Pinniped electroencephalography: Methodology and findings in California sea lions (Zalophus californianus).

This study was designed to identify abnormalities in the electroencephalograms (EEGs) recorded from stranded California sea lions (Zalophus californianus) with suspected domoic acid (DA) toxicosis. Recordings from animals presenting for non-neurological issues were also obtained to better understand the normal EEG (background activity and transient events) in this species, as, to date, studies have focused on examining natural sleep in pinnipeds. Most animals were sedated for electrode placement and EEG acquisition with some receiving antiepileptic medications or isoflurane during the procedure. A total of 103 recordings were read and scored from 0 (normal) to 3 (severely abnormal). Epileptiform discharges, consisting of spikes, sharp waves, slow waves, and/or spike waves, were present in all EEGs with scores of 1, 2, or 3. The distribution of these events over the scalp varied. While often generalized, others were lateralized over one hemisphere, bifrontal, bioccipital, and/or bitemporal, while some discharges were multifocal. Findings were different between sea lions and occasionally changed within the EEG on a given sea lion. No clinical seizures were observed during the recording but a few sea lions had findings consistent with electroencephalographic seizures. When available, supporting diagnostic results obtained from magnetic resonance imaging (MRI) and/or necropsy/histopathology were described, as well as the status of those sea lions that recovered and were released with satellite tags.

Ventral hippocampal formation is the primary epileptogenic zone in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy is common, but mechanisms of seizure initiation are unclear. We evaluated seizure initiation in female and male rats that had been systemically treated with pilocarpine, a widely used model of temporal lobe epilepsy. Local field potential recordings from many brain regions revealed variable sites of earliest recorded seizure activity, but mostly the ventral hippocampal formation. To test whether inactivation of the ventral hippocampal formation would reduce seizures, mini-osmotic pumps were used to continually and focally deliver tetrodotoxin. High doses of tetrodotoxin infused unilaterally into the ventral hippocampal formation blocked seizures reversibly but also reduced local field potential amplitudes in remote brain regions, indicating distant effects. A lower dose did not reduce local field potential amplitudes in remote brain regions but did not reduce seizures when infused unilaterally. Instead, seizures tended to initiate in the contralateral ventral hippocampal formation. Bilateral infusion of the lower dose into the ventral hippocampal formation reduced seizure frequency 85%. Similar bilateral treatment in the amygdala was not effective. Bilateral infusion of the dorsal hippocampus reduced seizure frequency, but only 17%. Together, these findings reveal that the ventral hippocampal formation is a primary bilaterally independent epileptogenic zone, and the dorsal hippocampus is a secondary epileptogenic zone in pilocarpine-treated rats. This is consistent with many human patients, and the results further validate the local field potential method for identifying seizure onset zones. Finally, the findings are more consistent with a focal mechanism of ictogenesis rather than one involving a network of interdependent nodes.SIGNIFICANCE STATEMENT:To better understand how seizures start, investigators need to know where seizures start in the animal models they study. In the widely used pilocarpine-treated rat model of temporal lobe epilepsy, earliest seizure activity was most frequently recorded in the ventral hippocampal formation. Confirming the primary role of the ventral hippocampal formation, seizure frequency was reduced most effectively when it was inactivated focally, bilaterally, and continually with infused tetrodotoxin. These findings suggest the ventral hippocampal formation is the primary site of seizure initiation in this animal model of temporal lobe epilepsy, consistent with findings in many human patients.

Lack of Hyperinhibition of Oriens Lacunosum-Moleculare Cells by Vasoactive Intestinal Peptide-Expressing Cells in a Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy remains a common disorder with no cure and inadequate treatments, potentially because of an incomplete understanding of how seizures start. CA1 pyramidal cells and many inhibitory interneurons increase their firing rate in the seconds-minutes before a spontaneous seizure in epileptic rats. However, some interneurons fail to do so, including those identified as putative interneurons with somata in oriens and axons targeting lacunosum-moleculare (OLM cells). Somatostatin-containing cells, including OLM cells, are the primary target of inhibitory vasoactive intestinal polypeptide and calretinin-expressing (VIP/CR) bipolar interneuron-selective interneurons, type 3 (ISI-3). The objective of this study was to test the hypothesis that in epilepsy inhibition of OLM cells by ISI-3 is abnormally increased, potentially explaining the failure of OLM recruitment when needed most during the ramp up of activity preceding a seizure. Stereological quantification of VIP/CR cells in a model of temporal lobe epilepsy demonstrated that they survive in epileptic mice, despite a reduction in their somatostatin-expressing (Som) cell targets. Paired recordings of unitary IPSCs (uIPSCs) from ISI-3 to OLM cells did not show increased connection probability or increased connection strength, and failure rate was unchanged. When miniature postsynaptic currents in ISI-3 were compared, only mIPSC frequency was increased in epileptic hippocampi. Nevertheless, spontaneous and miniature postsynaptic potentials were unchanged in OLM cells of epileptic mice. These results are not consistent with the hypothesis of hyperinhibition from VIP/CR bipolar cells impeding recruitment of OLM cells in advance of a seizure.

Non-invasive, neurotoxic surgery reduces seizures in a rat model of temporal lobe epilepsy.

Surgery can be highly effective for treating certain cases of drug resistant epilepsy. The current study tested a novel, non-invasive, surgical strategy for treating seizures in a rat model of temporal lobe epilepsy. The surgical approach uses magnetic resonance-guided, low-intensity focused ultrasound (MRgFUS) in combination with intravenous microbubbles to open the blood-brain barrier (BBB) in a transient and focal manner. During the period of BBB opening, a systemically administered neurotoxin (Quinolinic Acid: QA) that is normally impermeable to the BBB gains access to a targeted area in the brain, destroying neurons where the BBB has been opened. This strategy is termed Precise Intracerebral Non-invasive Guided Surgery (PING). Spontaneous recurrent seizures induced by pilocarpine were monitored behaviorally prior to and after PING or under control conditions. Seizure frequency in untreated animals or animals treated with MRgFUS without QA exhibited expected seizure rate fluctuations frequencies between the monitoring periods. In contrast, animals treated with PING targeting the intermediate-temporal aspect of the hippocampus exhibited substantial reductions in seizure frequency, with convulsive seizures being eliminated entirely in two animals. These findings suggest that PING could provide a useful alternative to invasive surgical interventions for treating drug resistant epilepsy, and perhaps for treating other neurological disorders in which aberrant neural circuitries play a role.

Ictal onset sites and γ-aminobutyric acidergic neuron loss in epileptic pilocarpine-treated rats.

OBJECTIVE: The present study tested whether ictal onset sites are regions of more severe interneuron loss in epileptic pilocarpine-treated rats, a model of human temporal lobe epilepsy. METHODS: Local field potential recordings were evaluated to identify ictal onset sites. Electrode sites were visualized in Nissl-stained sections. Adjacent sections were processed with proximity ligation in situ hybridization for glutamic acid decarboxylase 2 (Gad2). Gad2 neuron profile numbers at ictal onset sites were compared to contralateral regions. Other sections were processed with immunocytochemistry for reelin or nitric oxide synthase (NOS), which labeled major subtypes of granule cell layer-associated interneurons. Stereology was used to estimate numbers of reelin and NOS granule cell layer-associated interneurons per hippocampus. RESULTS: Ictal onset sites varied between and within rats but were mostly in the ventral hippocampus and were frequently bilateral. There was no conclusive evidence of more severe Gad2 neuron profile loss at sites of earliest seizure activity compared to contralateral regions. Numbers of granule cell layer-associated NOS neurons were reduced in the ventral hippocampus. SIGNIFICANCE: In epileptic pilocarpine-treated rats, ictal onset sites were mostly in the ventral hippocampus, where there was loss of granule cell layer-associated NOS interneurons. These findings suggest the hypothesis that loss of granule cell layer-associated NOS interneurons in the ventral hippocampus is a mechanism of temporal lobe epilepsy.

Proportional loss of parvalbumin-immunoreactive synaptic boutons and granule cells from the hippocampus of sea lions with temporal lobe epilepsy.

One in 26 people develop epilepsy and in these temporal lobe epilepsy (TLE) is common. Many patients display a pattern of neuron loss called hippocampal sclerosis. Seizures usually start in the hippocampus but underlying mechanisms remain unclear. One possibility is insufficient inhibition of dentate granule cells. Normally parvalbumin-immunoreactive (PV) interneurons strongly inhibit granule cells. Humans with TLE display loss of PV interneurons in the dentate gyrus but questions persist. To address this, we evaluated PV interneuron and bouton numbers in California sea lions (Zalophus californianus) that naturally develop TLE after exposure to domoic acid, a neurotoxin that enters the marine food chain during harmful algal blooms. Sclerotic hippocampi were identified by the loss of Nissl-stained hilar neurons. Stereological methods were used to estimate the number of granule cells and PV interneurons per dentate gyrus. Sclerotic hippocampi contained fewer granule cells, fewer PV interneurons, and fewer PV synaptic boutons, and the ratio of granule cells to PV interneurons was higher than in controls. To test whether fewer boutons was attributable to loss versus reduced immunoreactivity, expression of synaptotagmin-2 (syt2) was evaluated. Syt2 is also expressed in boutons of PV interneurons. Sclerotic hippocampi displayed proportional losses of syt2-immunoreactive boutons, PV boutons, and granule cells. There was no significant difference in the average numbers of PV- or syt2-positive boutons per granule cell between control and sclerotic hippocampi. These findings do not address functionality of surviving synapses but suggest reduced granule cell inhibition in TLE is not attributable to anatomical loss of PV boutons.

Testing Different Combinations of Acoustic Pressure and Doses of Quinolinic Acid for Induction of Focal Neuron Loss in Mice Using Transcranial Low-Intensity Focused Ultrasound.

The goal of this study was to test different combinations of acoustic pressure and doses of quinolinic acid (QA) for producing a focal neuronal lesion in the murine hippocampus without causing unwanted damage to adjacent brain structures. Sixty male CD-1 mice were divided into 12 groups that underwent magnetic resonance-guided focused ultrasound at high (0.67 MPa), medium (0.5 MPa) and low (0.33 MPa) acoustic peak negative pressures and received QA at high (0.012 mmol), medium (0.006 mmol) and low (0.003 mmol) dosages. Neuronal loss occurred only when magnetic resonance-guided focused ultrasound with adequate acoustic power (0.67 or 0.5 MPa) was combined with QA. The animals subjected to the highest acoustic power had larger lesions than those treated with medium acoustic power, but two mice had evidence of bleeding. When the intermediate acoustic power was used, medium and high dosages of QA produced lesions larger than those produced by the low dosage.

A single subconvulsant dose of domoic acid at mid-gestation does not cause temporal lobe epilepsy in mice.

Harmful blooms of domoic acid (DA)-producing algae are a problem in oceans worldwide. DA is a potent glutamate receptor agonist that can cause status epilepticus and in survivors, temporal lobe epilepsy. In mice, one-time low-dose in utero exposure to DA was reported to cause hippocampal damage and epileptiform activity, leading to the hypothesis that unrecognized exposure to DA from contaminated seafood in pregnant women can damage the fetal hippocampus and initiate temporal lobe epileptogenesis. However, development of epilepsy (i.e., spontaneous recurrent seizures) has not been tested. In the present study, long-term seizure monitoring and histology was used to test for temporal lobe epilepsy following prenatal exposure to DA. In Experiment One, the previous study's in utero DA treatment protocol was replicated, including use of the CD-1 mouse strain. Afterward, mice were video-monitored for convulsive seizures from 2 to 6 months old. None of the CD-1 mice treated in utero with vehicle or DA was observed to experience spontaneous convulsive seizures. After seizure monitoring, mice were evaluated for pathological evidence of temporal lobe epilepsy. None of the mice treated in utero with DA displayed the hilar neuron loss that occurs in patients with temporal lobe epilepsy and in the mouse pilocarpine model of temporal lobe epilepsy. In Experiment Two, a higher dose of DA was administered to pregnant FVB mice. FVB mice were tested as a potentially more sensitive strain, because they have a lower seizure threshold, and some females spontaneously develop epilepsy. Female offspring were monitored with continuous video and telemetric bilateral hippocampal local field potential recording at 1-11 months old. A similar proportion of vehicle- and DA-treated female FVB mice spontaneously developed epilepsy, beginning in the fourth month of life. Average seizure frequency and duration were similar in both groups. Seizure frequency was lower than that of positive-control pilocarpine-treated mice, but seizure duration was similar. None of the mice treated in utero with vehicle or DA displayed hilar neuron loss or intense mossy fiber sprouting, a form of aberrant synaptic reorganization that develops in patients with temporal lobe epilepsy and in pilocarpine-treated mice. FVB mice that developed epilepsy (vehicle- and DA-treated) displayed mild mossy fiber sprouting. Results of this study suggest that a single subconvulsive dose of DA at mid-gestation does not cause temporal lobe epilepsy in mice.

Seizure frequency correlates with loss of dentate gyrus GABAergic neurons in a mouse model of temporal lobe epilepsy.

Epilepsy occurs in one of 26 people. Temporal lobe epilepsy is common and can be difficult to treat effectively. It can develop after brain injuries that damage the hippocampus. Multiple pathophysiological mechanisms involving the hippocampal dentate gyrus have been proposed. This study evaluated a mouse model of temporal lobe epilepsy to test which pathological changes in the dentate gyrus correlate with seizure frequency and help prioritize potential mechanisms for further study. FVB mice (n = 127) that had experienced status epilepticus after systemic treatment with pilocarpine 31-61 days earlier were video-monitored for spontaneous, convulsive seizures 9 hr/day every day for 24-36 days. Over 4,060 seizures were observed. Seizure frequency ranged from an average of one every 3.6 days to one every 2.1 hr. Hippocampal sections were processed for Nissl stain, Prox1-immunocytochemistry, GluR2-immunocytochemistry, Timm stain, glial fibrillary acidic protein-immunocytochemistry, glutamic acid decarboxylase in situ hybridization, and parvalbumin-immunocytochemistry. Stereological methods were used to measure hilar ectopic granule cells, mossy cells, mossy fiber sprouting, astrogliosis, and GABAergic interneurons. Seizure frequency was not significantly correlated with the generation of hilar ectopic granule cells, the number of mossy cells, the extent of mossy fiber sprouting, the extent of astrogliosis, or the number of GABAergic interneurons in the molecular layer or hilus. Seizure frequency significantly correlated with the loss of GABAergic interneurons in or adjacent to the granule cell layer, but not with the loss of parvalbumin-positive interneurons. These findings prioritize the loss of granule cell layer interneurons for further testing as a potential cause of temporal lobe epilepsy.

Hilar somatostatin interneuron loss reduces dentate gyrus inhibition in a mouse model of temporal lobe epilepsy.

OBJECTIVE: In patients with temporal lobe epilepsy, seizures usually start in the hippocampus, and dentate granule cells are hyperexcitable. Somatostatin interneurons are a major subpopulation of inhibitory neurons in the dentate gyrus, and many are lost in patients and animal models. However, surviving somatostatin interneurons sprout axon collaterals and form new synapses, so the net effect on granule cell inhibition remains unclear. METHODS: The present study uses optogenetics to activate hilar somatostatin interneurons and measure the inhibitory effect on dentate gyrus perforant path-evoked local field potential responses in a mouse model of temporal lobe epilepsy. RESULTS: In controls, light activation of hilar somatostatin interneurons inhibited evoked responses up to 40%. Epileptic pilocarpine-treated mice exhibited loss of hilar somatostatin interneurons and less light-induced inhibition of evoked responses. SIGNIFICANCE: These findings suggest that severe epilepsy-related loss of hilar somatostatin interneurons can overwhelm the surviving interneurons' capacity to compensate by sprouting axon collaterals.

More Docked Vesicles and Larger Active Zones at Basket Cell-to-Granule Cell Synapses in a Rat Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy is a common and challenging clinical problem, and its pathophysiological mechanisms remain unclear. One possibility is insufficient inhibition in the hippocampal formation where seizures tend to initiate. Normally, hippocampal basket cells provide strong and reliable synaptic inhibition at principal cell somata. In a rat model of temporal lobe epilepsy, basket cell-to-granule cell (BC→GC) synaptic transmission is more likely to fail, but the underlying cause is unknown. At some synapses, probability of release correlates with bouton size, active zone area, and number of docked vesicles. The present study tested the hypothesis that impaired GABAergic transmission at BC→GC synapses is attributable to ultrastructural changes. Boutons making axosomatic symmetric synapses in the granule cell layer were reconstructed from serial electron micrographs. BC→GC boutons were predicted to be smaller in volume, have fewer and smaller active zones, and contain fewer vesicles, including fewer docked vesicles. Results revealed the opposite. Compared with controls, epileptic pilocarpine-treated rats displayed boutons with over twice the average volume, active zone area, total vesicles, and docked vesicles and with more vesicles closer to active zones. Larger active zones in epileptic rats are consistent with previous reports of larger amplitude miniature IPSCs and larger BC→GC quantal size. Results of this study indicate that transmission failures at BC→GC synapses in epileptic pilocarpine-treated rats are not attributable to smaller boutons or fewer docked vesicles. Instead, processes following vesicle docking, including priming, Ca(2+) entry, or Ca(2+) coupling with exocytosis, might be responsible. SIGNIFICANCE STATEMENT: One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported impaired inhibition in epileptic animals at basket cell-to-granule cell (BC→GC) synapses, which normally are reliable and strong. Electron microscopy was used to evaluate 3D ultrastructure of BC→GC synapses in a rat model of temporal lobe epilepsy. The hypothesis was that impaired synaptic transmission is attributable to smaller boutons, smaller synapses, and abnormally low numbers of synaptic vesicles. Results revealed the opposite. These findings suggest that impaired transmission at BC→GC synapses in epileptic rats is attributable to later steps in exocytosis following vesicle docking.

Unit Activity of Hippocampal Interneurons before Spontaneous Seizures in an Animal Model of Temporal Lobe Epilepsy.

Mechanisms of seizure initiation are unclear. To evaluate the possible roles of inhibitory neurons, unit recordings were obtained in the dentate gyrus, CA3, CA1, and subiculum of epileptic pilocarpine-treated rats as they experienced spontaneous seizures. Most interneurons in the dentate gyrus, CA1, and subiculum increased their firing rate before seizures, and did so with significant consistency from seizure to seizure. Identification of CA1 interneuron subtypes based on firing characteristics during theta and sharp waves suggested that a parvalbumin-positive basket cell and putative bistratified cells, but not oriens lacunosum moleculare cells, were activated preictally. Preictal changes occurred much earlier than those described by most previous in vitro studies. Preictal activation of interneurons began earliest (>4 min before seizure onset), increased most, was most prevalent in the subiculum, and was minimal in CA3. Preictal inactivation of interneurons was most common in CA1 (27% of interneurons) and included a putative ivy cell and parvalbumin-positive basket cell. Increased or decreased preictal activity correlated with whether interneurons fired faster or slower, respectively, during theta activity. Theta waves were more likely to occur before seizure onset, and increased preictal firing of subicular interneurons correlated with theta activity. Preictal changes by other hippocampal interneurons were largely independent of theta waves. Within seconds of seizure onset, many interneurons displayed a brief pause in firing and a later, longer drop that was associated with reduced action potential amplitude. These findings suggest that many interneurons inactivate during seizures, most increase their activity preictally, but some fail to do so at the critical time before seizure onset.

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.

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.

Preictal activity of subicular, CA1, and dentate gyrus principal neurons in the dorsal hippocampus before spontaneous seizures in a rat model of temporal lobe epilepsy.

Previous studies suggest that spontaneous seizures in patients with temporal lobe epilepsy might be preceded by increased action potential firing of hippocampal neurons. Preictal activity is potentially important because it might provide new opportunities for predicting when a seizure is about to occur and insight into how spontaneous seizures are generated. We evaluated local field potentials and unit activity of single, putative excitatory neurons in the subiculum, CA1, CA3, and dentate gyrus of the dorsal hippocampus in epileptic pilocarpine-treated rats as they experienced spontaneous seizures. Average action potential firing rates of neurons in the subiculum, CA1, and dentate gyrus, but not CA3, increased significantly and progressively beginning 2-4 min before locally recorded spontaneous seizures. In the subiculum, CA1, and dentate gyrus, but not CA3, 41-57% of neurons displayed increased preictal activity with significant consistency across multiple seizures. Much of the increased preictal firing of neurons in the subiculum and CA1 correlated with preictal theta activity, whereas preictal firing of neurons in the dentate gyrus was independent of theta. In addition, some CA1 and dentate gyrus neurons displayed reduced firing rates preictally. These results reveal that different hippocampal subregions exhibit differences in the extent and potential underlying mechanisms of preictal activity. The finding of robust and significantly consistent preictal activity of subicular, CA1, and dentate neurons in the dorsal hippocampus, despite the likelihood that many seizures initiated in other brain regions, suggests the existence of a broader neuronal network whose activity changes minutes before spontaneous seizures initiate.

Does mossy fiber sprouting give rise to the epileptic state?

Many patients with temporal lobe epilepsy display structural changes in the seizure initiating zone, which includes the hippocampus. Structural changes in the hippocampus include granule cell axon (mossy fiber) sprouting. The role of mossy fiber sprouting in epileptogenesis is controversial. A popular view of temporal lobe epileptogenesis contends that precipitating brain insults trigger transient cascades of molecular and cellular events that permanently enhance excitability of neuronal networks through mechanisms including mossy fiber sprouting. However, recent evidence suggests there is no critical period for mossy fiber sprouting after an epileptogenic brain injury. Instead, findings from stereological electron microscopy and rapamycin-delayed mossy fiber sprouting in rodent models of temporal lobe epilepsy suggest a persistent, homeostatic mechanism exists to maintain a set level of excitatory synaptic input to granule cells. If so, a target level of mossy fiber sprouting might be determined shortly after a brain injury and then remain constant. Despite the static appearance of synaptic reorganization after its development, work by other investigators suggests there might be continual turnover of sprouted mossy fibers in epileptic patients and animal models. If so, there may be opportunities to reverse established mossy fiber sprouting. However, reversal of mossy fiber sprouting is unlikely to be antiepileptogenic, because blocking its development does not reduce seizure frequency in pilocarpine-treated mice. The challenge remains to identify which, if any, of the many other structural changes in the hippocampus are epileptogenic.

Hippocampal neuropathology of domoic acid-induced epilepsy in California sea lions (Zalophus californianus).

California sea lions (Zalophus californianus) are abundant human-sized carnivores with large gyrencephalic brains. They develop epilepsy after experiencing status epilepticus when naturally exposed to domoic acid. We tested whether sea lions previously exposed to DA (chronic DA sea lions) display hippocampal neuropathology similar to that of human patients with temporal lobe epilepsy. Hippocampi were obtained from control and chronic DA sea lions. Stereology was used to estimate numbers of Nissl-stained neurons per hippocampus in the granule cell layer, hilus, and pyramidal cell layer of CA3, CA2, and CA1 subfields. Adjacent sections were processed for somatostatin immunoreactivity or Timm-stained, and the extent of mossy fiber sprouting was measured stereologically. Chronic DA sea lions displayed hippocampal neuron loss in patterns and extents similar but not identical to those reported previously for human patients with temporal lobe epilepsy. Similar to human patients, hippocampal sclerosis in sea lions was unilateral in 79% of cases, mossy fiber sprouting was a common neuropathological abnormality, and somatostatin-immunoreactive axons were exuberant in the dentate gyrus despite loss of immunopositive hilar neurons. Thus, hippocampal neuropathology of chronic DA sea lions is similar to that of human patients with temporal lobe epilepsy.

High-dose rapamycin blocks mossy fiber sprouting but not seizures in a mouse model of temporal lobe epilepsy.

PURPOSE: The role of granule cell axon (mossy fiber) sprouting in temporal lobe epileptogenesis is unclear and controversial. Rapamycin suppresses mossy fiber sprouting, but its reported effects on seizure frequency are mixed. The present study used high-dose rapamycin to more completely block mossy fiber sprouting and to measure the effect on seizure frequency. METHODS: Mice were treated with pilocarpine to induce status epilepticus. Beginning 24 h later and continuing for 2 months, vehicle or rapamycin (10 mg/kg/day) was administered. Starting 1 month after status epilepticus, mice were monitored by video 9 h per day, every day, for 1 month to measure the frequency of spontaneous motor seizures. At the end of seizure monitoring, a subset of mice was prepared for anatomic analysis. Mossy fiber sprouting was measured as the proportion of the granule cell layer and molecular layer that displayed black labeling in Timm-stained sections. KEY FINDINGS: Extensive mossy fiber sprouting developed in mice that experienced status epilepticus and were treated with vehicle. In rapamycin-treated mice, mossy fiber sprouting was blocked almost to the level of naive controls. Seizure frequency was similar in vehicle-treated and rapamycin-treated mice. SIGNIFICANCE: These findings suggest that mossy fiber sprouting is not necessary for epileptogenesis in the mouse pilocarpine model. They also reveal that rapamycin does not have antiseizure or antiepileptogenic effects in this model.

Early activation of ventral hippocampus and subiculum during spontaneous seizures in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy is the most common form of epilepsy in adults. The pilocarpine-treated rat model is used frequently to investigate temporal lobe epilepsy. The validity of the pilocarpine model has been challenged based largely on concerns that seizures might initiate in different brain regions in rats than in patients. The present study used 32 recording electrodes per rat to evaluate spontaneous seizures in various brain regions including the septum, dorsomedial thalamus, amygdala, olfactory cortex, dorsal and ventral hippocampus, substantia nigra, entorhinal cortex, and ventral subiculum. Compared with published results from patients, seizures in rats tended to be shorter, spread faster and more extensively, generate behavioral manifestations more quickly, and produce generalized convulsions more frequently. Similarities to patients included electrographic waveform patterns at seizure onset, variability in sites of earliest seizure activity within individuals, and variability in patterns of seizure spread. Like patients, the earliest seizure activity in rats was recorded most frequently within the hippocampal formation. The ventral hippocampus and ventral subiculum displayed the earliest seizure activity. Amygdala, olfactory cortex, and septum occasionally displayed early seizure latencies, but not above chance levels. Substantia nigra and dorsomedial thalamus demonstrated consistently late seizure onsets, suggesting their unlikely involvement in seizure initiation. The results of the present study reveal similarities in onset sites of spontaneous seizures in patients with temporal lobe epilepsy and pilocarpine-treated rats that support the model's validity.