Fiber Sprouting Research Articles

Electroacupuncture at ST36-ST37 and at ear ameliorates hippocampal mossy fiber sprouting in kainic acid-induced epileptic seizure rats.

Our previous study showed that mossy fiber sprouting can occur in the hippocampus region in rats 6 wk after kainic acid-induced epileptic seizure, and this mossy fiber sprouting can facilitate epileptogenesis. Transcutaneous auricular vagal nerve stimulation (VNS), which is similar to cervical VNS, can reduce the occurrence of epileptic seizure in intractable epilepsy patients. Greater parasympathetic nerve activity can be caused by 2 Hz electroacupuncture (EA). Therefore, we investigated the effect of 2 Hz EA at ST-36-ST37 and at the ear on mossy fiber sprouting in kainic-treated Sprague-Dawley rats. The results indicated that applying 2 Hz EA at ST36-ST37 and at the ear for 3 d per week over 6 consecutive weeks can ameliorate mossy fiber sprouting in the hippocampus region of rats. These results indicated that applying 2 Hz EA at ST36-ST37 and at the ear might be beneficial for the treatment and prevention of epilepsy in humans.

Enhanced Mossy Fiber Sprouting and Synapse Formation in Organotypic Hippocampal Cultures Following Transient Domoic Acid Excitotoxicity

We have previously reported evidence of BDNF upregulation and increased neurogenesis in rat organotypic hippocampal slice cultures (OHSC) after a transient excitotoxic injury to the hippocampal CA1 area induced by low concentrations of the AMPA/kainate receptor agonist domoic acid (DOM). The changes observed in OHSC were consistent with observations in vivo, where low concentrations of DOM administered to rats during perinatal development caused increased BDNF and TrkB expression in the resulting adult animals. The in vivo low dose-DOM treatment also results in permanent alterations in hippocampal structure and function, including abnormal formation of dentate granule cell axons projecting to area CA3 (mossy fiber sprouting). Our objective in the current study is to determine if low concentrations of DOM induce mossy fiber sprouting and/or synaptogenesis in OHSC in order to facilitate future studies on the mechanisms of structural hippocampal plasticity induced by DOM. We report herein that application of a low concentration of DOM (2 μM) for 24 h followed by recovery induced a significant increase in the expression of the mossy fiber marker ZnT3 that progressed over time in culture. The DOM insult (2 μM, 24 h) also resulted in a significant upregulation of both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. All of the observed effects were fully antagonized by co-administration of the AMPA/kainate antagonists CNQX or NBQX but only partly by the NMDA antagonist CPP and not by the calcium channel blocker nifedipine. We conclude that exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity can induce a progressive change in hippocampal structure that can effectively model DOM effects in vivo.

Thymoquinone Attenuates Astrogliosis, Neurodegeneration, Mossy Fiber Sprouting, and Oxidative Stress in a Model of Temporal Lobe Epilepsy

Temporal lobe epilepsy (TLE) is a rather common and difficult-to-treat variant of epilepsy. Nearly one third of people with epilepsy do not respond effectively to currently available anticonvulsants. In this study, we evaluated the protective effect of thymoquinone (TQ), the main constituent of black seed with antioxidant and anti-inflammatory effects, in the intrahippocampal kainate model of TLE in rat. Following kainate injection, seizure activity was observed that was significantly diminished by TQ pretreatment at a dose of 10 mg/kg, p.o. Intrahippocampal kainate also increased malondialdehyde (MDA), nitrite, and nitrate levels and decreased activity of superoxide dismutase and TQ only significantly attenuated MDA. In addition, intrahippocampal kainate caused a significant reduction of neurons in CA1, CA3 and the hilar regions, and TQ significantly attenuated these changes. Timm histochemistry showed a marked mossy fiber sprouting (MFS) in the dentate gyrus of kainate-lesioned rats, and TQ significantly lowered MFS intensity. Meanwhile, a number of reactive astrocytes (astrogliosis) increased significantly in the kainate group, and TQ pretreatment significantly decreased it. These data suggest that TQ pretreatment could attenuate seizure activity and lipid peroxidation, lower hippocampal neuronal loss and MFS, and mitigate astrogliosis in kainate model of TLE.

Sympathetic Fiber Sprouting in Inflamed Joints and Adjacent Skin Contributes to Pain-Related Behavior in Arthritis

Although chronic pain is the most common symptom of arthritis, relatively little is known about the mechanisms driving it. Recently, a sprouting of autonomic sympathetic fibers into the upper dermis of the skin, an area that is normally devoid of them, was found in the skin following chronic inflammation of the rat hindpaw. While this sprouting only occurred when signs of joint and bone damage were present, it remained to be clarified whether it was a consequence of the chronic inflammation of the skin or of the arthritis and whether it also occurred in the joint. In the present study, we used a model of arthritis in which complete Freund's adjuvant (CFA) was injected into the rat ankle joint. At 4 weeks following CFA treatment, there was an increase in sympathetic and peptidergic fiber density in the ankle joint synovium. We also observed a sympathetic, but not peptidergic, fiber sprouting in the skin over the joint, which may be a consequence of the increased levels of mature nerve growth factor levels in skin, as revealed by Western blot analysis. The pharmacological suppression of sympathetic fiber function with systemic guanethidine significantly decreased the pain-related behavior associated with arthritis. Guanethidine completely suppressed the heat hyperalgesia and attenuated mechanical and cold hypersensitivity. These results suggest that transmitters released from the sprouted sympathetic fibers in the synovial membrane and upper dermis contribute to the pain-related behavior associated with arthritis. Blocking the sympathetic fiber sprouting may provide a novel therapeutic approach to alleviate pain in arthritis.

Coenzyme Q10 Ameliorates Neurodegeneration, Mossy Fiber Sprouting, and Oxidative Stress in Intrahippocampal Kainate Model of Temporal Lobe Epilepsy in Rat

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults and the most resistant type to treatment. Novel treatment approaches are strongly required to prevent or even reverse the cellular and molecular mechanisms of epileptogenesis. In this study, we investigated the possible neuroprotective effect of coenzyme Q10 (CoQ10) in an intrahippocampal kainate model of TLE in rat. Kainate injection caused a higher seizure severity during status epilepticus and spontaneous seizure phases, and CoQ10 pretreatment significantly attenuated its severity and incidence rate. Intrahippocampal kainate also led to elevation of malondialdehyde (MDA) and nitrite, and CoQ10 significantly attenuated the increased MDA and nitrite content. In addition, intrahippocampal kainate induced a significant degeneration of neurons in CA1, CA3, and hilar regions of the hippocampus, and CoQ10 significantly attenuated these changes in CA1 and CA3 regions. Timm's staining data showed a robust mossy fiber sprouting (MFS) in dentate gyrus of kainate-lesioned rats and CoQ10 significantly lowered MFS intensity. These data suggest that CoQ10 pretreatment could attenuate spontaneous recurrent seizures and inhibit hippocampal neuronal loss and aberrant MFS in kainate-induced model of TLE in rat, and part of its beneficial effect is due to its potential to mitigate oxidative stress.

Role of morphological changes in newly born granule cells of hippocampus after status epilepticus induced by pilocarpine in hyperexcitability

Newly born dentate gyrus (DG) granule cells (GCs) after Status Epilecticus induced by pilocarpine (PILO) exhibit morphological changes including narrower arborizations, greater number of branches and more endings in the inner molecular layer (IML) [1]. The increased concentration of dendrites in the IML where granule cell axons (mossy fibers) sprout in epileptic animals and make extensive recurrent excitatory synapses may contribute to enhance the DG hyperexcitability. A previous DG network model has shown that mossy fiber sprouting has a crucial role on hyperexcitability with only 10% sprouting being enough to generate spread of activity to all GCs in the network [2]. However, the additional effect of GC morphological changes on DG hyperexcitability is as yet unknown. Here we used the DG model [2] to evaluate the effect of different GC morphologies on the network activity. We replaced all reduced GC models of [2] with morphologically detailed models coming from tridimensional reconstructions of newly born doublecortin-positive DG GCs [1]. The scheme of the network connections is shown in Figure ​Figure1A.1A. Our sample of morphologically reconstructed GCs includes 20 from PILO-treated rats and 20 from control rats. Our cell models were constructed in NEURON and their ionic channels and maximum conductance distributions were the same as [2] taking into consideration the shorter arborizations of the PILO-treated cells. Our “control” network model is exactly the same as the topographic ring network of [2] with 10% sprouting available in ModelDB with the 500 original GC models replaced by morphologically reconstructed cells randomly chosen from our control sample. From our control network model we constructed two other models: one with 10% of the GCs (chosen randomly) replaced by cells chosen randomly from our PILO-treated sample and the other with the fraction of PILO-treated GC cells increased to 50%. The networks were submitted to focal perforant-path stimulations as in [2] and to obtain averages we ran 100 simulations for each network (connections between cells were created anew before each run). Our control network model produced a response similar to the one of the original model [2], showing that the insertion of GCs with realistic morphologies did not affect the 10% sprouting DG hyperexcitability (Figure 1 B, 100% C). In contrast, when a few amount of PILO-treated GCs were inserted the excitability of the network increased (Figure ​(Figure1B1B , 90% C – 10% P) and the increase was higher when the amount of PILO-treated GCs was larger (Figure 1 B, 50% C – 50% P). However, the effect of the insertion of PILO-treated GCs was only visible in combination with mossy fiber sprouting. Our results suggest that changes in GC morphology alone are not enough to affect the DG hyperexcitability but when these changes occur in the presence of other alterations such as mossy fiber sprouting they could enhance the DG hyperexcitability. Figure 1 Basic circuit diagram and effect of PILO-treated GC morphological changes on the average GCs response. A. Schematic view of the network (adapted from [1]). GC: granule cell; BC: basket cell; HC: HIPP cell; MC: mossy cell. DD: distal dendrites; MD: medial ...

Sprouting of Nervous Fibers and Upregulation of C‐X‐C Chemokine Receptor Type 4 Expression in Hippocampal Formation of Rats with Enhanced Spatial Learning and Memory

Hippocampal mossy fiber sprouting following training in the Morris water maze (MWM) is associated with spatial learning, memory and neural plasticity. The C-X-C chemokine receptor type 4 (CXCR4) is the main receptor for stromal cell-derived factor-1 (SDF-1), which is a chemokine that can regulate axonal elongation. This study aimed to investigate the relationship between the morphological plasticity of hippocampal formation and CXCR4 expression. A model of spatial learning and memory was established in rats by training using the MWM. Mossy fiber sprouting in the striatum oriens of the CA3 area of the hippocampus was found in trained rats by Neo-Timm's method. As shown by immunohistochemistry, the CXCR4 immunopositive neurons were distributed in all layers and areas of hippocampal formation. There were no differences among groups regarding the distribution or shape of the immunopositive neurons. However, the immunoreactive staining intensity was increased in trained rats as compared with the control rats. Both CXCR4 gene transcription and translation were significantly upregulated in the trained group as compared with the control group (P < 0.01). Morphological plasticity in the form of axonal sprouting in the hippocampal formation can be induced by enhanced spatial learning and memory activity, and CXCR4 mRNA and protein expression is upregulated, indicating a positive correlation between CXCR4 expression and axonal sprouting.

Neuroprotective Effect ofUncaria rhynchophyllain Kainic Acid-Induced Epileptic Seizures by Modulating Hippocampal Mossy Fiber Sprouting, Neuron Survival, Astrocyte Proliferation, and S100B Expression

Uncaria rhynchophylla (UR), which is a traditional Chinese medicine, has anticonvulsive effect in our previous studies, and the cellular mechanisms behind this are still little known. Because of this, we wanted to determine the importance of the role of UR on kainic acid- (KA-) induced epilepsy. Oral UR for 6 weeks can successfully attenuate the onset of epileptic seizure in animal tests. Hippocampal mossy fiber sprouting dramatically decreased, while neuronal survival increased with UR treatment in hippocampal CA1 and CA3 areas. Furthermore, oral UR for 6 weeks significantly attenuated the overexpression of astrocyte proliferation and S100B proteins but not γ-aminobutyric acid A (GABAA) receptors. These results indicate that oral UR for 6 weeks can successfully attenuate mossy fiber sprouting, astrocyte proliferation, and S100B protein overexpression and increase neuronal survival in KA-induced epileptic rat hippocampus

Transplantation of Neural Stem Cells Overexpressing Cardiotrophin-1 Inhibits Sprouting of Hippocampal Mossy Fiber in a Rat Model of Status Epilepticus

In this study, we studied the effects of hippocampal transplantation of neural stem cells (NSCs) overexpressing cardiotrophin 1 (CT1) on hippocampal mossy fiber sprouting (MFS) in a rat model of status epilepticus (SE). SE rats (lithium-pilocarpine model) were randomized into four study groups (18 rats per group): CT1-NSCs group, NSCs group, SE control group, and normal control group. Six rats were randomly chosen from each group at 1, 4, and 8 weeks after transplantation. MFS in hippocampal dentate gyrus was scored (Timm staining) at these time points. The MFS scores were as follows: CT1-NSCs 0.77 ± 0.04, 2.48 ± 0.89, and 2.39 ± 0.82 (1, 4, and 8 weeks after transplantation, respectively); NSCs 1.12 ± 0.62, 3.17 ± 0.64, and 3.88 ± 0.51; SE control 1.32 ± 0.35, 3.28 ± 0.75, and 4.32 ± 1.55; and normal control 0.37 ± 0.06, 0.34 ± 0.07, and 0.43 ± 0.04. Compared to SE control group and NSCs group, the scores of MFS in CT1-NSCs group were significantly lower (P < 0.05). In conclusion, transplantation with NSCs overexpressing CT1 inhibits hippocampal MFS and facilitates reduction of recurrent seizures.

Rapamycin Suppresses Mossy Fiber Sprouting But Not Seizure Frequency in a Mouse Model of Temporal Lobe Epilepsy

Temporal lobe epilepsy is prevalent and can be difficult to treat effectively. Granule cell axon (mossy fiber) sprouting is a common neuropathological finding in patients with mesial temporal lobe epilepsy, but its role in epileptogenesis is unclear and controversial. Focally infused or systemic rapamycin inhibits the mammalian target of rapamycin (mTOR) signaling pathway and suppresses mossy fiber sprouting in rats. We tested whether long-term systemic treatment with rapamycin, beginning 1 d after pilocarpine-induced status epilepticus in mice, would suppress mossy fiber sprouting and affect the development of spontaneous seizures. Mice that had experienced status epilepticus and were treated for 2 months with rapamycin displayed significantly less mossy fiber sprouting (42% of vehicle-treated animals), and the effect was dose dependent. However, behavioral and video/EEG monitoring revealed that rapamycin- and vehicle-treated mice displayed spontaneous seizures at similar frequencies. These findings suggest mossy fiber sprouting is neither pro- nor anti-convulsant; however, there are caveats. Rapamycin treatment also reduced epilepsy-related hypertrophy of the dentate gyrus but did not significantly affect granule cell proliferation, hilar neuron loss, or generation of ectopic granule cells. These findings are consistent with the hypotheses that hilar neuron loss and ectopic granule cells might contribute to temporal lobe epileptogenesis.

Effects of Pilocarpine-induced Seizures on the Neuronal Cell Death, Mossy Fiber Sprouting and Neurogensis in the Hippocampus of Mice

Effects of Pilocarpine-induced Seizures on the Neuronal Cell Death, Mossy Fiber Sprouting and Neurogensis in the Hippocampus of Mice

Increased excitability and spontaneous activity of rat sensory neurons following in vitro stimulation of sympathetic fiber sprouts in the isolated dorsal root ganglion

Increased excitability and spontaneous activity of rat sensory neurons following in vitro stimulation of sympathetic fiber sprouts in the isolated dorsal root ganglion

Increased excitability and spontaneous activity of rat sensory neurons following in vitro stimulation of sympathetic fiber sprouts in the isolated dorsal root ganglion

Many chronic pain conditions including complex regional pain syndrome are exacerbated by sympathetic activity. In animal models, sympathetic fibers sprout into the dorsal root ganglia (DRG) after peripheral nerve injury, forming abnormal connections with sensory neurons. However, functional studies of sympathetic-sensory connections have been limited largely to in vivo studies. This study describes a new method for studying sympathetic-sensory connections in an isolated whole DRG preparation in the rat spinal nerve ligation (SNL) model. Three days after ligation of the ventral ramus of the spinal nerve (SNL), sympathetic fibers sprouting into the DRG were observed to originate largely in the intact dorsal ramus of the spinal nerve, which at the lumbar level is a small branch of the spinal nerve separating from the ventral ramus near the intervertebral foramen. In whole DRG isolated 3days after SNL, microelectrode recordings of sensory neurons showed that repeated stimulation of the dorsal ramus enhanced spontaneous activity in large and medium diameter neurons and reduced rheobase in large neurons. These effects, which were slow and long lasting, were attributed to stimulation of the sympathetic sprouts because: stimulation had no effect in uninjured DRG; and effects could be reduced or eliminated by a “cocktail” of antagonists of norepinephrine and ATP receptors, by pretreatment with the sympathetic release blocker bretylium, or by pre-cutting the grey ramus through which sympathetic fibers coursed to the ligated DRG. The latter treatment, a relatively minimal form of sympathectomy, was also highly effective in reducing mechanical pain ipsilateral to the SNL.

Intraspinal sprouting of unmyelinated pelvic afferents after complete spinal cord injury is correlated with autonomic dysreflexia induced by visceral pain

Autonomic dysreflexia is a potentially life-threatening hypertensive syndrome following high thoracic (T) spinal cord injury (SCI). It is commonly triggered by noxious pelvic stimuli below the injury site that correlates with increased sprouting of primary afferent C-fibers into the lumbosacral (L/S) spinal cord. We have recently demonstrated that injury-induced plasticity of (L/S) propriospinal neurons, which relay pelvic visceral sensations to thoracolumbar sympathetic preganglionic neurons, is also correlated with the development of this syndrome. To determine the phenotype of pelvic afferent fiber sprouts after SCI, cholera toxin subunit beta (CTb) was injected into the distal colon 2 weeks post–T4 transection/sham to label colonic visceral afferents. After 1 week of transport, the (L/S) spinal cords were cryosectioned and immunohistochemically stained for CTb, the nociceptive-specific marker calcitonin gene-related peptide (CGRP), and the myelinated fiber marker RT97. Quantitative analysis showed that the density of CGRP+ afferent fibers was significantly increased in the L/S dorsal horns of T4-transected versus sham rats, whereas RT97+ afferent fiber density showed no change. Importantly, CTb-labeled pelvic afferent fibers were co-localized with CGRP+ fibers, but not with RT97+ fibers. These results suggest that the sprouting of unmyelinated nociceptive pelvic afferents following high thoracic SCI, but not myelinated fibers, contributes to hypertensive autonomic dysreflexia induced by pelvic visceral pain.

Effect of Electroacupuncture Stimulation of Hindlimb on Seizure Incidence and Supragranular Mossy Fiber Sprouting in a Rat Model of Epilepsy

Recently, electroacupuncture (EA) has been gaining more and more attention as a treatment for epilepsy. However, concrete evidence is needed to better understand its antiepileptic effect and the mechanism underlying this effect. The present study was designed to assess the effect of EA stimulation of hindlimb on the incidence of behavioral seizures (spontaneous recurrent seizures, [SRS]) and electroencephalogram (EEG) seizures, and the extent of supragranular mossy fiber sprouting (MFS) using the lithium-pilocarpine rat model of epilepsy. Sham EA at the same point without electrical stimulation was set as the control. EA and the sham EA were performed bilaterally (at the symmetrical Zusanli acupoints on both hind legs) 30 times every two days. The numbers of behavioral seizures and EEG seizures were then analyzed to evaluate the antiepileptic effect. After confirmation of the antiepileptic effect, MFS in the dentate gyrus (DG) supragranular layer was investigated by Timm's staining. The results showed that the EA stimulation of hindlimb significantly reduced the behavioral seizures, EEG seizures, and supragranular MFS; however, the sham EA without electrical stimulation showed no significant effect on seizures or supragranular MFS. The findings indicate that EA stimulation of hindlimb possesses an antiepileptic effect, which is probably related to its suppressive effect on aberrant MFS in DG.

Autonomic Fiber Sprouting in the Skin in Chronic Inflammation

Pain is a major symptom associated with chronic inflammation. In previous work from our laboratory, we have shown that in animal models of neuropathic pain there is a sprouting of sympathetic fibers into the upper dermis, a territory normally devoid of them. However, it is not known whether such sympathetic spouting, which is likely trophic factor mediated, also occurs in chronic inflammation and arthritis. In the present study, we used a rat model of chronic inflammation in which a small single dose of complete Freund's adjuvant (CFA) was injected subcutaneously, unilaterally, into the plantar surface of the hindpaw. This led to a localized long-term skin inflammation and arthritis in all joints of the hindpaw. Animals were perfused with histological fixatives at 1, 2, 3 or 4 weeks after the injection. Experimental animals treated with CFA were compared to saline-injected animals. We then investigated the changes in the pattern of peripheral innervation of the peptidergic nociceptors and sympathetic fibers in rat glabrous hindpaw skin. Antibodies directed towards calcitonin gene-related peptide (CGRP) and dopamine beta-hydroxylase (DBH) were used for the staining of peptidergic and sympathetic fibers, respectively. Immunofluorescence was then used to analyze the different nerve fiber populations of the upper dermis. At 4 weeks following CFA treatment, DBH-immunoreactive (IR) fibers were found to sprout into the upper dermis, in a pattern similar to the one we had observed in animals with a chronic constriction injury of the sciatic nerve in a previous publication. There was also a significant increase in the density of CGRP-IR fibers in the upper dermis in CFA treated animals at 2, 3 and 4 weeks post-injection. The increased peptidergic fiber innervation and the ectopic autonomic fibers found in the upper dermis may have a role in the pain-related behavior displayed by these animals.

Prolonged Infusion of Cycloheximide Does Not Block Mossy Fiber Sprouting in a Model of Temporal Lobe Epilepsy

The role of protein synthesis in mossy fiber sprouting is unclear. Conflicting reports exist on whether a single dose of the protein synthesis-blocker cycloheximide administered around the time of an epileptogenic injury can block the eventual development of mossy fiber sprouting. In rats, osmotic minipumps and cannulae were implanted to deliver 8 mg/ml cycloheximide to one dentate gyrus and vehicle to the other. This method has been used to block protein synthesis in the infused region for up to 5 days with minimal neurotoxic effects (Taha and Stryker, Neuron 2002;34:425-36). After 2 days of infusion, rats were treated with pilocarpine to induce status epilepticus. Pumps were removed 3 days later. Thirty days after pilocarpine treatment, rats were perfused, and hippocampal sections were processed for Timm staining. Timm staining revealed aberrant mossy fiber sprouting in the inner molecular layer regardless of whether hippocampi were treated with cycloheximide or vehicle. Cycloheximide-treated hippocampi displayed more aberrant Timm staining and more tissue damage around the infusion site than did vehicle-treated hippocampi. Prolonged infusion of cycloheximide, spanning the period of pilocarpine treatment, did not block mossy fiber sprouting. This finding suggests that protein-dependent mechanisms around the time of an epileptogenic injury are not necessary for the eventual development of synaptic reorganization.

Sympathetic Fiber Sprouting in Chronically Compressed Dorsal Root Ganglia Without Peripheral Axotomy

Sympathetic axonal sprouting in axotomized dorsal root ganglia (DRG) has been shown to be a major phenomenon implicated in neuropathic pain. However, it is not known whether sympathetic sprouting can occur in pathologic ganglia without peripheral axotomy. We thus examined presence and density of sympathetic axonal sprouting within DRG of rats subjected to a persistent compressive injury by inserting a stainless steel metal rod into L(4) and L(5) lumbar intervertebral foramen. Sympathetic axons were identified by immunohistochemical staining with anti-tyrosine hydroxylase antibodies. Results indicate that progressive increase in sympathetic axonal sprouting occurred in the bilateral DRGs between postoperative days 2 and 28. The sympathetic fiber density was greater on the lesion side than the contralateral side. In conclusion, chronic compressive injury of the DRG results in sympathetic sprouting in the non-axotomized ganglion and may partially contribute to the development and maintenance of certain pathological pain states.

Induction of Corneal Lesion and Nerve Fiber Sprouting by Neonatal Capsaicin Application Depends on the Dose of the Drug and Survival Time after Treatment

The effects of capsaicin on the rat cornea and its NsAchE (non specific acetylcholinesterase)-positive nerve fibers were investigated after long and short survival periods following subcutaneous (s.c) injection of the drug. Sixteen rats were injected once s.c with capsaicin on postnatal day 2 at a dose of 50 mg/kg. Age-matched rats were injected only vehicle, and served as control (n = 16). After 4 (n = 6), 8 (n = 6) and 12 (n = 4) mos., both sides of the corneas were examined under a binocular microscope to look for corneal abnormalities. Immediately after the enucleation, bilateral corneas were excised with a thin scleral margin and their ciliary body and iris were removed in DPBS solution. Then, they were fixed in a 4% paraformaldehyde solution containing 8% sucrose for 1 h at 4 degrees C, and processed for staining by the NsAchE method. For comparison, 15 rats were injected 3 times with capsaicin (total dose: 150 mg/kg) at a dose of 50 mg/kg on days 1, 2 and 3 after birth. Age-matched rats (n = 16) were injected vehicle as controls. Five (n = 4), 17 (n = 4), 45 (n = 3) and 75 (n = 4) days later, their corneas were similarly handled as described above. Corneal lesions and sprouting of the NsAchE-positive subepithelial nerve fibers appeared 4 mos. after the treatment with capsaicin (50 mg/kg). In particular, all the treated corneas (8/8) at 12 mos. showed corneal abnormalities. Contrary to the results from the single injection of capsaicin, corneal changes had already appeared on day 17 after treatment with capsaicin at a high dose (150 mg/kg). These data reinforce the suggestion that yielding of corneal lesions is closely associated with the longer survival time at lower doses and with the short survival time at a high dose of the drug. Further, the relationship between sprouting of corneal nerve fibers and corneal wounds was discussed on the basis of the previous reports.

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.