Reassessment of the Effects of Cycloheximide on Mossy Fiber Sprouting and Epileptogenesis in the Pilocarpine Model of Temporal Lobe Epilepsy

A feature of animal models of temporal lobe epilepsy and the human disorder is hippocampal sclerosis and Timm stain in the inner molecular layer (IML) of the dentate gyrus, which represents synaptic reorganization and may be important in epileptogenesis. We reassessed the hypothesis that pre-treatment with cycloheximide (CHX) prevents Timm staining in the IML following pilocarpine (PILO)-induced status epilepticus (a multifocal model of temporal lobe epilepsy), but allows epileptogenesis (i.e., chronic spontaneous seizures) after a latent period. Hippocampal slices from PILO-treated rats without Timm stain in the IML after CHX treatment were hypothesized to lack the electrophysiological abnormalities suggestive of recurrent excitation. The primary experimental groups were as follows: 1) CHX (1 mg/kg) 30–45 min prior to administration of PILO (320 mg/kg ip, 2) only PILO, and 3) only saline (0.5 ml, IP). The CHX pre-treatment significantly decreased the number of rats that responded to PILO with status epilepticus compared to rats that received only PILO. Pre-treatment with CHX did not significantly alter the spontaneous motor seizure rate post-treatment compared to treatment with PILO alone in those animals from each group that developed status epilepticus during PILO treatment. Timm stain in the IML was not significantly different between the PILO- and PILO+CHX-treated rats. Using quantitative methods, CHX did not prevent hilar, CA1, or CA3 neuronal loss compared to the PILO-treated rats. Extracellular responses to hilar stimulation in 30 μM bicuculline and 6 mM [K+]o demonstrated all-or-none bursting in both the CHX+PILO- and PILO-treated rats but not in control rats. Whole cell recordings from granule cells, using glutamate flash photolysis to activate other granule cells, showed that both the CHX+PILO- and PILO-treated rats had excitatory synaptic interactions in the granule cell layer, which were not found after saline treatment. Some rats responded to PILO (with or without CHX pre-treatment) with only one or a few seizures at treatment, and some of these animals ( n = 4) demonstrated spontaneous motor seizures within 2 mo after treatment. Timm staining and neuron loss in this group were not clearly different from saline-treated rats. These results suggest that in the PILO model, pre-treatment with CHX does not affect mossy fiber sprouting in the IML of epileptic rats and does not prevent the formation of recurrent excitatory circuits. However, the develoment of spontaneous motor seizures, in a small number of rats, could occur without detectable hippocampal neuron loss or mossy fiber sprouting, as assessed by the Timm stain method.

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Regionally Localized Recurrent Excitation in the Dentate Gyrus of a Cortical Contusion Model of Posttraumatic Epilepsy

Journal of Neurophysiology ◽

10.1152/jn.00957.2009 ◽

2010 ◽

Vol 103 (3) ◽

pp. 1490-1500 ◽

Cited By ~ 46

Author(s):

Robert F. Hunt ◽

Stephen W. Scheff ◽

Bret N. Smith

Keyword(s):

Head Injury ◽

Dentate Gyrus ◽

Action Potentials ◽

Granule Cells ◽

Mossy Fiber ◽

Granule Cell Layer ◽

Mossy Fiber Sprouting ◽

Posttraumatic Epilepsy ◽

Dentate Granule Cells ◽

Cortical Contusion

Posttraumatic epilepsy is a frequent consequence of brain trauma, but relatively little is known about how neuronal circuits are chronically altered after closed head injury. We examined whether local recurrent excitatory synaptic connections form between dentate granule cells in mice 8–12 wk after cortical contusion injury. Mice were monitored for behavioral seizures shortly after brain injury and ≤10 wk postinjury. Injury-induced seizures were observed in 15% of mice, and spontaneous seizures were observed weeks later in 40% of mice. Timm's staining revealed mossy fiber sprouting into the inner molecular layer of the dorsal dentate gyrus ipsilateral to the injury in 95% of mice but not contralateral to the injury or in uninjured controls. Whole cell patch-clamp recordings were made from granule cells in isolated hippocampal brain slices. Cells in slices with posttraumatic mossy fiber sprouting had an increased excitatory postsynaptic current (EPSC) frequency compared with cells in slices without sprouting from injured and control animals ( P < 0.001). When perfused with Mg2+-free artificial cerebrospinal fluid containing 100 μM picrotoxin, these cells had spontaneous bursts of EPSCs and action potentials. Focal glutamate photostimulation of the granule cell layer evoked a burst of EPSCs and action potentials indicative of recurrent excitatory connections in granule cells of slices with mossy fiber sprouting. In granule cells of slices without sprouting from injured animals and controls, spontaneous or photostimulation-evoked epileptiform activity was never observed. These results suggest that a new regionally localized excitatory network forms between dentate granule cells near the injury site within weeks after cortical contusion head injury.

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Status Epilepticus-Induced Neurogenesis and Neuron Loss in the Kainate Model of Temporal Lobe Epilepsy

Journal of the Japan Epilepsy Society ◽

10.3805/jjes.21.157 ◽

2003 ◽

Vol 21 (2) ◽

pp. 157-166

Author(s):

Takashi Ninomiya ◽

Kiyoshi Morimoto ◽

Takemi Watanabe ◽

Tohru Hirao ◽

Osamu Yasuhara ◽

...

Keyword(s):

Status Epilepticus ◽

Temporal Lobe Epilepsy ◽

Temporal Lobe ◽

Neuron Loss

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Spontaneous Excitatory Currents and κ-Opioid Receptor Inhibition in Dentate Gyrus Are Increased in the Rat Pilocarpine Model of Temporal Lobe Epilepsy

Journal of Neurophysiology ◽

10.1152/jn.1997.78.4.1860 ◽

1997 ◽

Vol 78 (4) ◽

pp. 1860-1868 ◽

Cited By ~ 22

Author(s):

Michele L. Simmons ◽

Gregory W. Terman ◽

Charles Chavkin

Keyword(s):

Temporal Lobe Epilepsy ◽

Dentate Gyrus ◽

Opioid Receptor ◽

Temporal Lobe ◽

Mossy Fiber ◽

Perforant Path ◽

Mossy Fiber Sprouting ◽

Receptor Inhibition ◽

Excitatory Activity ◽

Pilocarpine Model

Simmons, Michele L., Gregory W. Terman, and Charles Chavkin. Spontaneous excitatory currents and κ-opioid receptor inhibition in dentate gyrus are increased in the rat pilocarpine model of temporal lobe epilepsy. J. Neurophysiol. 78: 1860–1868, 1997. Temporal lobe epilepsy is associated with a characteristic pattern of synaptic reorganization in the hippocampal formation, consisting of neuronal loss and aberrant growth of mossy fiber collaterals into the dentate gyrus inner molecular layer. We have used the rat pilocarpine model of temporal lobe epilepsy to study the functional consequences of mossy fiber sprouting on excitatory activity and κ-opioid receptor-mediated inhibition. Using the whole cell voltage-clamp technique, we found that abnormal excitatory activity was evident in granule cells of the dentate gyrus from pilocarpine-treated rats. The frequency of spontaneous excitatory postsynaptic currents (EPSCs) was increased greatly in cells from tissue in which significant mossy fiber sprouting had developed. In the presence of bicuculline, giant spontaneous EPSCs, with large amplitudes and long durations, were seen only in association with mossy fiber sprouting. Giant EPSCs also could be evoked by low-intensity stimulation of the perforant path. Mossy fibers release not only excitatory amino acids, but also opioid peptides. κ-Opioid receptor-mediated inhibition in normal Sprague-Dawley rats was seen only in hippocampal sections from the ventral pole. In pilocarpine-treated rats, however, kappa receptor-mediated effects were seen in both ventral and more dorsal sections. Thus in this model of temporal lobe epilepsy, several types of abnormal excitatory activity were observed, thereby supporting the idea that mossy fiber sprouting leads to recurrent excitatory connections. At the same time, inhibition of excitatory activity by κ-opioid receptors was increased, perhaps representing an endogenous anticonvulsant mechanism.

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Optical Recording Study of Granule Cell Activities in the Hippocampal Dentate Gyrus of Kainate-Treated Rats

Journal of Neurophysiology ◽

10.1152/jn.2000.83.4.2421 ◽

2000 ◽

Vol 83 (4) ◽

pp. 2421-2430 ◽

Cited By ~ 12

Author(s):

Yo Otsu ◽

Eiichi Maru ◽

Hisayuki Ohata ◽

Ichiro Takashima ◽

Riichi Kajiwara ◽

...

Keyword(s):

Dentate Gyrus ◽

Granule Cell ◽

Granule Cells ◽

Mossy Fiber ◽

Molecular Layer ◽

Mossy Fibers ◽

Optical Recording ◽

Granule Cell Layer ◽

Gabaergic Inhibition ◽

Mossy Fiber Sprouting

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca2+. The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizurelike activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.

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Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy

Hippocampus ◽

10.1002/hipo.22543 ◽

2015 ◽

Vol 26 (5) ◽

pp. 577-588 ◽

Cited By ~ 21

Author(s):

Ute Häussler ◽

Katrin Rinas ◽

Antje Kilias ◽

Ulrich Egert ◽

Carola A. Haas

Keyword(s):

Mouse Model ◽

Temporal Lobe Epilepsy ◽

Temporal Lobe ◽

Pyramidal Cell ◽

Mossy Fiber ◽

Mossy Fiber Sprouting ◽

Cell Dispersion

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

Epilepsia ◽

10.1111/j.1528-1167.2005.04605.x ◽

2005 ◽

Vol 46 (7) ◽

pp. 1017-1020 ◽

Cited By ~ 13

Author(s):

Izumi Toyoda ◽

Paul S. Buckmaster

Keyword(s):

Temporal Lobe Epilepsy ◽

Temporal Lobe ◽

Mossy Fiber ◽

Prolonged Infusion ◽

Mossy Fiber Sprouting

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Transient downregulation of sema3a mrna in a rat model for temporal lobe epilepsy A novel molecular event potentially contributing to mossy fiber sprouting

Experimental Neurology ◽

10.1016/s0014-4886(03)00035-9 ◽

2003 ◽

Vol 182 (1) ◽

pp. 142-150 ◽

Cited By ~ 59

Author(s):

A Holtmaat

Keyword(s):

Temporal Lobe Epilepsy ◽

Temporal Lobe ◽

Rat Model ◽

Mossy Fiber ◽

Molecular Event ◽

Mossy Fiber Sprouting

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Intrinsic neurophysiological properties of hilar ectopic and normotopic dentate granule cells in human temporal lobe epilepsy and a rat model

Journal of Neurophysiology ◽

10.1152/jn.00835.2014 ◽

2015 ◽

Vol 113 (4) ◽

pp. 1184-1194 ◽

Cited By ~ 25

Author(s):

A. L. Althaus ◽

O. Sagher ◽

J. M. Parent ◽

G. G. Murphy

Keyword(s):

Temporal Lobe Epilepsy ◽

Temporal Lobe ◽

Granule Cells ◽

Salient Feature ◽

Granule Cell Layer ◽

Disease Stage ◽

Resting Membrane Potential ◽

Firing Rates ◽

Dentate Granule Cells ◽

Human Temporal Lobe

Hilar ectopic dentate granule cells (DGCs) are a salient feature of aberrant plasticity in human temporal lobe epilepsy (TLE) and most rodent models of the disease. Recent evidence from rodent TLE models suggests that hilar ectopic DGCs contribute to hyperexcitability within the epileptic hippocampal network. Here we investigate the intrinsic excitability of DGCs from humans with TLE and the rat pilocarpine TLE model with the objective of comparing the neurophysiology of hilar ectopic DGCs to their normotopic counterparts in the granule cell layer (GCL). We recorded from 36 GCL and 7 hilar DGCs from human TLE tissue. Compared with GCL DGCs, hilar DGCs in patient tissue exhibited lower action potential (AP) firing rates, more depolarized AP threshold, and differed in single AP waveform, consistent with an overall decrease in excitability. To evaluate the intrinsic neurophysiology of hilar ectopic DGCs, we made recordings from retrovirus-birthdated, adult-born DGCs 2–4 mo after pilocarpine-induced status epilepticus or sham treatment in rats. Hilar DGCs from epileptic rats exhibited higher AP firing rates than normotopic DGCs from epileptic or control animals. They also displayed more depolarized resting membrane potential and wider AP waveforms, indicating an overall increase in excitability. The contrasting findings between disease and disease model may reflect differences between the late-stage disease tissue available from human surgical specimens and the earlier disease stage examined in the rat TLE model. These data represent the first neurophysiological characterization of ectopic DGCs from human hippocampus and prospectively birthdated ectopic DGCs in a rodent TLE model.

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