Differential actions of nerve growth factor receptors TrkA and p75NTR in a rat model of epileptogenesis

Kindling, an experimental model of epileptogenesis, and activation-induced synaptic reorganization are modulated by nerve growth factor (NGF), but whether NGF acts via its high-affinity receptor TrkA and/or the common neurotrophin receptor p75NTR is unknown. We previously demonstrated, and confirmed in this study, that inhibition of NGF binding to both TrkA and p75NTR inhibited kindling and decreased kindling-induced mossy fiber sprouting. We now report specific inhibition of TrkA.NGF binding, but not p75NTR.NGF binding, retarded perforant path kindling progression. However, mossy fiber sprouting was inhibited by either selective TrkA.NGF or p75NTR.NGF antagonists. Our results suggest that TrkA, but not p75NTR, plays a role in kindling, while both receptors modulate kindling-induced mossy fiber sprouting. This implicates different mechanisms of neurotrophin action on kindling (mediated by TrkA) and neuronal sprouting (mediated by both TrkA and p75NTR) and suggests that sprouting involves kindling-independent neurotrophin action via p75NTR.     

The cholinergic system modulates kindling and kindling-induced mossy fiber sprouting

In a previous study, our laboratory demonstrated that the intraventricular infusion of nerve growth factor (NGF) accelerated kindling rates and enhanced mossy fiber sprouting in the absence of noticeable kindling-associated neuronal loss. The purpose of the present study was to investigate whether these NGF effects were mediated via the cholinergic system. This study evaluated the effects of the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine on kindling rates and kindling-induced mossy fiber sprouting in adult rats. The results showed that pilocarpine accelerated kindling rates and enhanced kindling-induced mossy fiber sprouting in the CA3 region of the hippocampus, whereas scopolamine retarded kindling rates and blocked kindling-induced mossy fiber sprouting in the CA3 and IML regions. These findings suggest that the cholinergic system may contribute to the long-term structural and functional alterations that are characteristic of the kindled state. Moreover, these data provide support for the hypothesis that NGF infusions may mediate kindling and kindling-induced mossy fiber sprouting via regulation of the cholinergic system.     

EphA5 and ephrin-A2 expression during optic nerve regeneration: a 'two-edged sword'

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.     

Transient up-regulation of retinal EphA3 and EphA5, but not ephrin-A2, coincides with re-establishment of a topographic map during optic nerve regeneration in goldfish

Eph tyrosine kinase receptors and their ligands, the ephrins, play a key role in the establishment of retinotectal topography during development. Tectal up-regulation of ephrin-A2 in goldfish, coincident with the reestablishment of a retinotectal map, suggests a similar role during optic nerve regeneration. Here we report a complementary study of EphA3, EphA5 and ephrin-A2 expression in the retina. EphA3 and EphA5 are transiently up-regulated as ascending naso-temporal gradients, whereas ephrin-A2 remains uniform. The expression profiles differ from those in developing chick and mouse, suggesting that different combinations of retinal Eph receptors and ligands can generate topographic guidance information.     

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.     

Strain differences affect the induction of status epilepticus and seizure-induced morphological changes

Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance between excitation and inhibition, and ultimately result in seizures. Rodents show similar genetic determinants of seizure induction. To test whether seizure‐prone phenotypes exhibit increased seizure‐related morphological changes, we compared two standard rat strains (Long–Evans hooded and Wistar) and two specially bred strains following status epilepticus. The special strains, namely the kindling‐prone (FAST) and kindling‐resistant (SLOW) strains, were selectively bred based on their amygdala kindling rate. Although the Wistar and Long–Evans hooded strains experienced similar amounts of seizure activity, Wistar rats showed greater mossy fiber sprouting and hilar neuronal loss than Long–Evans hooded rats. The mossy fiber system was affected differently in FAST and SLOW rats. FAST animals showed more mossy fiber granules in the naïve state, but were more resistant to seizure‐induced mossy fiber sprouting than SLOW rats. These properties of the FAST strain are consistent with those observed in juvenile animals, further supporting the hypothesis that the FAST strain shares circuit properties similar to those seen in immature animals. Furthermore, the extent of mossy fiber sprouting was not well correlated with sensitivity to status epilepticus, but was positively correlated with the frequency of spontaneous recurrent seizures in the FAST rats only, suggesting a possible role for axonal sprouting in the development of spontaneous seizures in these animals. We conclude that genetic factors clearly affect seizure development and related morphological changes in both standard laboratory strains and the selectively bred seizure‐prone and seizure‐resistant strains.     

EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins-A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon-axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli.     

Amygdala kindling develops without mossy fiber sprouting and hippocampal neuronal degeneration in rats

Repeated electrical stimulation of limbic structures has been reported to produce the kindling effect together with morphological changes in the hippocampus such as mossy fiber sprouting and/or neuronal loss. However, to argue against a causal role of these neuropathological changes in the development of kindling-associated seizures, we examined mossy fiber sprouting in amygdala (AM)-kindled rats using Timm histochemical staining, and evaluated the hippocampal neuronal degeneration in AM-kindled rats by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labelling (TUNEL). Amygdala kindling was established by 10.3 +/- 0.7 electrical stimulations, and no increase in Timm granules (neuronal sprouting) was observed up to the time of acquisition of a fully kindled state. However, the density and distribution of Timm granules increased significantly in the dentate gyrus compared with unkindled rats after 29 after-discharges or more than 10 kindled convulsions. In addition, no significant increase in TUNEL-positive cells was found in the hilar polymorphic neurons or in CA3 pyramidal neurons of the kindled rats that had fewer than 29 after-discharges. However, a significant increase of TUNEL-positive cells was found in the granule cell layer in the dentate gyrus of the stimulated side after 18 after-discharges or 10 kindled convulsions. Our result show that AM kindling develops without evidence of mossy fiber sprouting, and that mossy fiber sprouting may appear after repeated kindled convulsions, following death of the granule cells in the dentate gyrus.     

Absence of hippocampal mossy fiber sprouting in transgenic mice overexpressing brain-derived neurotrophic factor

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain-derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain-derived neurotrophic factor. Despite 2--3-fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild-type mice. In addition, no changes of the mRNA expression of two growth-associated proteins, GAP-43 and SCG-10 were found. These data suggest that early and persistent elevations of brain-derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus.     

Localized overexpression of FGF-2 and BDNF in hippocampus reduces mossy fiber sprouting and spontaneous seizures up to 4 weeks after pilocarpine-induced status epilepticus

Purpose: We have recently reported that viral vector–mediated supplementation of fibroblast growth factor‐2 (FGF‐2) and brain‐derived neurotrophic factor (BDNF) in a lesioned, epileptogenic rat hippocampus limits neuronal damage, favors neurogenesis, and reduces spontaneous recurrent seizures. To test if this treatment can also prevent hippocampal circuit reorganization, we examined here its effect on mossy fiber sprouting, the best studied form of axonal plasticity in epilepsy. Methods: A herpes‐based vector expressing FGF‐2 and BDNF was injected into the rat hippocampus 3 days after an epileptogenic insult (pilocarpine‐induced status epilepticus). Continuous video–electroencephalography (EEG) monitoring was initiated 7 days after status epilepticus, and animals were sacrificed at 28 days for analysis of cell loss (measured using NeuN immunofluorescence) and mossy fiber sprouting (measured using dynorphin A immunohistochemistry). Key Findings: The vector expressing FGF‐2 and BDNF decreased both mossy fiber sprouting and the frequency and severity of spontaneous seizures. The effect on sprouting correlated strictly with the cell loss in the terminal fields of physiologic mossy fiber innervation (mossy cells in the dentate gyrus hilus and CA3 pyramidal neurons). Significance: These data suggest that the supplementation of FGF‐2 and BDNF in an epileptogenic hippocampus may prevent epileptogenesis by decreasing neuronal loss and mossy fiber sprouting, that is, reducing some forms of circuit reorganization.     

Anesthesia induced retrograde amnesia is ameliorated by ephrinA5-IgG in mice: EphA receptor tyrosine kinases are involved in mammalian memory

EphA receptors and their ephrin-A ligands were previously thought to play a role only in embryonic development of the brain. Recently, however, these proteins were shown to be expressed in the adult mouse brain, primarily in the hippocampus, and were implicated in hippocampal synaptic plasticity and learning. What aspects of learning EphA receptors mediate have not been studied? Using the fear conditioning paradigm we demonstrate that EphA receptors play roles in memory. We show that post-training surgical anesthesia leads to robust context specific retrograde amnesia in mice, and post-anesthesia activation of EphA receptors induces a significant amelioration of this amnesia. As acquisition was left unaffected and performance factors were found unaltered, we suggest that the amelioration was due to changes in cognition leading to improved memory. Our data represent the first pieces of evidence for the involvement of EphA receptor tyrosine kinase receptors in mammalian memory, a finding that opens a new avenue into the functional analysis of the largest receptor tyrosine kinase subfamily in the brain.     

Mossy fiber synaptic reorganization induced by kindling: time course of development, progression, and permanence

Recent studies have revealed that mossy fiber axons of granule cells in the dentate gyrus undergo reorganization of their terminal projections in both animal models of epilepsy and human epilepsy. This synaptic reorganization has been demonstrated by the Timm method, a histochemical technique that selectively labels synaptic terminals of mossy fibers because of their high zinc content. It has been generally presumed that the reorganization of the terminal projections of the mossy fiber pathway is a consequence of axonal sprouting and synaptogenesis by mossy fibers. To evaluate this possibility further, the time course for development of Timm granules, which correspond ultrastructurally to mossy fiber synaptic terminals, was examined in the supragranular layer of the dentate gyrus at the initiation of kindling stimulation with an improved scoring method for assessment of alterations in Timm histochemistry. The progression and permanence of this histological alteration were similarly evaluated during the behavioral and electrographic evolution of kindling evoked by perforant path, amygdala, or olfactory bulb stimulation. Mossy fiber synaptic terminals developed in the supragranular region of the dentate gyrus by 4 d after initiation of kindling stimulation in a time course compatible with axon sprouting. The induced alterations in the terminal projections of the mossy fiber pathway progressed with the evolution of behavioral kindled seizures, became permanent in parallel with the development of longlasting susceptibility to evoked seizures, and were observed as long as 8 months after the last evoked kindled seizure. The results demonstrated a strong correlation between mossy fiber synaptic reorganization and the development, progression, and permanence of the kindling phenomenon.     

The effects of fimbria/fornix transections on perforant path kindling and mossy fiber sprouting

Various clinical and experimental studies of epilepsy have described synaptic reorganization in the dentate gyrus of hippocampus, in the form of collateral sprouting of the mossy fibers. These reports have led to the hypothesis that reorganized mossy fibers form a functional excitatory feedback circuit that contributes to local circuit hyperexcitability and chronic seizures. Much of the evidence supporting the sprouting hypothesis has been derived from kindling. We recently reported that transection of the fimbria/fornix (FF), which produces chronic epileptiform activity in the hippocampus, also induces mossy fiber sprouting in the inner molecular layer of the dentate gyrus. In the present study, we attempted to determine whether mossy fiber sprouting contributes to epileptiform activity, by examining the effects FF transections on perforant path (PP) kindling and associated mossy fiber sprouting. We found that FF transections and PP kindling produced moderate levels of sprouting, whereas the combination of the two treatments produced significantly denser sprouting. FF transections had mixed effects on kindling: afterdischarge thresholds were decreased and clonus and afterdischarge durations were increased, suggesting increased local excitation, whereas the kindling of behavioral seizures was delayed, suggesting decreased epileptogenesis.     

Long-term potentiation trains induce mossy fiber sprouting

It has been shown that both amygdaloid and hippocampal kindling induce sprouting of the mossy fibers in the dentate gyrus. In this study, we investigated whether non-epileptogenic stimulation could also induce mossy fiber sprouting. Long-term potentiation (LTP) was induced in the dentate gyrus by the application of brief, high-frequency trains to the perforant path. The potentiating stimulation was applied each day for 10 days, and the tissue was prepared for Timm labelling 7 days later. Sprouting was significantly increased in the LTP group compared to the implanted control rats. These results suggest that mossy fiber sprouting is not damage-induced and is dependent on neuronal activation.     

New model of retinocollicular mapping predicts the mechanisms of axonal competition and explains the role of reverse molecular signaling during development

Precise connections in the brain result from elaborate processes during development. In the visual system, axonal projections from retinal ganglion cells (RGCs) onto the superior colliculus form a precise retinotopic map. Studies have revealed that the development of retinocollicular maps involves three main factors: graded expression of molecular guidance cues such as EphAs and ephrin-As, activity-dependent processes driven by spontaneous activity in RGCs, and different forms of axonal competition. In this study, we developed a new, versatile model including these factors. We first modeled the selective arborization of RGC axons, mediated by EphA/ephrin-A signaling, without assuming that this initial process instructed the map's final topology. We also derived an integro-differential equation modeling a second, dynamic phase in which activity-dependent plasticity of axonal arbors combined with their competition for collicular resources can deeply remodel the topology of immature maps. Our model hence challenges the view that retinotopic maps are instructed by matching molecular gradients and then merely refined by activity-dependent processes. We reproduce fine features of retinotopic map development in wild-type and various transgenic mice, allowing a new understanding of the underlying mechanisms. Our model predicts that competition is not based on comparisons of axonal EphA receptor levels but rather relies on the optimization of collicular resources mediated by neurotrophic receptors such as p75(NTR). Our model finally clarifies the elusive role of reverse signaling between retinal ephrin-As and collicular EphAs by reproducing for the first time the phenotypes of two mouse genotypes in which this function is altered.     

Alteration of long-lasting structural and functional effects of kainic acid in the hippocampus by brief treatment with phenobarbital.

Kainic acid, an analog of the excitatory amino acid L-glutamate, induces acute hyperexcitability and permanent structural alterations in the hippocampal formation of the adult rat. Administration of kainic acid is followed by acute seizures in hippocampal pathways, neuronal loss in CA3 and the hilus of the dentate gyrus, and reorganization of the synaptic connections of the mossy fiber pathway. Rats with these hippocampal structural alterations have increased susceptibility to kindling. To evaluate the role of the acute seizures and associated hippocampal structural alterations in the development of this long-lasting susceptibility, rats that received intraventricular kainic acid were cotreated with phenobarbital (60 mg/kg, s.c., once daily). Treatment with this dose for 5 d after administration of kainic acid suppressed acute seizure activity, protected against excitotoxic damage in the dentate gyrus, reduced mossy fiber sprouting, and completely abolished the increased susceptibility to kindling associated with kainic acid. Brief treatment with phenobarbital modified the pattern of damage and synaptic reorganization in the dentate gyrus in response to seizure-induced injury, and altered the long-lasting functional effects associated with hippocampal damage. As phenobarbital treatment did not protect against neuronal damage in CA3 or other regions of the hippocampus, the circuitry of the dentate gyrus was implicated as a locus of cellular alterations that influenced the development of kindling. These observations are evidence that pharmacological intervention can prevent the development of epilepsy in association with acquired structural lesions, and suggest that pharmacological modification of cellular responses to injury can favorably alter long-term functional effects of CNS damage.     

Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells

Ephrin-As are repulsive axonal guidance cues that regulate retinotectal projection. EphA tyrosine kinases, which are the receptors of ephrin-As, activate signaling cascades leading to cytosckeleton reorganization. Here, we address the role of cyclin-dependent kinase (Cdk) 5 in Eph receptor signaling induced by ephrin-A5. Ephrin-A5 induced a cell morphological response in PC-3M cells that endogenously express Cdk5 and EphA2, a receptor for ephrin-A5. This response was augmented by the transfection of p35, which is a neuronal regulator of Cdk5. While the morphological response of native PC-3M cells was not affected by olomoucine, an inhibitor of Cdk, the response was inhibited in the p35-transfected cells. In retinal ganglion cells, either olomoucine at 20 microM or Y-27632 at 10 microM, an inhibitor of Rho-kinase/ROKalpha/ROCKII, showed maximum inhibitory effect against ephrin-A5 (10 microg/ml)-induced growth cone collapse. Combined application of olomoucine and Y-27632 further suppressed the ephrin-A5-induced response. Ephrin-A5 evoked phosphorylation of Cdk5 at Tyr15 and tau, a substrate of Cdk5 in retinal growth cones. Recombinant herpes simplex virus expressing Cdk5 mutant (kinase-negative or Tyr15 to Ala) showed a dominant-negative effect on the ephrin-A5-induced growth cone collapse. These findings demonstrate that both Cdk5 and the Rho kinase pathway independently contribute to the downstream of ephrin-A-induced signaling in retinal ganglion cells.     

Restoring axonal localization and transport of transmembrane receptors to promote repair within the injured CNS:a critical step in CNS regeneration

Each neuronal subtype is distinct in how it develops, responds to environmental cues, and whether it is capable of mounting a regenerative response following injury. Although the adult central nervous system (CNS) does not regenerate, several experimental interventions have been trialled with successful albeit lim-ited instances of axonal repair. We highlight here some of these approaches including extracellular matrix (ECM) modiifcation, cellular gratfing, gene therapy-induced replacement of proteins, as well as application of biomaterials. We also review the recent report demonstrating the failure of axonal localization and trans-port of growth-promoting receptors within certain classes of mature neurons. More speciifcally, we discuss an inability of integrin receptors to localize within the axonal compartment of mature motor neurons such as in the corticospinal and rubrospinal tracts, whereas in immature neurons of those pathways and in mature sensory tracts such as in the optic nerve and dorsal column pathways these receptors readily local-ize within axons. Furthermore we assert that this failure of axonal localization contributes to the intrinsic inability of axonal regeneration. We conclude by highlighting the necessity for both combined therapies as well as a targeted approach speciifc to both age and neuronal subtype will be required to induce substantial CNS repair.