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1.  Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex 
Neuropharmacology  2012;63(5):806-817.
Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3–36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.
doi:10.1016/j.neuropharm.2012.06.009
PMCID: PMC3409872  PMID: 22722023
Temporal lobe epilepsy; Subiculum; Entorhinal cortex; Animal epilepsy model; EEG; Epileptogenesis
2.  Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex 
Neuropharmacology  2012;63(5):806-817.
Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3–36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.
Highlights
► The onset of spontaneous seizures was highly variable (3–36 days) with an average latency of 15 days. ► Massive neurodegeneration was already present 24 h after KA-induced seizures. ► Neurons of the proximal subiculum and EC layer III preferentially degenerate. ► Distribution of reactive gliosis roughly matches the pattern of neurodegeneration. ► Time course of reactive gliosis parallels that of epileptogenesis.
doi:10.1016/j.neuropharm.2012.06.009
PMCID: PMC3409872  PMID: 22722023
Temporal lobe epilepsy; Subiculum; Entorhinal cortex; Animal epilepsy model; EEG; Epileptogenesis; DG, dentate gyrus; FJ-C, Fluoro Jade C; GAD65 and GAD67, glutamate decarboxylase 65 and 67; GFAP, glial fibrillary acidic protein; KA, kainic acid; NeuN, neuron-specific nuclear protein; SE, status epilepticus; Sub, subiculum; TLE, temporal lobe epilepsy; VGLUT1, vesicular glutamate transporter 1
3.  Chronic Cellular Hyperexcitability in Elderly Epileptic Rats with Spontaneous Seizures Induced by Kainic Acid Status Epilepticus while Young Adults 
Aging and Disease  2011;2(4):332-338.
Emerging data indicate that age-related brain changes alter seizure susceptibility, seizure-associated neurodegeneration, and responsiveness to AEDs. The present study assessed long-term animal survival in the Kainic Acid (KA) model along with in-vivo spontaneous seizure frequency, cellular hyperexcitability in CA1 in-vitro and in-vivo in subiculum, and responsiveness of in-vitro CA1 hyperexcitability to topiramate. Sprague-Dawley male rats were given KA to induce convulsive status epilepticus (KA-SE) at 2–3 months of age. The one-month mortality after KA-SE was 27%. One-month survivor rats had 37% sudden unexplained late mortality after KA-SE as compared to none in saline controls during their second year of life. In-vivo seizure frequency was examined prior to terminal experiments. The diurnal average seizure frequency in the KA-SE group at age 2 years was 1.06 ± 0.24 seizures/hour while no seizures were observed in the saline age-matched controls (p<0.001). In-vitro recordings of CA1 pyramidal neurons revealed that depolarizing current injection from −60 mV evoked an increased number of action potentials in the aged KA-SE group compared to controls (p<0.002). Topiramate exhibited dose-dependent inhibition of action potential firing evoked by current injections into CA1 pyramidal neurons of KA-SE rats. In subiculum, KA-SE rats had frequent interictal spikes associated with high frequency oscillations while only rare spontaneous EPSPs were recorded in saline controls. Our experiments revealed that the hippocampal formation of aged epileptic rats shares features of hyperexcitability previously described in young adult epileptic rats using the KA model.
PMCID: PMC3295074  PMID: 22396885
Topiramate; Aged; Epilepsy; Bursting; Hippocampal slice; SUDEP
4.  Seizure Control of Current Shunt on Rats with Temporal Lobe Epilepsy and Neocortical Epilepsy 
PLoS ONE  2014;9(1):e86477.
Purpose
To examine the effects of current shunt on rats with temporal lobe epilepsy and neocortex epilepsy.
Experimental Design
A kainic acid (KA)-induced model of temporal lobe seizure and a penicillin-induced model of neocortical partial seizure were used in this study. Rats of each model were randomly allocated into two groups: control and model groups. The model group was further divided into the KA or penicillin group, sham conduction group and conduction group. The current shunt was realized through the implantation of a customized conduction electrode. After surgery, electroencephalogram (EEG) was recorded for two hours for each rat under anesthesia. Subsequently, the rats were video monitored for 72 h to detect the occurrence of behavioral seizures upon awakening. The average number and duration of seizures on EEG and the number of behavioral seizures were measured.
Results
In KA model, the number of total EEG seizures in conduction group (9.57±2.46) was significantly less than that in sham conduction group (15.13±3.45) (p<0.01). The duration of EEG seizures in conduction group (26.13±7.81 s) was significantly shorter than that in sham conduction group (34.17±7.25 s) (p = 0.001). A significant reduction of behavioral seizures was observed in the conduction group compared with KA (p = 0.000) and sham conduction groups (p = 0.000). In penicillin model, there was a 61% reduction in total EEG seizures in conduction group compared with sham conduction group (p<0.01), and the duration of EEG seizures in conduction group (6.29±2.64 s) was significantly shorter than that in the sham conduction group (12.07±3.81 s) (p = 0.002). A significant reduction of behavioral seizures was observed in conduction group compared with penicillin (p<0.01) and sham conduction groups (p<0.01).
Conclusion
Current shunt effectively reduces the onset and severity of seizures. Current shunt therapy could be an effective alternative minimally invasive approach for temporal lobe epilepsy and neocortex epilepsy.
doi:10.1371/journal.pone.0086477
PMCID: PMC3907408  PMID: 24497949
5.  Seizure suppression by EEG-guided repetitive transcranial magnetic stimulation in the rat 
Objective
To test the anticonvulsive potential of a range of repetitive transcranial magnetic stimulation (rTMS) frequencies by novel methods for simultaneous EEG and rTMS in a rat seizure model.
Methods
Seizures were triggered by intraperitoneal kainic acid (KA; 10 mg/kg). Rats (n = 21) were divided into three groups in which individual seizures were treated with rTMS trains at one of three frequencies: 0.25, 0.5 or 0.75 Hz. EEG was continuously viewed by an operator who identified each seizure onset. Consecutive seizures in each animal were (1) treated with active rTMS, (2) treated with sham rTMS, or (3) were untreated. EEG was re-analyzed post hoc by visual inspection, and seizure durations were compared within and between treatment groups.
Results
KA-induced seizures were abbreviated by 0.75 Hz (P = 0.019) and 0.5 Hz (P = 0.033) active EEG-guided rTMS. In contrast, neither active 0.25 Hz rTMS nor the control conditions affected seizure duration (P > 0.2).
Conclusions
We demonstrate that EEG-guided rTMS can suppress seizures in the rat KA epilepsy model, and that the effect is frequency dependent, with 0.75 and 0.5 Hz rTMS being superior to 0.25 Hz rTMS.
Significance
These data support the use of rat seizure models in translational research aimed at evaluation and development of effective rTMS anticonvulsive protocols. We also offer a proof of principle that real-time analysis of EEG can be used to guide rTMS to suppress individual seizures.
doi:10.1016/j.clinph.2008.09.003
PMCID: PMC2668608  PMID: 18977170
Transcranial magnetic stimulation; Seizure; Rat; EEG
6.  Changes in the expression of GABAA receptor subunit mRNAs in parahippocampal areas after kainic acid induced seizures 
The parahippocampal areas including the subiculum, pre- and parasubiculum, and notably the entorhinal cortex (EC) are intimately involved in the generation of limbic seizures in temporal lobe epilepsy. We investigated changes in the expression of 10 major GABAA receptor subunit mRNAs in subfields of the ventral hippocampus, ventral subiculum, EC, and perirhinal cortex (PRC) at different intervals (1, 8, 30, and 90 days) after kainic acid (KA)-induced status epilepticus priming epileptogenesis in the rat. The most pronounced and ubiquitous changes were a transient (24 h after KA only) down-regulation of γ2 mRNA and lasting decreases in subunit α5, β3, and δ mRNAs that were prominent in all hippocampal and parahippocampal areas. In the subiculum similarly as in sectors CA1 and CA3, levels of subunit α1, α2, α4, and γ2 mRNAs decreased transiently (1 day after KA-induced status epilepticus). They were followed by increased expression of subunit α1 and α3 mRNAs in the dentate gyrus (DG) and sectors CA1 and CA3, and subunit α1 also in the EC layer II (30 and 90 days after KA). We also observed sustained overexpression of subunits α4 and γ2 in the subiculum and in the Ammon’s horn. Subunit γ2 mRNA was also increased in sector CA1 at the late intervals after KA. Taken together, our results suggest distinct regulation of mRNA expression for individual GABAA receptor subunits. Especially striking was the wide-spread down-regulation of the often peri- or extrasynaptically located subunits α5 and δ. These subunits are often associated with tonic inhibition. Their decrease could be related to decreased tonic inhibition or may merely reflect compensatory changes. In contrast, expression of subunit α4 that may also mediate tonic inhibition when associated with the δ-subunit was significantly upregulated in the DG and in the proximal subiculum at late intervals. Thus, concomitant up-regulation of subunit γ2, α1 and α4 mRNAs (and loss in δ-subunits) ultimately indicates significant rearrangement of GABAA receptor composition after KA-induced seizures.
doi:10.3389/fncir.2013.00142
PMCID: PMC3776158  PMID: 24065890
epilepsy; tonic inhibition; GABAA-receptor; temporal lobe epilepsy; subiculum; entorhinal cortex; epileptogenesis
7.  Parvalbumin interneurons and calretinin fibers arising from the thalamic nucleus reuniens degenerate in the subiculum after kainic acid-induced seizures 
Neuroscience  2011;189(1-2):316-329.
The subiculum is the major output area of the hippocampus. It is closely interconnected with the entorhinal cortex and other parahippocampal areas. In animal models of temporal lobe epilepsy (TLE) and in TLE patients it exerts increased network excitability and may crucially contribute to the propagation of limbic seizures. Using immunohistochemistry and in situ-hybridization we now investigated neuropathological changes affecting parvalbumin and calretinin containing neurons in the subiculum and other parahippocampal areas after kainic acid-induced status epilepticus. We observed prominent losses in parvalbumin containing interneurons in the subiculum and entorhinal cortex, and in the principal cell layers of the pre- and parasubiculum. Degeneration of parvalbumin-positive neurons was associated with significant precipitation of parvalbumin-immunoreactive debris 24 h after kainic acid injection. In the subiculum the superficial portion of the pyramidal cell layer was more severely affected than its deep part. In the entorhinal cortex, the deep layers were more severely affected than the superficial ones. The decrease in number of parvalbumin-positive neurons in the subiculum and entorhinal cortex correlated with the number of spontaneous seizures subsequently experienced by the rats. The loss of parvalbumin neurons thus may contribute to the development of spontaneous seizures. On the other hand, surviving parvalbumin neurons revealed markedly increased expression of parvalbumin mRNA notably in the pyramidal cell layer of the subiculum and in all layers of the entorhinal cortex. This indicates increased activity of these neurons aiming to compensate for the partial loss of this functionally important neuron population. Furthermore, calretinin-positive fibers terminating in the molecular layer of the subiculum, in sector CA1 of the hippocampus proper and in the entorhinal cortex degenerated together with their presumed perikarya in the thalamic nucleus reuniens. In addition, a significant loss of calretinin containing interneurons was observed in the subiculum. Notably, the loss in parvalbumin positive neurons in the subiculum equaled that in human TLE. It may result in marked impairment of feed-forward inhibition of the temporo-ammonic pathway and may significantly contribute to epileptogenesis. Similarly, the loss of calretinin-positive fiber tracts originating from the nucleus reuniens thalami significantly contributes to the rearrangement of neuronal circuitries in the subiculum and entorhinal cortex during epileptogenesis.
Graphical Abstract
•••
Highlights
▶A subpopulation of PV neurons degenerates in subiculum and entorhinal cortex after KA seizures. ▶Surviving PV neurons exhibit increased PV mRNA expression. ▶The loss in PV neurons in subiculum and entorhinal cortex correlates to spontaneous seizures. ▶Degeneration of PV neurons in the subiculum may be related to seizure-induced loss of feed-forward inhibition. ▶CR-ir neurons in the N. reuniens thalami and their projections to the subiculum degenerate.
doi:10.1016/j.neuroscience.2011.05.021
PMCID: PMC3152681  PMID: 21616128
status epilepticus; temporal lobe epilepsy; epileptogenesis; entorhinal cortex; epilepsy models; CR, calretinin; EC, entorhinal cortex; -ir, immunoreactive; KA, kainic acid; NeuN, neuron specific nuclear protein; O-LM, oriens-lacunosum moleculare; PV, parvalbumin; ROD, relative optical densities; SE, status epilepticus; TBS, tris-buffered saline; TLE, temporal lobe epilepsy
8.  Synaptic reorganization in subiculum and CA3 after early-life status epilepticus in the kainic acid rat model 
Epilepsy research  2006;73(2):156-165.
Purpose
The immature rat brain is highly susceptible to seizures, but has a resistance to pathological changes induced by seizures as compared to adult rats. However, prolonged seizures during early-life enhance cellular injury and hyperexcitability induced by convulsive insults later in adulthood. The mechanisms underlying these phenomena are not understood. In adult models, the CA1 axons reorganize their projections to subiculum. Seizure induced plasticity in this pathway has not been investigated in immature seizure models, and may contribute to the vulnerability to later seizures.
Methods
On postnatal day 15, rats experienced convulsive status epilepticus with kainic acid (KA). Seizure induced plasticity was examined with Timm histochemistry and iontophoretic injections of sodium selenite, a retrograde tracer. Cellular injury was evaluated with Fluoro-Jade B histochemistry.
Results
Retrograde tracing experiments determined a 67% larger dorsoventral extent of retrograde labeling in the CA1 pyramidal region after tracer injections in subiculum. The synaptic reorganization of the CA1 projection to subiculum was noted in the absence of overt neuronal injury in subiculum or CA1. In contrast, mossy fiber sprouting was detected into the stratum oriens of CA3 with limited neuronal injury to CA3 pyramidal neurons. No mossy fiber sprouting into the inner molecular layer of the dentate gyrus, or CA1 sprouting into the stratum moleculare of CA1 were noted.
Conclusions
The results indicate that the developing brain has distinct mechanisms of seizure induced reorganization as compared to the adult brain. Our experiments show that the concept of “resistance of the immature brain to excitotoxicity” is considerably more complicated than generally believed. Morphological plasticity in the immature brain appears more extensive in distal, but not proximal, projections of hippocampal pathways, and across hippocampal lamellae. The abnormal connectivity between hippocampal lamellae might play a role in the increased susceptibility to injury and hyperexcitability associated with later convulsive insults.
doi:10.1016/j.eplepsyres.2006.09.004
PMCID: PMC1876715  PMID: 17070016
sprouting; seizures; immature; retrograde tracing; Zinc
9.  Increased GABA-ergic Inhibition in the Midline Thalamus Affects Signaling and Seizure Spread in the Hippocampus-Prefrontal Cortex Pathway 
Epilepsia  2011;52(3):523-530.
Purpose
The midline thalamus is an important component of the circuitry in limbic seizures, but it is unclear how synaptic modulation of the thalamus affects that circuitry. In this study, we wished to understand how synaptic modulation of the thalamus can affect inter-regional signaling and seizure spread in the limbic network.
Methods
We examined the effect of GABA modulation of the mediodorsal (MD) region of the thalamus on responses in the prefrontal cortex (PFC) by stimulation of the subiculum (SB). Muscimol, a GABA-A agonist, was injected into the MD, and the effect on local responses to subiculum stimulation were examined. Evoked potentials were induced in the MD and the PFC by low frequency stimulation of the SB, and seizures were generated in the subiculum by repeated 20 Hz stimulations. The effect of muscimol in the MD on the evoked potentials and seizures was measured.
Key Findings
Thalamic responses to stimulation of the subiculum were reduced in the presence of muscimol. Reduction of the amplitudes of evoked potentials in the MD resulted in an attenuation of the late, thalamic components of the responses in the PFC, as well as of seizure durations.
Significance
Activation of GABA- A receptors in the midline thalamus not only causes changes within the thalamus, but has broader effects on the limbic network. This work provides further evidence that synaptic modulation within the midline thalamus alters system excitability more broadly and reduces seizure activity.
doi:10.1111/j.1528-1167.2010.02919.x
PMCID: PMC3058300  PMID: 21204829
GABA; mediodorsal nucleus; subiculum; prefrontal cortex; seizures; limbic system
10.  Hippocampal CA1/subiculum-prefrontal cortical pathways induce plastic changes of nociceptive responses in cingulate and prelimbic areas 
BMC Neuroscience  2010;11:100.
Background
Projections from hippocampal CA1-subiculum (CA1/SB) areas to the prefrontal cortex (PFC), which are involved in memory and learning processes, produce long term synaptic plasticity in PFC neurons. We examined modifying effects of these projections on nociceptive responses recorded in the prelimbic and cingulate areas of the PFC.
Results
Extracellular unit discharges evoked by mechanical noxious stimulation delivered to the rat-tail and field potentials evoked by a single stimulus pulse delivered to CA1/SB were recorded in the PFC. High frequency stimulation (HFS, 100 Hz) delivered to CA1/SB, which produced long-term potentiation (LTP) of field potentials, induced long-term enhancement (LTE) of nociceptive responses in 78% of cases, while, conversely, in 22% responses decreased (long-term depression, LTD). These neurons were scattered throughout the cingulate and prelimbic areas. The results obtained for field potentials and nociceptive discharges suggest that CA1/SB-PFC pathways can produce heterosynaptic potentiation in PFC neurons. HFS had no effects on Fos expression in the cingulated cortex. Low frequency stimulation (LFS, 1 Hz, 600 bursts) delivered to the CA1/SB induced LTD of nociceptive discharges in all cases. After recovery from LTD, HFS delivered to CA1/SB had the opposite effect, inducing LTE of nociceptive responses in the same neuron. The bidirectional type of plasticity was evident in these nociceptive responses, as in the homosynaptic plasticity reported previously. Neurons inducing LTD are found mainly in the prelimbic area, in which Fos expression was also shown to be inhibited by LFS. The electrophysiological results closely paralleled those of immunostaining. Our results indicate that CA1/SB-PFC pathways inhibit excitatory pyramidal cell activities in prelimbic areas.
Conclusion
Pressure stimulation (300 g) applied to the rat-tail induced nociceptive responses in the cingulate and prelimbic areas of the PFC, which receives direct pathways from CA1/SB. HFS and LFS delivered to the CA1/SB induced long-term plasticity of nociceptive responses. Thus, CA1/SB-PFC projections modulate the nociceptive responses of PFC neurons.
doi:10.1186/1471-2202-11-100
PMCID: PMC2936438  PMID: 20716327
11.  Automated identification of multiple seizure-related and interictal epileptiform event types in the EEG of mice 
Scientific Reports  2013;3:1483.
Visual scoring of murine EEG signals is time-consuming and subject to low inter-observer reproducibility. The Racine scale for behavioral seizure severity does not provide information about interictal or sub-clinical epileptiform activity. An automated algorithm for murine EEG analysis was developed using total signal variation and wavelet decomposition to identify spike, seizure, and other abnormal signal types in single-channel EEG collected from kainic acid-treated mice. The algorithm was validated on multi-channel EEG collected from γ-butyrolacetone-treated mice experiencing absence seizures. The algorithm identified epileptiform activity with high fidelity compared to visual scoring, correctly classifying spikes and seizures with 99% accuracy and 91% precision. The algorithm correctly identifed a spike-wave discharge focus in an absence-type seizure recorded by 36 cortical electrodes. The algorithm provides a reliable and automated method for quantification of multiple classes of epileptiform activity within the murine EEG and is tunable to a variety of event types and seizure categories.
doi:10.1038/srep01483
PMCID: PMC3604748  PMID: 23514826
12.  The Cause of the Imbalance in the Neuronal Network Leading to Seizure Activity Can Be Predicted by the Electrographic Pattern of the Seizure Onset 
This study investigates the temporal dynamics of ictal electrical activity induced by injection of the GABAA receptor antagonist bicuculline, and the glutamate agonist kainic acid, into the CA3 area of hippocampus. Experiments were conducted in freely moving adult Wistar rats implanted with microelectrodes in multiple brain areas. Wide-band electrical activity (0.1–3000 Hz) was recorded, and the latency of seizure onset as well as the pattern of electrical activity were investigated for each drug. The latencies between injection and the occurrence of first epileptiform events were 3.93 ± 2.76 (±STD) min for bicuculline and 6.37 ± 7.66 min for kainic acid, suggesting the existence of powerful seizure-suppressive mechanisms in the brain. Bicuculline evoked high-amplitude rhythmic epileptiform events at the site of injection which resembled interictal EEG spikes and rapidly propagated to adjacent and remote brain areas. Kainic acid evoked a completely different pattern with a gradual increase in the amplitude of 30–80 Hz activity. Whereas there was strong temporal correlation between EEG events at the site of bicuculline injection and discharges in distant areas, much less correlation was seen with kainic acid injection. Both patterns were followed by generalized ictal EEG discharges and behavioral seizures. Our results illustrate that the same area of the brain can trigger seizures with different electrographic patterns. The knowledge of the network mechanisms underlying these two distinct electrographic patterns might be helpful in designing differential strategies for preventing seizure occurrence.
doi:10.1523/JNEUROSCI.5309-08.2009
PMCID: PMC2688438  PMID: 19295168
13.  Early Activation of Ventral Hippocampus and Subiculum during Spontaneous Seizures in a Rat Model of Temporal Lobe Epilepsy 
The Journal of Neuroscience  2013;33(27):11100-11115.
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.
doi:10.1523/JNEUROSCI.0472-13.2013
PMCID: PMC3718374  PMID: 23825415
14.  Sprouting and Synaptic Reorganization in the Subiculum and CA1 Region of the Hippocampus in Acute and Chronic Models of Partial-Onset Epilepsy 
Neuroscience  2004;126(3):677-688.
Repeated seizures induce permanent alterations in the hippocampal circuitry in experimental models and patients with intractable temporal lobe epilepsy (TLE). Most studies have concentrated their attention on seizure-induced reorganization of the mossy fiber pathway. The present study examined the projection pathway of the CA1 pyramidal neurons to the Subiculum, which is the output of the hippocampal formation in five models of TLE. We examined the laminar pattern of Timm's histochemistry in the stratum lacunosum-moleculare of CA1 in three acute and two chronic models of TLE: intraventricular kainic acid (KA), systemic KA, systemic pilocarpine, chronic electric kindling and chronic intraperitoneal pentylenetetrazol. The laminar pattern of Timm histochemistry in the stratum moleculare of CA1 was permanently remodeled in epileptic models suggesting sprouting of Timm containing terminals from the adjacent stratum lacunosum. Ultrastructural examination confirmed that Timm granules were localized in synaptic terminals. As the source of Timm labeled terminals in this region was not known, sodium selenite, a selective retrograde tracer for Zinc-containing terminals, was iontophoretically injected in-vivo in rats exposed to systemic pilocarpine, systemic KA or chronic PTZ. The normal projection of CA1 pyramidal neurons to the Subiculum is topographically organized in a lamellar fashion. In normal rats, the extent of the injection site (terminals) and the retrogradely labeled pyramidal neurons (cell soma) corresponded to the same number of lamellas. In epileptic rats, the retrograde labeling extended 81–130% farther than the normal dorso-ventral extent including lamellas above and below the expected. This is direct evidence for sprouting of CA1 pyramidal axons into the Subiculum and stratum lacunosum-moleculare of the CA1 region confirming the alterations of the laminar pattern of Timm's histochemistry. Sprouting of the CA1 projection to Subiculum across hippocampal lamellas might lead to translamellar hyperexcitability, and to amplification and synchronization of epileptic discharges in the output gate of the hippocampal formation.
doi:10.1016/j.neuroscience.2004.04.014
PMCID: PMC3179906  PMID: 15183517
hippocampus; plasticity; seizures; tracing; Timm staining
15.  Spontaneous and Evoked High Frequency Oscillations in the Tetanus Toxin Model of Epilepsy 
Epilepsia  2010;51(11):2289-2296.
Summary
Purpose
High-frequency oscillations (HFOs) are an emerging biomarker for epileptic tissue. Yet the mechanism by which HFOs are produced is unknown, and their rarity makes them difficult to study. Our objective was to examine the occurrence of HFOs in relation to action potentials (APs) and the effect of microstimulation in the tetanus toxin (TT) model of epilepsy, a non-lesional model with a short latency to spontaneous seizures.
Methods
Rats were injected with TT into dorsal hippocampus and implanted with a 16 channel (8 × 2) multielectrode array, one row each in CA3 and CA1. After onset of spontaneous seizures (3-9 days), recordings were begun of APs and local field potentials, analyzed for the occurrence of interictal spikes and HFOs. Recordings were made during microstimulation of each electrode using customized, open-source software.
Results
Population bursts of APs during interictal spikes were phase-locked with HFOs, which were observable almost exclusively with high-amplitude interictal spikes. Further, HFOs could reliably be produced by microstimulation of the hippocampus, providing evidence that these oscillations can be controlled temporally by external means.
Discussion
We show for the first time the occurrence of HFOs in the TT epilepsy model, an attractive preparation for their experimental investigation and, importantly, one with a different etiology than status models, providing further evidence of the generality of HFOs. The ability to provoke HFOs with microstimulation may prove useful for better understanding HFOs by directly evoking them in the lab, and designing high-throughput techniques for pre-surgical localization of the epileptic focus.
doi:10.1111/j.1528-1167.2010.02753.x
PMCID: PMC3010348  PMID: 20946126
Oscillations; microelectrode; stimulation; interictal spike; electrocorticography; animal model
16.  Seizure entrainment with polarizing low frequency electric fields in a chronic animal epilepsy model 
Journal of neural engineering  2009;6(4):046009.
Neural activity can be modulated by applying a polarizing low frequency (≪ 100 Hz) electric field (PLEF). Unlike conventional pulsed stimulation, PLEF stimulation has a graded, modulatory effect on neuronal excitability, and permits the simultaneous recording of neuronal activity during stimulation suitable for continuous feedback control. We tested a prototype system that allows for simultaneous PLEF stimulation with minimal recording artifact in a chronic tetanus toxin animal model (rat) of hippocampal epilepsy with spontaneous seizures. Depth electrode local field potentials recorded during seizures revealed a characteristic pattern of field postsynaptic potentials (fPSPs). Sinusoidal voltage-controlled PLEF stimulation (0.5–25 Hz) was applied in open-loop cycles radially across the CA3 of ventral hippocampus. For stimulated seizures, fPSPs were transiently entrained with the PLEF waveform. Statistical significance of entrainment was assessed with Thomson’s harmonic F-test, with 45/132 stimulated seizures in 4 animals individually demonstrating significant entrainment (p < 0.04). Significant entrainment for multiple presentations at the same frequency (p < 0.01) was observed in 3 of 4 animals in 42/64 stimulated seizures. This is the first demonstration in chronically implanted freely behaving animals of PLEF modulation of neural activity with simultaneous recording.
doi:10.1088/1741-2560/6/4/046009
PMCID: PMC3057918  PMID: 19602730
Seizure control; low frequency; electric fields; modulation; temporal lobe epilepsy; multitaper
17.  Ictal and interictal high frequency oscillations in patients with focal epilepsy 
Objective
High frequency oscillations (HFOs) can be recorded with depth electrodes in focal epilepsy patients. They occur during seizures and interictally and seem important in seizure genesis. We investigated whether interictal and ictal HFOs occur in the same regions and how they relate to epileptiform spikes.
Methods
In 25 patients, spikes, ripples (80–250 Hz) and fast ripples (FR: 250–500 Hz) and their co-occurrences were marked during interictal slow wave sleep (5–10 min), during 10 preictal seconds and 5 s following seizure onset. We compared occurrence and spatial distribution between these periods.
Results
HFOs and spikes increased from interictal to ictal periods: the percentage of time occupied by ripples increased from 2.3% to 6.5%, FR from 0.2% to 0.8%, spikes from 1.1% to 4.8%. HFOs increased from interictal to preictal periods in contrast to spikes. Spikes were in different channels in the interictal, preictal and ictal periods whereas HFOs largely remained in the same channels.
Conclusions
HFOs remain confined to the same, possibly epileptogenic, area, during interictal and ictal periods, while spikes are more widespread during seizures than interictally.
Significance
Ictal and interictal HFOs represent the same (epileptogenic) area and are probably similar phenomena.
doi:10.1016/j.clinph.2010.09.021
PMCID: PMC3771929  PMID: 21030302 CAMSID: cams3344
High frequency oscillations; Focal epilepsy; Epilepsy surgery; Depth EEG; Ictogenesis
18.  High frequency stimulation can suppress globally seizures induced by 4-AP in the rat hippocampus: An acute in vivo study 
Brain stimulation  2012;6(2):180-189.
Background
High frequency stimulation (HFS) on the hippocampus can locally suppress epileptiform activity in-vitro and decrease seizure frequency in vivo. In-vitro HFS on the ventral commissural tract, a novel target, was shown to block the axonal conduction and suppress activity in the CA1 and CA3 neuron.
Objective
To study the spatial extent of seizure suppression by HFS applied on the tract and focus site in an in vivo experiment.
Methods
Five adult Sprague–Dawley rats were used for the study. Six electrodes were placed on the septal, middle, and temporal hippocampus bilaterally to simultaneously record seizure activity in the entire hippocampus. Seizure activity was induced by injecting 4-aminopyridine (4-AP) into the right middle part of the hippocampus. Following induction, HFS (100 Hz) was applied to the tract and the focus site at 100, 300 and 500 μA.
Results
The induced seizure activity was dominated by two patterns, high frequency spiking and pseudo-periodic spikes. Either tract or focus site stimulation could generate suppression of only the pseudo-periodic spikes. The suppression rates were dependent on stimulation amplitude (P < 0.005, chi square test). However, HFS also caused conversion of the seizure pattern. The conversion rates increased with higher stimulation amplitudes and were higher with focus site stimulation (P < 0.01, Fisher’s exact test).
Conclusions
The results of this study have two practical implications [1], both tract and focus site stimulation can produce global suppression of hippocampus and [2] the choice of stimulation parameters is critical in order to produce suppression, but not conversion, of seizure pattern.
doi:10.1016/j.brs.2012.04.008
PMCID: PMC3624017  PMID: 22621942
High frequency electrical stimulation; Hippocampus; Seizure suppression
19.  High-Frequency Stimulation of Nucleus Accumbens Changes in Dopaminergic Reward Circuit 
PLoS ONE  2013;8(11):e79318.
Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is a potential remedial therapy for drug craving and relapse, but the mechanism is poorly understood. We investigated changes in neurotransmitter levels during high frequency stimulation (HFS) of the unilateral NAc on morphine-induced rats. Sixty adult Wistar rats were randomized into five groups: the control group (administration of saline), the morphine-only group (systematic administration of morphine without electrode implantation), the morphine-sham-stimulation group (systematic administration of morphine with electrode implantation but not given stimulation), the morphine-stimulation group (systematic administration of morphine with electrode implantation and stimulation) and the saline-stimulation group (administration of saline with electrode implantation and stimulation). The stimulation electrode was stereotaxically implanted into the core of unilateral NAc and microdialysis probes were unilaterally lowered into the ipsilateral ventral tegmental area (VTA), NAc, and ventral pallidum (VP). Samples from microdialysis probes in the ipsilateral VTA, NAc, and VP were analyzed for glutamate (Glu) and γ-aminobutyric acid (GABA) by high-performance liquid chromatography (HPLC). The levels of Glu were increased in the ipsilateral NAc and VP of morphine-only group versus control group, whereas Glu levels were not significantly changed in the ipsilateral VTA. Furthermore, the levels of GABA decreased significantly in the ipsilateral NAc, VP, and VTA of morphine-only group when compared with control group. The profiles of increased Glu and reduced GABA in morphine-induced rats suggest that the presence of increased excitatory neurotransmission in these brain regions. The concentrations of the Glu significantly decreased while the levels of GABA increased in ipsilateral VTA, NAc, and VP in the morphine-stimulation group compared with the morphine-only group. No significant changes were seen in the morphine-sham stimulation group compared with the morphine-only group. These findings indicated that unilateral NAc stimulation inhibits the morphine-induced rats associated hyperactivation of excitatory neurotransmission in the mesocorticolimbic reward circuit.
doi:10.1371/journal.pone.0079318
PMCID: PMC3828386  PMID: 24244479
20.  Systemic Injection of Kainic Acid Differently Affects LTP Magnitude Depending on its Epileptogenic Efficiency 
PLoS ONE  2012;7(10):e48128.
Seizures have profound impact on synaptic function and plasticity. While kainic acid is a popular method to induce seizures and to potentially affect synaptic plasticity, it can also produce physiological-like oscillations and trigger some forms of long-term potentiation (LTP). Here, we examine whether induction of LTP is altered in hippocampal slices prepared from rats with different sensitivity to develop status epilepticus (SE) by systemic injection of kainic acid. Rats were treated with multiple low doses of kainic acid (5 mg/kg; i.p.) to develop SE in a majority of animals (72–85% rats). A group of rats were resistant to develop SE (15–28%) after several accumulated doses. Animals were subsequently tested using chronic recordings and object recognition tasks before brain slices were prepared for histological studies and to examine basic features of hippocampal synaptic function and plasticity, including input/output curves, paired-pulse facilitation and theta-burst induced LTP. Consistent with previous reports in kindling and pilocapine models, LTP was reduced in rats that developed SE after kainic acid injection. These animals exhibited signs of hippocampal sclerosis and developed spontaneous seizures. In contrast, resistant rats did not become epileptic and had no signs of cell loss and mossy fiber sprouting. In slices from resistant rats, theta-burst stimulation induced LTP of higher magnitude when compared with control and epileptic rats. Variations on LTP magnitude correlate with animals’ performance in a hippocampal-dependent spatial memory task. Our results suggest dissociable long-term effects of treatment with kainic acid on synaptic function and plasticity depending on its epileptogenic efficiency.
doi:10.1371/journal.pone.0048128
PMCID: PMC3485282  PMID: 23118939
21.  Structural and Functional Asymmetry in the Normal and Epileptic Rat Dentate Gyrus 
The rat dentate gyrus is usually described as relatively homogeneous. Here, we present anatomic and physiological data which demonstrate that there are striking differences between the supra- and infrapyramidal blades after status epilepticus and recurrent seizures. These differences appear to be an accentuation of a subtle asymmetry present in normal rats. In both pilocarpine and kainic acid models, there was greater mossy fiber sprouting in the infrapyramidal blade. This occurred primarily in the middle third of the hippocampus. Asymmetric sprouting was evident both with Timm stain as well as antisera to brain-derived neurotrophic factor (BDNF) or neuropeptide Y (NPY). In addition, surviving NPY-immunoreactive hilar neurons were distributed preferentially in the suprapyramidal region of the hilus. Extracellular recordings from infrapyramidal sites in hippocampal slices of pilocarpine-treated rats showed larger population spikes and weaker paired-pulse inhibition in response to perforant path stimulation relative to suprapyramidal recordings. A single stimulus could evoke burst discharges in infrapyramidal granule cells but not suprapyramidal blade neurons. BDNF exposure led to spontaneous epileptiform discharges that were larger in amplitude and longer lasting in the infrapyramidal blade. Stimulation of the infrapyramidal molecular layer evoked larger responses in area CA3 than suprapyramidal stimulation. In slices from the temporal pole, in which anatomic evidence of asymmetry waned, there was little evidence of physiological asymmetry either. Of interest, some normal rats also showed signs of greater evoked responses in the infrapyramidal blade, and this could be detected with both microelectrode recording and optical imaging techniques. Although there were no signs of hyperexcitability in normal rats, the data suggest that there is some asymmetry in the normal dentate gyrus and this asymmetry is enhanced by seizures. Taken together, the results suggest that supra- and infrapyramidal blades of the dentate gyrus could have different circuit functions and that the infrapyramidal blade may play a greater role in activating the hippocampus.
doi:10.1002/cne.10449
PMCID: PMC2519114  PMID: 12455007
neuropeptide Y (NPY); brain-derived neurotrophic factor (BDNF); voltage-dependent dyes; pilocarpine; mossy fiber sprouting; seizure
22.  Loss of Hippocampal Neurons after Kainate Treatment Correlates with Behavioral Deficits 
PLoS ONE  2014;9(1):e84722.
Treating rats with kainic acid induces status epilepticus (SE) and leads to the development of behavioral deficits and spontaneous recurrent seizures later in life. However, in a subset of rats, kainic acid treatment does not induce overt behaviorally obvious acute SE. The goal of this study was to compare the neuroanatomical and behavioral changes induced by kainate in rats that developed convulsive SE to those who did not. Adult male Wistar rats were treated with kainic acid and tested behaviorally 5 months later. Rats that had experienced convulsive SE showed impaired performance on the spatial water maze and passive avoidance tasks, and on the context and tone retention tests following fear conditioning. In addition, they exhibited less anxiety-like behaviors than controls on the open-field and elevated plus-maze tests. Histologically, convulsive SE was associated with marked neuron loss in the hippocampal CA3 and CA1 fields, and in the dentate hilus. Rats that had not experienced convulsive SE after kainate treatment showed less severe, but significant impairments on the spatial water maze and passive avoidance tasks. These rats had fewer neurons than control rats in the dentate hilus, but not in the hippocampal CA3 and CA1 fields. Correlational analyses revealed significant relationships between spatial memory indices of rats and neuronal numbers in the dentate hilus and CA3 pyramidal field. These results show that a part of the animals that do not display intense behavioral seizures (convulsive SE) immediately after an epileptogenic treatment, later in life, they may still have noticeable structural and functional changes in the brain.
doi:10.1371/journal.pone.0084722
PMCID: PMC3883667  PMID: 24409306
23.  Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model 
Brain research  2009;1282:162-172.
Early-life seizures result in increased susceptibility to seizures and greater neurologic injury with a second insult in adulthood. The mechanisms which link seizures in early-life to increased susceptibility to neurologic injury following a ‘second hit’ are not known. We examined the contribution of microglial activation and increased proinflammatory cytokine production to the subsequent increase in susceptibility to neurologic injury using a kainic acid (KA)-induced, established ‘two-hit’ seizure model in rats. Postnatal day (P)15 rats were administered intraperitoneal KA (early-life seizures) or saline, followed on P45 with either a ‘second hit’ of KA, a first exposure to KA (adult seizures), or saline. We measured the levels of proinflammatory cytokines (IL-1β, TNF-α, and S100B), the chemokine CCL2, microglial activation, seizure susceptibility and neuronal outcomes in adult rats 12 hours and 10 days after the second hit on P45. The ‘two-hit’ group exposed to KA on both P15 and P45 had higher levels of cytokines, greater microglial activation, and increased susceptibility to seizures and neurologic injury compared to the adult seizures group. Treatment after early-life seizures with Minozac, a small molecule experimental therapeutic that targets up-regulated proinflammatory cytokine production, attenuated the enhanced microglial and cytokine responses, the increased susceptibility to seizures, and the greater neuronal injury in the ‘two-hit’ group. These results implicate microglial activation as one mechanism by which early-life seizures contribute to increased vulnerability to neurologic insults in adulthood, and indicate the potential longer term benefits of early-life intervention with therapies that target up-regulation of proinflammatory cytokines.
doi:10.1016/j.brainres.2009.05.073
PMCID: PMC2739829  PMID: 19501063
microglia; cytokines; epilepsy; therapeutic; chemokine
24.  Antioxidants and free radical scavengers do not consistently delay seizure onset in animal models of acute seizures 
Epilepsy & behavior : E&B  2008;13(1):77-82.
Summary
A number of herbal compounds with direct antioxidant activity slow the onset, or completely block, the appearance of seizures. This increase in latency has been proposed to be due to the antioxidant activity. This hypothesis was directly tested by determining the effects of Trolox®, a vitamin E analog, vitamin C, melatonin, and α-lipoic acid on the latency to acute seizures induced with pilocarpine, kainic acid or pentylenetetrazol (PTZ sc) in adult rats. Trolox®, vitamin C, and α-lipoic acid had significant anticonvulsant activity against pilocarpine, but there were no acute changes in reduced glutathione levels at 15 or 120 minutes. Other than a reduced mortality with vitamin C in the PTZ model, none of the antioxidants had a significant effect against PTZ, or kainic acid-induced seizures. The lack of consistent anticonvulsant effect suggests that antioxidant activity of the herbal preparations cannot account for the delay in seizure onset.
doi:10.1016/j.yebeh.2008.03.002
PMCID: PMC2486491  PMID: 18396108
Trolox; vitamin E; melatonin; vitamin C; α-lipoic acid; kainic acid; pilocarpine; pentylenetetrazol
25.  Simultaneous fMRI and local field potential measurements during epileptic seizures in medetomidine sedated rats using RASER pulse sequence 
Simultaneous electrophysiological and functional magnetic resonance imaging (fMRI) measurements of animal models of epilepsy are methodologically challenging, but essential to better understand abnormal brain activity and hemodynamics during seizures. In the present study, fMRI of medetomidine sedated rats was performed using novel Rapid Acquisition by Sequential Excitation and Refocusing (RASER) fast imaging pulse sequence and simultaneous local field potential (LFP) measurements during kainic acid (KA) induced seizures. The image distortion caused by the hippocampal measuring electrode was clearly seen in echo planar imaging (EPI) images, whereas no artifact was seen in RASER images. Robust blood oxygenation level dependent (BOLD) responses were observed in the hippocampus during KA induced seizures. The recurrent epileptic seizures were detected in the LFP signal after KA injection. The presented combination of deep electrode LFP measurements and fMRI under medetomidine anesthesia, that does not significantly suppress KA induced seizures, provides a unique tool for studying abnormal brain activity in rats.
doi:10.1002/mrm.22508
PMCID: PMC2946452  PMID: 20725933
fMRI; RASER; Medetomidine; Local field potential; Epilepsy; Kainic acid

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