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.

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.

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.

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.

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.

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.

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.

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.

Rat strain differences in seizure frequency and hilar neuron loss after systemic treatment with pilocarpine.

At least 3 months after systemic treatment with pilocarpine to induce status epilepticus, Long-Evans and Sprague-Dawley rats were video-EEG monitored for seizures continuously for 1 month. Rats were then perfused, hippocampi were processed for Nissl staining, and hilar neurons were quantified. Seizure frequency in Long-Evans rats was 1/10th of that in Sprague-Dawley rats, and more variable. Hilar neuron loss was also less severe in Long-Evans rats. However, there was no correlation between hilar neuron loss and seizure frequency in either strain. The low and variable seizure frequency suggests limited usefulness of pilocarpine-treated Long-Evans rats for some epilepsy experiments.

Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy.

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in 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.

Stress coping stimulates hippocampal neurogenesis in adult monkeys.

Coping with intermittent social stress is an essential aspect of living in complex social environments. Coping tends to counteract the deleterious effects of stress and is thought to induce neuroadaptations in corticolimbic brain systems. Here we test this hypothesis in adult squirrel monkey males exposed to intermittent social separations and new pair formations. These manipulations simulate conditions that typically occur in male social associations because of competition for limited access to residency in mixed-sex groups. As evidence of coping, we previously confirmed that cortisol levels initially increase and then are restored to prestress levels within several days of each separation and new pair formation. Follow-up studies with exogenous cortisol further established that feedback regulation of the hypothalamic-pituitary-adrenal axis is not impaired. Now we report that exposure to intermittent social separations and new pair formations increased hippocampal neurogenesis in squirrel monkey males. Hippocampal neurogenesis in rodents contributes to spatial learning performance, and in monkeys we found that spatial learning was enhanced in conditions that increased hippocampal neurogenesis. Corresponding changes were discerned in the expression of genes involved in survival and integration of adult-born granule cells into hippocampal neural circuits. These findings support recent indications that stress coping stimulates hippocampal neurogenesis in adult rodents. Psychotherapies designed to promote stress coping potentially have similar effects in humans with major depression.

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.

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.

Mossy cell dendritic structure quantified and compared with other hippocampal neurons labeled in rats in vivo.

Mossy cells are likely to contribute to normal hippocampal function and to the pathogenesis of neurologic disorders that involve the hippocampus, including epilepsy. Mossy cells are the least well-characterized excitatory neurons in the hippocampus. Their somatic and dendritic morphology has been described qualitatively but not quantitatively. In the present study rat mossy cells were labeled intracellularly with biocytin in vivo. Somatic and dendritic structure was reconstructed three-dimensionally. For comparison, granule cells, CA3 pyramidal cells, and CA1 pyramidal cells were labeled and analyzed using the same approach. Among the four types of hippocampal neurons, granule cells had the smallest somata, fewest primary dendrites and dendritic branches, and shortest total dendritic length. CA1 pyramidal cells had the most dendritic branches and longest total dendritic length. Mossy cells and CA3 pyramidal cells both had large somata and similar total dendritic lengths. However, mossy cell dendrites branched less than CA3 pyramidal cells, especially close to the soma. These findings suggest that mossy cells have dendritic features that are not identical to any other type of hippocampal neuron. Therefore, electrotonic properties that depend on soma-dendritic structure are likely to be distinct in mossy cells compared to other neurons.

Identification of new epilepsy treatments: issues in preclinical methodology.

Preclinical research has facilitated the discovery of valuable drugs for the symptomatic treatment of epilepsy. Yet, despite these therapies, seizures are not adequately controlled in a third of all affected individuals, and comorbidities still impose a major burden on quality of life. The introduction of multiple new therapies into clinical use over the past two decades has done little to change this. There is an urgent demand to address the unmet clinical needs for: (1) new symptomatic antiseizure treatments for drug-resistant seizures with improved efficacy/tolerability profiles, (2) disease-modifying treatments that prevent or ameliorate the process of epileptogenesis, and (3) treatments for the common comorbidities that contribute to disability in people with epilepsy. New therapies also need to address the special needs of certain subpopulations, that is, age- or gender-specific treatments. Preclinical development in these treatment areas is complex due to heterogeneity in presentation and etiology, and may need to be formulated with a specific seizure, epilepsy syndrome, or comorbidity in mind. The aim of this report is to provide a framework that will help define future guidelines that improve and standardize the design, reporting, and validation of data across preclinical antiepilepsy therapy development studies targeting drug-resistant seizures, epileptogenesis, and comorbidities.

Rapamycin suppresses axon sprouting by somatostatin interneurons in a mouse model of temporal lobe epilepsy.

PURPOSE: In temporal lobe epilepsy many somatostatin interneurons in the dentate gyrus die. However, some survive and sprout axon collaterals that form new synapses with granule cells. The functional consequences of γ-aminobutyric acid (GABA)ergic synaptic reorganization are unclear. Development of new methods to suppress epilepsy-related interneuron axon sprouting might be useful experimentally. METHODS: Status epilepticus was induced by systemic pilocarpine treatment in green fluorescent protein (GFP)-expressing inhibitory nerurons (GIN) mice in which a subset of somatostatin interneurons expresses GFP. Beginning 24 h later, mice were treated with vehicle or rapamycin (3 mg/kg intraperitoneally) every day for 2 months. Stereologic methods were then used to estimate numbers of GFP-positive hilar neurons per dentate gyrus and total length of GFP-positive axon in the molecular layer plus granule cell layer. GFP-positive axon density was calculated. The number of GFP-positive axon crossings of the granule cell layer was measured. Regression analyses were performed to test for correlations between GFP-positive axon length versus number of granule cells and dentate gyrus volume. KEY FINDINGS: After pilocarpine-induced status epilepticus, rapamycin- and vehicle-treated mice had approximately half as many GFP-positive hilar neurons as did control animals. Despite neuron loss, vehicle-treated mice had over twice the GFP-positive axon length per dentate gyrus as controls, consistent with GABAergic axon sprouting. In contrast, total GFP-positive axon length was similar in rapamycin-treated mice and controls. GFP-positive axon length correlated most closely with dentate gyrus volume. SIGNIFICANCE: These findings suggest that rapamycin suppressed axon sprouting by surviving somatostatin/GFP-positive interneurons after pilocarpine-induced status epilepticus in GIN mice. It is unclear whether the effect of rapamycin on axon length was on interneurons directly or secondary, for example, by suppressing growth of granule cell dendrites, which are synaptic targets of interneuron axons. The mammalian target of rapamycin (mTOR) signaling pathway might be a useful drug target for influencing GABAergic synaptic reorganization after epileptogenic treatments, but additional side effects of rapamycin treatment must be considered carefully.

Is there a critical period for mossy fiber sprouting in a mouse model of temporal lobe epilepsy?

PURPOSE: Dentate granule cell axon (mossy fiber) sprouting creates an aberrant positive-feedback circuit that might be epileptogenic. Presumably, mossy fiber sprouting is initiated by molecular signals, but it is unclear whether they are expressed transiently or persistently. If transient, there might be a critical period when short preventative treatments could permanently block mossy fiber sprouting. Alternatively, if signals persist, continuous treatment would be necessary. The present study tested whether temporary treatment with rapamycin has long-term effects on mossy fiber sprouting. METHODS: Mice were treated daily with 1.5 mg/kg rapamycin or vehicle (i.p.) beginning 24 h after pilocarpine-induced status epilepticus. Mice were perfused for anatomic evaluation immediately after 2 months of treatment ("0 delay") or after an additional 6 months without treatment ("6-month delay"). One series of sections was Timm-stained, and an adjacent series was Nissl-stained. Stereologic methods were used to measure the volume of the granule cell layer plus molecular layer and the Timm-positive fraction. Numbers of Nissl-stained hilar neurons were estimated using the optical fractionator method. KEY FINDINGS: At 0 delay, rapamycin-treated mice had significantly less black Timm staining in the granule cell layer plus molecular layer than vehicle-treated animals. However, by 6-month delay, Timm staining had increased significantly in mice that had been treated with rapamycin. Percentages of the granule cell layer plus molecular layer that were Timm-positive were high and similar in 0 delay vehicle-treated, 6-month delay vehicle-treated, and 6-month delay rapamycin-treated mice. Extent of hilar neuron loss was similar among all groups that experienced status epilepticus and, therefore, was not a confounding factor. Compared to naive controls, average volume of the granule cell layer plus molecular layer was larger in 0 delay vehicle-treated mice. The hypertrophy was partially suppressed in 0 delay rapamycin-treated mice. However, 6-month delay vehicle- and 6-month delay rapamycin-treated animals had similar average volumes of the granule cell layer plus molecular layer that were significantly larger than those of all other groups. SIGNIFICANCE: Status epilepticus-induced mossy fiber sprouting and dentate gyrus hypertrophy were suppressed by systemic treatment with rapamycin but resumed after treatment ceased. These findings suggest that molecular signals that drive mossy fiber sprouting and dentate gyrus hypertrophy might persist for >2 months after status epilepticus in mice. Therefore, prolonged or continuous treatment might be required to permanently suppress mossy fiber sprouting.

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.