Prolonged infusion of cycloheximide does not block mossy fiber sprouting in a model of temporal lobe epilepsy

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Prolonged infusion of inhibitors of calcineurin or L-type calcium channels does not block mossy fiber sprouting in a model of temporal lobe epilepsy

Purpose:   It would be useful to selectively block granule cell axon (mossy fiber) sprouting to test its functional role in temporal lobe epileptogenesis. Targeting axonal growth cones may be an effective strategy to block mossy fiber sprouting. L-type calcium channels and calcineurin, a calcium-activated phosphatase, are critical for normal growth cone function. Previous studies have provided encouraging evidence that blocking L-type calcium channels or inhibiting calcineurin during epileptogenic treatments suppresses mossy fiber sprouting.
Methods:   Rats were treated systemically with pilocarpine to induce status epilepticus, which lasted at least 2 h. Then, osmotic pumps and cannulae were implanted to infuse calcineurin inhibitors (FK506 or cyclosporin A) or an L-type calcium channel blocker (nicardipine) into the dorsal dentate gyrus. After 28 days of continuous infusion, extent of mossy fiber sprouting was evaluated with Timm staining and stereological methods.
Results:   Percentages of volumes of the granule cell layer plus molecular layer that were Timm-positive were similar in infused and noninfused hippocampi.
Conclusions:   These findings suggest inhibiting calcineurin or L-type calcium channels does not block mossy fiber sprouting in the pilocarpine-treated rat model of temporal lobe epilepsy.     

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.     

颞叶癫痈大鼠海马内Rnd1 mRNA及蛋白表达变化

目的：动态观察小分子GTPase Rho家族的Rnd1 mRNA及其蛋白在氯化锂-毛果芸香碱（匹罗卡品）致痫大鼠模型海马中的表达变化，探讨其在颞叶癫痫发生发展中的作用。方法：在氯化锂-毛果芸香碱颞叶癫痫模型中应用逆转录聚合酶链反应（RT—PCR）检测癫痫持续发作（SE）后各时间点海马内Rnd1 mRNA的表达变化，并运用免疫组织化学染色法及Neo—Timm染色法分别检测齿状回门区、CA1区及CA3区中该蛋白在不同时间点的表达变化及苔藓纤维出芽（MFS）情况。结果：实验发现模型组于SE后8h内即出现Rnd1表达上调，SE后约1d达高峰，7d左右回复至对照组水平，此后其mRNA表达水平与对照组相似；而免疫组化染色发现Rnd1蛋白表达从SE后8h内即开始上调，约3d达高峰，至7d虽略有回落，但仍高于对照组水平，且这种情况可一直持续至慢性期。结论：急性期海马齿状回门区Rnd1表达上调可能通过促进MFS的发生参与了颞叶癫痫的发生。     

Locus Coeruleus and Neuronal Plasticity in a Model of Focal Limbic Epilepsy

Summary:  Purpose: A lesion of the noradrenergic nucleus Locus Coeruleus (LC) converts sporadic seizures evoked by microinfusion of bicuculline into the anterior piriform cortex (APC) of rats into limbic status epilepticus (SE). The purpose of this study was to evaluate the chronic effects of this new model of SE on the onset of secondary epileptogenesis. We further related the loss of noradrenaline (NE) with hippocampal mossy fiber sprouting.
Methods: Male Sprague Dawley rats were treated with systemic saline or DSP-4 (a neurotoxin selective for noradrenergic terminals originating from the LC), microinfused with bicuculline into the APC three days later, and sacrificed after 45 days. Naïve and DSP-4 pretreated sham-operated rats served as respective controls. The following evaluations were performed: (a) monitoring of acute seizures and delayed occurrence of spontaneous recurrent seizures (SRS); (b) NE levels in the hippocampus, frontal and olfactory cortex; (c) occurrence of mossy fiber sprouting into the inner molecular layer of the dentate gyrus of the dorsal hippocampus.
Results: In 30% of rats lacking noradrenergic terminals, SE evoked from the APC was followed by SRS. Conversely, seizures evoked in intact rats did not result in chronic epileptogenesis. Seizures/SE did not modify NE levels as compared with baseline levels both in naïve and DSP-4-pretreated rats. Rats undergoing SE following DSP-4 + bicuculline developed SRS which were accompanied by hippocampal mossy fiber sprouting.
Conclusions: Noradrenergic loss converts focally induced sporadic seizures into an epileptogenic SE, which is accompanied by mossy fiber sprouting within the dentate gyrus.     

Dissociation of mossy fiber sprouting and electrically-induced seizure sensitivity: rapid kindling versus adaptation

It has been shown that massed stimulation (MS) of the amygdala or hippocampus does not result in seizure progression but in the 'phenomenon of adaptation', whereas alternate day rapid kindling (ADRK) produces reliable kindling (Lothman, E.W., Williamson, J.M., 1994. Brain Res. 649, 71-84). The goal of the present experiment was to determine if the two different effects are due to differences in mossy fiber sprouting and/or different seizure and postictal spike propagation patterns. Nine rats underwent MS (66-70 stimulations separated by 5-min interstimulus interval), six were exposed to ADRK (12 stimulations/day, every 30 min, with 4 stimulus days, each separated by 1 stimulus-free day), five rats served as control. All rats had electrodes implanted bilaterally in dorsal and ventral hippocampi (VH) and 14 of them had additional electrodes in the piriform cortex. Animals were stimulated in the left VH at afterdischarge threshold. There was no potentiation in seizure response 4-7 weeks after MS. In contrast, ADRK produced not only kindling but also ongoing epileptogenesis resulting 4-7 weeks later in spontaneous seizures and development of a prolonged convulsive state in response to the initially subconvulsive stimulus. Epileptiform activity during MS was mostly restricted to VH, whereas during ADRK it spread widely among studied structures including piriform cortex. Afterdischarges during MS were elicited frequently but seizures did not progress beyond stage 2-3. During ADRK, afterdischarges were evoked less frequently but seizures reached stage 4-7 by the end of the 3rd and 4th stimulus days. The fully kindled state was not reached at this time, but epileptogenic changes continued to progress. Seven weeks after the initial stimulation, both groups demonstrated mossy fiber sprouting of similar intensity in VH. We suggest, (1) frequent but predominantly local hippocampal afterdischarges induce mossy fiber sprouting, but this is not sufficient to produce significant enhancement in seizure susceptibility, and (2) the involvement of extra-hippocampal structures, possibly piriform cortex, and formation of an aberrant hippocampal-para-hippocampal circuit is required to result in a condition of progressive epileptogenesis.     

Effectiveness of muscimol-containing microparticles against pilocarpine-induced focal seizures

PURPOSE: To investigate the efficacy of in situ lipid-protein-sugar particles (LPSPs) in mitigating the epileptogenic and histologic effects of intrahippocampal pilocarpine in rats. METHODS: LPSPs with and without muscimol were produced by spray-drying, sized by Coulter counter, and muscimol content determined by high-pressure liquid chromatography (HLPC). Particles, free muscimol or saline, were injected into the hippocampi of Sprague-Dawley rats before 40 mM pilocarpine, and seizure activity was scored. The trajectories of behavioral scores between groups were compared with two-way repeated measures analysis of variance. Animals were killed after 2 weeks. Brain sections were stained (Timm and thionin) and scored. RESULTS: LPSPs were 4 to 5 microm in diameter, and contained 0 or 2% (wt/wt) muscimol. In vitro, muscimol was released over a 5-day period. Intrahippocampal injections of normal saline and blank LPSPs did not deter seizure activity from pilocarpine. The rise of the trajectory in behavior scores in animals given LPSPs containing 5 microg muscimol was significantly slower than in those receiving saline, blank particles, or 5 microg of unencapsulated muscimol. There was less apparent neuronal injury and CA3 and supragranular mossy fiber sprouting in hippocampi of animals receiving muscimol-containing particles than in animals that did not receive muscimol. Hippocampi of animals that received 5 microg of encapsulated muscimol showed less supragranular sprouting than did those receiving 5 microg of unencapsulated muscimol, but showed no difference in cell loss or CA3 sprouting. CONCLUSIONS: Focally delivered biodegradable microparticles loaded with muscimol are effective in reducing seizure activity from pilocarpine in animals and mitigate the histologic effects.     

Effects of vigabatrin treatment on status epilepticus-induced neuronal damage and mossy fiber sprouting in the rat hippocampus

Selective neuronal damage and mossy fiber sprouting may underlie epileptogenesis and spontaneous seizure generation in the epileptic hippocampus. It may be beneficial to prevent their development after cerebral insults that are known to be associated with a high risk of epilepsy later in life in humans. In the present study, we investigated whether chronic treatment with an anticonvulsant, vigabatrin (gamma-vinyl GABA), would prevent the damage to hilar neurons and the development of mossy fiber sprouting. Vigabatrin treatment was started either 1 h, or 2 or 7 days after the beginning of kainic acid-induced (9 mg/kg, i.p.) status epilepticus and continued via subcutaneous osmotic minipumps for 2 months (75 mg/kg per day). Thereafter, rats were perfused for histological analyses. One series of horizontal sections was stained with thionine to estimate the total number of hilar neurons by unbiased stereology. One series was prepared for somatostatin immunohistochemistry and another for Timm histochemistry to detect mossy fiber sprouting. Our data show that vigabatrin treatment did not prevent the decrease in the total number of hilar cells, nor the decrease in hilar somatostatin-immunoreactive (SOM-ir) neurons when SOM-ir neuronal numbers were averaged from all septotemporal levels. However, when vigabatrin was administered 2 days after the onset of status epilepticus, we found a mild neuroprotective effect on SOM-ir neurons in the septal end of the hippocampus (92% SOM-ir neurons remaining; P < 0.05 compared to the vehicle group). Vigabatrin did not prevent mossy fiber sprouting regardless of when treatment was started. Rather, sprouting actually increased in the septal end of the hippocampus when vigabatrin treatment began 1 h after the onset of status epilepticus (P < 0.05 compared to the vehicle group). Our data show that chronic elevation of brain GABA levels after status epilepticus does not have any substantial effects on neuronal loss or mossy fiber sprouting in the rat hippocampus.     

Cell damage and neurogenesis in the dentate granule cell layer of adult rats after pilocarpine- or kainate-induced status epilepticus

Dentate granule cells are generally considered to be relatively resistant to excitotoxicity and have been associated with robust synaptogenesis after neuronal damage. Synaptic reorganization of dentate granule cell axons, the mossy fibers, has been suggested to be relevant for hyperexcitability in human temporal lobe epilepsy and animal models. A recent hypothesis suggested that mossy-fiber sprouting is dependent on newly formed dentate granule cells. However, we recently demonstrated that cycloheximide (CHX) can block the mossy-fiber sprouting that would otherwise be induced by different epileptogenic agents and does not interfere with epileptogenesis in those models. Here, we investigated cell damage and neurogenesis in the dentate gyrus of pilocarpine- or kainate-treated animals with or without coadministration of CHX. Dentate granule cells were highly vulnerable to pilocarpine induced-status epilepticus (SE), but were hardly damaged by kainate-induced SE. CHX pretreatment markedly reduced the number of injured neurons after pilocarpine-induced SE. Induction of SE dramatically increased the mitotic rate of KA- and KA + CHX-treated animals. Induction of SE in animals injected with pilocarpine alone led to 2-7-fold increases in the mitotic rate of dentate granule cells as compared to 5- and 30-fold increases for pilocarpine + CHX animals. We suggest that such increased mitotic rates might be associated with a protection of a vulnerable precursor cell population that would otherwise degenerate after pilocarpine-induced SE. We further suggest that mossy-fiber sprouting and neurogenesis of granule cells are not necessarily linked to one another.     

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.     

Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers.

Morphological data from humans with temporal lobe epilepsy and from animal models of epilepsy suggest that seizure-induced damage to dentate hilar neurons causes granule cells to sprout new axon collaterals that innervate other granule cells. This aberrant projection has been suggested to be an anatomical substrate for epileptogenesis. This hypothesis was tested in the present study with intra- and extracellular recordings from granule cells in hippocampal slices removed from rats 1-4 months after kainate treatment. In this animal model, hippocampal cell loss leads to sprouting of mossy fiber axons from the granule cells into the inner molecular layer of the dentate gyrus. Unexpectedly, when slices with mossy fiber sprouting were examined in normal medium, extracellular stimulation of the hilus or perforant path evoked relatively normal responses. However, in the presence of the GABAA-receptor antagonist, bicuculline, low-intensity hilar stimulation evoked delayed bursts of action potentials in about one-quarter of the slices. In one-third of the bicuculline-treated slices with mossy fiber sprouting, spontaneous bursts of synchronous spikes were superimposed on slow negative field potentials. Slices from normal rats or kainate-treated rats without mossy fiber sprouting never showed delayed bursts to weak hilar stimulation or spontaneous bursts in bicuculline. These data suggest that new local excitatory circuits may be suppressed normally, and then emerge functionally when synaptic inhibition is blocked. Therefore, after repeated seizures and excitotoxic damage in the hippocampus, synaptic reorganization of the mossy fibers is consistently associated with normal responses; however, in some preparations, the mossy fibers may form functional recurrent excitatory connections, but synaptic inhibition appears to mask these potentially epileptogenic alterations.     

Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.     

Surviving mossy cells enlarge and receive more excitatory synaptic input in a mouse model of temporal lobe epilepsy

Numerous hypotheses of temporal lobe epileptogenesis have been proposed, and several involve hippocampal mossy cells. Building on previous hypotheses we sought to test the possibility that after epileptogenic injuries surviving mossy cells develop into super‐connected seizure‐generating hub cells. If so, they might require more cellular machinery and consequently have larger somata, elongate their dendrites to receive more synaptic input, and display higher frequencies of miniature excitatory synaptic currents (mEPSCs). To test these possibilities pilocarpine‐treated mice were evaluated using GluR2‐immunocytochemistry, whole‐cell recording, and biocytin‐labeling. Epileptic pilocarpine‐treated mice displayed substantial loss of GluR2‐positive hilar neurons. Somata of surviving neurons were 1.4‐times larger than in controls. Biocytin‐labeled mossy cells also were larger in epileptic mice, but dendritic length per cell was not significantly different. The average frequency of mEPSCs of mossy cells recorded in the presence of tetrodotoxin and bicuculline was 3.2‐times higher in epileptic pilocarpine‐treated mice as compared to controls. Other parameters of mEPSCs were similar in both groups. Average input resistance of mossy cells in epileptic mice was reduced to 63% of controls, which is consistent with larger somata and would tend to make surviving mossy cells less excitable. Other intrinsic physiological characteristics examined were similar in both groups. Increased excitatory synaptic input is consistent with the hypothesis that surviving mossy cells develop into aberrantly super‐connected seizure‐generating hub cells, and soma hypertrophy is indirectly consistent with the possibility of axon sprouting. However, no obvious evidence of hyperexcitable intrinsic physiology was found. Furthermore, similar hypertrophy and hyper‐connectivity has been reported for other neuron types in the dentate gyrus, suggesting mossy cells are not unique in this regard. Thus, findings of the present study reveal epilepsy‐related changes in mossy cell anatomy and synaptic input but do not strongly support the hypothesis that mossy cells develop into seizure‐generating hub cells. © 2014 Wiley Periodicals, Inc.     

Effects of early-life LiCl-pilocarpine-induced status epilepticus on memory and anxiety in adult rats are associated with mossy fiber sprouting and elevated CSF S100B protein

Purpose: This study investigated putative correlations among behavioral changes and: (1) neuronal loss, (2) hippocampal mossy fiber sprouting, and (3) reactive astrogliosis in adult rats submitted to early‐life LiCl‐pilocarpine‐induced status epilepticus (SE). Methods: Rats (P15) received LiCl (3 mEq/kg, i.p.) 12–18 h prior pilocarpine (60 mg/kg; s.c.). At adulthood, animals were submitted to behavioral tasks and after the completion of tasks biochemical and histological analysis were performed. Results: In SE group, it was observed an increased number of degenerating neurons in the CA1 subfield and in the hilus of animals 24 h after SE. At adulthood, SE group presented an aversive memory deficit in an inhibitory avoidance task and the animals that presented lower latency to the step down showed a higher score for mossy fiber sprouting. In the light‐dark exploration task, SE rats returned less and spent less time in the light compartment and present an increased number of risk assessment behavior (RA). There was a negative correlation between the time spent in the light compartment and the score for mossy fiber sprouting and a positive correlation between score for mossy fiber sprouting and number of RA. LiCl‐pilocarpine‐treated animals showed higher levels of S100B immunocontent in the CSF as well as a positive correlation between the score for sprouting and the GFAP immunocontent in the CA1 subfield, suggesting an astrocytic response to neuronal injury. Conclusions: We showed that LiCl‐pilocarpine‐induced SE during development produced long‐lasting behavioral abnormalities, which might be associated with mossy fiber sprouting and elevated CSF S100B levels at adulthood.     

Kainic acid induction of mossy fiber sprouting: dependence on mouse strain

After seizures caused by kindling or kainic acid (KA), hippocampal granule-cell axons, the mossy fibers, sprout into the supragranular layer of the rat. The mechanisms underlying this phenomenon remain elusive, but excitotoxic loss of hilar cells, which project to this supragranular layer, is suspected to be a critical determinant. Consistent with this hypothesis, we previously reported that while rats show mossy fiber sprouting after kainate, ICR mice do not. This may be associated with the observation that ICR mice, unlike rats, do not appear to show hilar cell death after KA (McNamara et al., Mol Brain Res 1996;40:177-187). Other strains of mice, however, such as 129/SvEMS, do show hilar cell death after KA (Schauwecker and Steward, Proc Natl Acad Sci USA 1997;94:4103-4108). We examined the possibility that the 129/SvEMS mouse strain would show granule-cell sprouting, in contrast to ICR mice. After administration of KA, mossy fiber sprouting was indeed observed in strain 129/SvEMS, but only in animals displaying evident hilar cell death. In contrast, neither hilar cell death nor mossy fiber sprouting was observed in ICR mice, confirming previous results. Both mouse strains demonstrated comparable behavioral seizures. These results strengthen the view that hilar cell death, together with epileptogenesis, triggers reactive synaptogenesis and mossy fiber sprouting.     

The Role of Mossy Cell Death and Activation of Protein Synthesis in the Sprouting of Dentate Mossy Fibers: Evidence from Calretinin and Neo-Timm Staining in Pilocarpine-Epileptic Mice

Summary: Mossy fiber sprouting is a major anatomical reorganization seen in patients with temporal lobe epilepsy and animal models of epilepsy. The final outcome of this reorganization is viewed by many as epileptogenic. Yet, important and relevant data from both human and animal models of epilepsy challenge this prevailing view. Regardless of the outcome of this debate, understanding of the mechanisms that underlie mossy fiber sprouting (MFS) might contribute to our understanding of both the adaptive and maladaptive changes that take place in the nervous system after injury. Available evidence suggests that two events might be crucial for mossy fibers to sprout in epilepsy: the death of mossy cells and the synthesis of trophic factors. The availability of means that prevent MFS, which is normally triggered after induction of status epilepticus, allow for the testing of hypotheses regarding the need for and the sufficiency of specific events for mossy fibers to sprout. We present data on a specific marker for mossy cells, calretinin, in the pilocarpine model of epilepsy in mice. Our data suggest that in the presence of a protein synthesis inhibitor status epilepticus—induced death of mossy cells is not sufficient to trigger mossy fiber sprouting. We suggest that both events, mossy cell death and synthesis of trophic factors, might be necessary for robust MFS to ensue.     

Blockade of excitatory synaptogenesis with proximal dendrites of dentate granule cells following rapamycin treatment in a mouse model of temporal lobe epilepsy

Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine‐induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive‐feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate the numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine‐treated control mice, in mice 5 days after pilocarpine‐induced status epilepticus, and after status epilepticus and daily treatment beginning 24 hours later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl‐stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2,280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 days after status epilepticus, recovered to 93% of controls in vehicle‐treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin‐treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy. J. Comp. Neurol. 523:281–297, 2015. © 2014 Wiley Periodicals, Inc.     

Alterations of Neuronal Connectivity in Area CA1 of Hippocampal Slices from Temporal Lobe Epilepsy Patients and from Pilocarpine-Treated Epileptic Rats

Summary: Purpose : Neuronal network reorganization might be involved in epileptogenesis in human and rat limbic epilepsy. Apart from aberrant mossy fiber sprouting, a more wide-spread fiber rearrangement in the hippocampal formation might occur. Therefore, we studied sprouting in area CA1 because this region is most affected in human temporal lobe epilepsy.
Methods : In slices from hippocampi of patients operated on for temporal lobe epilepsy (n = 134), from pilocarpine-treated rats (n = 74), and from control rats (n = 15), viable neurons were labeled with fluorescent dextran amines.
Results : In human hippocampi as well as in pilocarpine-treated rats, the degree of nerve cell loss varied. In 67 of 134 slices from human specimens with distinct Ammon's horn sclerosis and in 23 of 74 slices from pilocarpine-treated rats, a severe shrunken area CA1 presented with a similar picture: few damaged neurons were labeled, and aberrant fiber connections were not visible. This was in contrast to human resected hippocampi and hippocampi from pilocarpine-treated rats with no or moderate loss of neurons. In these cases, pyramidal cells remote from the injection site were labeled (human tissue, n = 59 of 134; pilocarpine-treated rats, n = 39 of 74). In human resected hippocampi without obvious pathology and in control animals, no pyramidal neurons were labeled apart from the injection site.
Conclusions : Axon collaterals of CA1 pyramidal cells are increased in human temporal lobe epilepsy and in pilocarpine-treated rats. Adjacent CA1 pyramidal cells project via aberrant collaterals to the stratum pyramidale and the stratum radiatum of area CA1. This network reorganization can contribute to hyperexcitability via increased backward excitation.     

Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice

Several rodent models are available to study the cellular mechanisms associated with the development of temporal lobe epilepsy (TLE), but few have been successfully transferred to inbred mouse strains commonly used in genetic mutation studies. We examined spontaneous seizure development and correlative axon sprouting in the dentate gyrus of CD-1 and C57BL/6 mice after systemic injection of pilocarpine. Pilocarpine induced seizures and status epilepticus (SE) after systemic injection in both strains, although SE onset latency was greater for C57BL/6 mice. There were also animals of both strains which did not experience SE after pilocarpine treatment. After a period of normal behavior for several days after the pilocarpine treatment, spontaneous tonic-clonic seizures were observed in most CD-1 mice and all C57BL/6 that survived pilocarpine-induced SE. Robust mossy fiber sprouting into the inner molecular layer was observed after 4-8 weeks in mice from both strains which had experienced SE, and cell loss was apparent in the hippocampus. Mossy fiber sprouting and spontaneous seizures were not observed in mice that did not experience a period of SE. These results indicate that pilocarpine induces spontaneous seizures and mossy fiber sprouting in both CD-1 and C57BL/6 mouse strains. Unlike systemic kainic acid treatment, the pilocarpine model offers a potentially useful tool for studying TLE development in genetically modified mice raised on the C57BL/6 background.     

Castration in female rats modifies the development of the pilocarpine model of epilepsy

Previous studies have shown that the susceptibility to pilocarpine-induced status epilepticus (SE) in female rats changes according to estrous cycle phases. These studies have also shown that following pilocarpine administration changes occur in gonadal, hypophyseal and hypothalamic hormones that could contribute for the sequence of the epileptic events. Accordingly, the present work aimed to investigate the role of sexual hormones withdrawal on the development of the pilocarpine model of epilepsy in female rats. With this purpose, castrated and non-castrated adult female Wistar rats were injected with pilocarpine and some characteristic parameters of the experimental model were observed. The results showed increased mortality after pilocarpine injection in the castrated rats when compared with non-castrated females. The latency period for SE onset and for the first spontaneous seizure was decreased in castrated when compared with non-castrated animals. The mossy fiber sprouting measured by neo-Timm scale during the chronic period, reached grade 3 for castrated epileptic rats while the non-castrated epileptic rats showed grade 2. Our results indicate that castration interferes with the epileptogenesis in the pilocarpine model of epilepsy suggesting that female sexual hormones could have protective effects against pilocarpine-induced SE.