that dynorphin can be released from hippocampal dentate granule cells in an activity-dependent manner. Since actions of dynorphin may be important in limiting excitability in human epilepsy, we have investigated its effects on voltage-dependent Ca2+ channels in dentate granule cells isolated from hippocampi removed during epilepsy surgery. One group of patients showed classical Ammon's horn sclerosis characterized by segmental neuronal cell loss and astrogliosis. Prominent dynorphin-immunoreactive axon terminals were present in the inner molecular layer of the dentate gyrus, indicating pronounced recurrent mossy fiber sprouting. A second group displayed lesions in the temporal lobe that did not involve the hippocampus proper. All except one of these specimens showed a normal pattern of dynorphin immunoreactivity confined to dentate granule cell somata and their mossy fiber terminals in the hilus and CA3 region. In patients without mossy fiber sprouting the application of the kappa receptor selective opioid agonist dynorphin A ([D-Arg6]1-13, 1 microM) caused a reversible and dose-dependent depression of voltage-dependent Ca2+ channels in most granule cells. These effects could be antagonized by the non-selective opioid antagonist naloxone (1 microM). In contrast, significantly less dentate granule cells displayed inhibition of Ca2+ channels by dynorphin A in patients with mossy fiber sprouting (Chi-square test, P < 0.0005). The lack of dynorphin A effects in patients showing mossy fiber sprouting compares well to the loss of kappa receptors on granule cells in Ammon's horn sclerosis but not lesion-associated epilepsy. Our data suggest that a protective mechanism exerted by dynorphin release and activation of kappa receptors may be lost in hippocampi with recurrent mossy fiber sprouting.