Rapid volume pulsations of the extracellular space accompany epileptiform activity in trauma-injured neocortex and depend on the sodium-bicarbonate cotransporter NBCe1.

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.

4-AP challenge reveals that early intervention with brivaracetam prevents posttraumatic epileptogenesis in rats.

PURPOSE: There are currently no clinical treatments to prevent posttraumatic epilepsy (PTE). Recently, our group has shown that administration of levetiracetam (LEV) or brivaracetam (BRV) shortly after cortical neurotrauma prevents the development of epileptiform activity in rats, as measured ex vivo in neocortical slices. Due to the low incidence of spontaneous seizures in rodent-based models of traumatic brain injury (TBI), chemoconvulsants have been used to test injured animals for seizure susceptibility. We used a low dose of the voltage-gated potassium channel blocker 4-aminopyridine (4-AP) to evaluate posttraumatic epileptogenesis after controlled cortical impact (CCI) injury. We then used this assessment to further investigate the efficacy of BRV as an antiepileptogenic treatment. METHODS: Sprague-Dawley rats aged P24-35 were subjected to severe CCI injury. Following trauma, one group received BRV-21 mg/kg (IP) at 0-2 min after injury and the other BRV-100 mg/kg (IP) at 30 min after injury. Four to eight weeks after injury, animals were given a single, low dose of 4-AP (3.0-3.5 mg/kg, IP) and then monitored up to 90 min for stage 4/5 seizures. RESULTS: The chemoconvulsant challenge revealed that within four to eight weeks, CCI injury led to a two-fold increase in percentage of rats with 4-AP induced stage 4-5 seizures relative to sham-injured controls. Administration of a single dose of BRV within 30 min after trauma significantly reduced injury-induced seizure susceptibility, bringing the proportion of CCI-rats that exhibited evoked seizures down to control levels. CONCLUSIONS: This study is the first to use a low dose of 4-AP as a chemoconvulsant challenge to test epileptogenicity within the first two months after CCI injury in rats. Our findings show that a single dose of BRV administered within 30 min after TBI prevents injury-induced increases in seizure susceptibility. This supports our hypothesis that early intervention with BRV may prevent PTE.

Efficient Delivery of FMR1 across the Blood Brain Barrier Using AAVphp Construct in Adult FMR1 KO Mice Suggests the Feasibility of Gene Therapy for Fragile X Syndrome.

Background Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. Gene therapy may offer an efficient method to ameliorate the symptoms of this disorder. Methods An AAVphp.eb-hSyn-mFMR1IOS7 vector and an empty control were injected into the tail vein of adult Fmr1 knockout (KO) mice and wildtype (WT) controls. The KO mice were injected with 2 × 1013 vg/kg of the construct. The control KO and WT mice were injected with an empty vector. Four weeks following treatment, the animals underwent a battery of tests: open field, marble burying, rotarod, and fear conditioning. The mouse brains were studied for levels of the Fmr1 product FMRP. Results: No significant levels of FMRP were found outside the CNS in the treated animals. The gene delivery was highly efficient, and it exceeded the control FMRP levels in all tested brain regions. There was also improved performance in the rotarod test and partial improvements in the other tests in the treated KO animals. Conclusion: These experiments demonstrate efficient, brain-specific delivery of Fmr1 via peripheral administration in adult mice. The gene delivery led to partial alleviation of the Fmr1 KO phenotypical behaviors. FMRP oversupply may explain why not all behaviors were significantly affected. Since AAV.php vectors are less efficient in humans than in the mice used in the current experiment, studies to determine the optimal dose using human-suitable vectors will be necessary to further demonstrate feasibility.

Brivaracetam prevents the development of epileptiform activity when administered early after cortical neurotrauma in rats.

OBJECTIVES: There is no effective therapy to prevent the development of posttraumatic epilepsy (PTE). Recently, we reported that administration of the antiseizure medication (ASM) levetiracetam (LEV) shortly after trauma prevented the development of epileptiform activity in two experimental models of neurotrauma. However, the time window for effective intervention with LEV may be too narrow for most clinical settings. Using the controlled cortical impact (CCI) injury model, the current study tested whether early administration of brivaracetam (BRV), an ASM with 20 times the affinity of LEV for the SV2A synaptic vesicle protein, could improve upon the antiepileptogenic action observed with LEV. METHODS: Rats (postnatal day [P] 24-32) subjected to CCI injury were given a single dose of BRV (21 or 100 mg/kg, i.p.) at one of three post-injury time points: immediately (0-2 minutes), 30 minutes, or 60 minutes. Control animals received only vehicle (0.9% saline). Posttraumatic electrographic epileptiform activity was assayed ex vivo from coronal neocortical slices collected proximal to the injury (four per rat) 3-4 weeks after injury. In this model, ictal-like burst discharges occur spontaneously or can be evoked in an "all or none" manner with applied electrical stimulation within the first 2 weeks after injury. RESULTS: A single dose of BRV administered to rats up to 60 minutes after traumatic brain injury (TBI) significantly reduced the development of posttraumatic epileptiform activity by (1) inhibiting the development of both evoked and spontaneous epileptiform activity, (2) raising the threshold for stimulus-evoked epileptiform discharges, and (3) reducing the intensity of epileptiform bursts that arise after cortical neurotrauma. SIGNIFICANCE: Clinically there has been little success preventing the development of posttraumatic epilepsy. The results of this study support the hypothesis that early intervention with BRV has the potential to prevent or reduce posttraumatic epileptogenesis, and that there may be a limited time window for successful prophylactic intervention.

Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

Fragile X syndrome results from the absence of the FMR1 gene product-Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

Rapid volume pulsation of the extracellular space coincides with epileptiform activity in mice and depends on the NBCe1 transporter.

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3- cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3- cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.

Early intervention with levetiracetam prevents the development of cortical hyperexcitability and spontaneous epileptiform activity in two models of neurotrauma in rats.

This study examined the antiepileptogenic potential of the antiseizure drug (ASD) levetiracetam (LEV) using the in vitro traumatized-slice and in vivo controlled cortical impact (CCI) models of traumatic brain injury (TBI) in rats when administered early after the injury. For the in vitro model, acute coronal slices (400-450 µm) of rat neocortex (P21-32) were injured via a surgical cut that separated the superficial layers from the deeper regions. Persistent stimulus-evoked epileptiform activity developed within 1-2 h after trauma. In randomly selected slices, LEV (500 µM) was bath-applied for 1 h starting immediately or delayed by 30-80 min after injury. Treated and untreated slices were examined for epileptiform activity via intracellular and extracellular recordings. For the in vivo model, rats (P24-32) were subjected to a non-penetrating, focal, CCI injury targeting the neocortex (5.0 mm diameter; 2.0 mm depth). Immediately after injury, rats were given either a single dose of LEV (60-150 mg/kg, i.p.) or the saline vehicle. At 2-3 weeks after the injury, ex vivo cortical slices were examined for epileptiform activity. The results from the traumatized-slice experiments showed that in vitro treatment with LEV within 60 min of injury significantly reduced (> 50%) the proportion of slices that exhibited stimulus-evoked epileptiform activity. LEV treatment also increased the stimulus intensity required to trigger epileptiform bursts in injured slices by 2-4 fold. Consistent with these findings, LEV treatment of CCI-injured rats (n = 15) significantly reduced the proportion of animals that exhibited spontaneous and stimulus-evoked epileptiform bursts in ex vivo cortical slices compared to saline-treated controls (n = 15 rats), and also significantly increased the stimulus intensity required to evoke epileptiform bursts. These results suggest that early administration of LEV has the potential to prevent or reduce posttraumatic epileptogenesis and that there may be a narrow therapeutic window for successful prophylactic intervention.

Persistent Increases of PKMζ in Sensorimotor Cortex Maintain Procedural Long-Term Memory Storage.

Procedural motor learning and memory are accompanied by changes in synaptic plasticity, neural dynamics, and synaptogenesis. Missing is information on the spatiotemporal dynamics of the molecular machinery maintaining these changes. Here we examine whether persistent increases in PKMζ, an atypical protein kinase C (PKC) isoform, store long-term memory for a reaching task in rat sensorimotor cortex that could reveal the sites of procedural memory storage. Specifically, perturbing PKMζ synthesis (via antisense oligodeoxynucleotides) and blocking atypical PKC activity (via zeta inhibitory peptide [ZIP]) in S1/M1 disrupts and erases long-term motor memory maintenance, indicating atypical PKCs and specifically PKMζ store consolidated long-term procedural memories. Immunostaining reveals that PKMζ increases in S1/M1 layers II/III and V as performance improved to an asymptote. After storage for 1 month without reinforcement, the increase in M1 layer V persists without decrement. Thus, the persistent increases in PKMζ that store long-term procedural memory are localized to the descending output layer of the primary motor cortex.

Controlled cortical impact-induced neurodegeneration decreases after administration of the novel calpain-inhibitor Gabadur.

One aspect of secondary injury in traumatic brain injury is the marked increase in intracellular calcium and resultant over-activation of the calcium-dependent neutral cysteine protease calpain. Gabadur is a novel protease inhibitor with calpain-inhibition properties formulated from the classic protease inhibitor leupeptin linked to a pregabalin carrier. This construction allows the entire compound to cross the blood-brain barrier after peripheral administration to better target the site of injury. In this study, a single intraperitoneal dose of Gabadur was administered immediately following controlled cortical impact injury in rats. Neocortical slices were examined at 48 h post-injury via Fluoro-Jade B staining, revealing an improvement in cortical neurodegeneration in Gabadur treated rats. Levels of detrimental active calpain-2 measured via western blot were also decreased in rats receiving Gabadur. This data supports the benefit of targeted protease inhibition in the treatment of traumatic brain injury.

Neuroanatomical characterization of the cellular and axonal architecture of subcortical band heterotopia in the BXD29-Tlr4lps-2J/J mouse cortex.

Subcortical band heterotopia (SBH) are malformations of the human cerebral cortex typically associated with epilepsy and cognitive delay/disability. Rodent models of SBH have demonstrated strong face validity as they are accompanied by both cognitive deficits and spontaneous seizures or reduced seizure threshold. BXD29-Tlr4lps-2J/J recombinant inbred mice display striking bilateral SBH, partial callosal agenesis, morphological changes in subcortical structures of the auditory pathway, and display sensory deficits in behavioral tests (Rosen et al., 2013; Truong et al., 2013, 2015). Surprisingly, these mice show no cognitive deficits and have a higher seizure threshold to chemi-convulsive treatment (Gabel et al., 2013) making them different than other rodent SBH models described previously. In the present report, we perform a detailed characterization of the cellular and axonal constituents of SBH in BXD29-Tlr4lps-2J/J mice and demonstrate that various types of interneurons and glia as well as cortical and subcortical projections are found in SBH. In addition, the length of neuronal cilia was reduced in SBH compared to neurons in the overlying and adjacent normotopic cortex. Finally, we describe additional and novel malformations of the hippocampus and neocortex present in BXD29-Tlr4lps-2J/J mice. Together, our findings in BXD29-Tlr4lps-2J/J mice are discussed in the context of the known neuroanatomy and phenotype of other SBH rodent models.

Aging-induced Seizure-related Changes to the Hippocampal Mossy Fiber Pathway in Forebrain Specific BDNF Overexpressing Mice.

Aging confers an increased risk for developing seizure activity, especially within brain regions that mediate learning and synaptic plasticity. Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family that has an important role in regulating growth and development of the nervous system. BDNF is upregulated after pharmacological seizure induction and this upregulation contributes to enhanced excitability of the hippocampal mossy fiber-CA3 pathway, which is accompanied by neuropeptide Y (NPY) upregulation. Mice overexpressing a BDNF transgene in forebrain neurons provide an avenue for understanding the role of neurotrophic support in the aged hippocampus. In this study BDNF transgenic (TG) mice were utilized to determine whether increased BDNF expression through genetic manipulation resulted in age-related changes in hippocampal excitability and NPY expression. Spontaneous behavioral seizures were observed in TG mice, but not WT mice, past 5 months of age and the severity of behavioral seizures increased with age. Electrophysiological investigation of hippocampal CA3 activity indicated that slices from aged TG mice (86%), but not age-matched WT mice, or young TG mice, showed epileptiform activity in response to either repeated paired pulse or high frequency (tetanic) stimulation. Electrophysiological results were supported by the observation of robust ectopic NPY immunoreactivity in hippocampal mossy fibers of most aged TG mice (57%), which was absent in age-matched WT mice and young TG mice. The results from this study indicate that forebrain restricted BDNF overexpression produces age-related changes in hyperexcitability and NPY immunoreactivity in mossy fiber-CA3 pathway. Together, these data suggest that the capability for BDNF to promote epileptogenesis is maintained, and may be enhanced, in the aging hippocampus.

Spontaneous epileptiform activity in rat neocortex after controlled cortical impact injury.

A hallmark of severe traumatic brain injury (TBI) is the development of post-traumatic epilepsy (PTE). However, the mechanisms underlying PTE remain poorly understood. In this study, we used a controlled cortical impact (CCI) model in rats to examine post-traumatic changes in neocortical excitability. Neocortical slices were prepared from rats at 7-9 days (week 1) and 14-16 days (week 2) after CCI injury. By week 2, we observed a substantial gray matter lesion with a cavity that extended to the hippocampal structure. Fluoro-Jade B staining of slices revealed active neuronal degeneration during weeks 1 and 2. Intracellular and extracellular recordings obtained from layer V revealed evoked and spontaneous epileptiform discharges in neocortices of CCI-injured rats. At week 1, intracellular recordings from pyramidal cells revealed evoked epileptiform firing that was synchronized with population events recorded extracellularly, suggestive of increased excitability. This activity was characterized by bursts of action potentials that were followed by recurrent, repetitive after-discharges. At week 2, both spontaneous and evoked epileptiform firing were recorded in slices from injured rats. The evoked discharges resembled those observed at week 1, but with longer burst durations. Spontaneous activity included prolonged, ictal-like discharges lasting up to 8-10 sec, and briefer interictal-like burst events (<1 sec). These results indicate that during the first 2 weeks following severe CCI injury, there is a progressive development of neocortical hyperexcitability that ultimately leads to spontaneous epileptiform firing, suggesting a rapid epileptogenic process.

Modest thyroid hormone insufficiency during development induces a cellular malformation in the corpus callosum: a model of cortical dysplasia.

There is a growing body of evidence that subtle decreases in maternal thyroid hormone during gestation can impact fetal brain development. The present study examined the impact of graded levels of thyroid hormone insufficiency on brain development in rodents. Maternal thyroid hormone insufficiency was induced by exposing timed-pregnant dams to propylthiouracil (PTU) at doses of 0, 1, 2, 3, and 10 ppm in the drinking water from gestational d 6 through weaning on postnatal d 30. An examination of Nissl-stained sections of the brains from developmentally hypothyroid offspring killed on postnatal d 23 revealed the presence of a heretofore unreported bilateral cellular malformation, a heterotopia, positioned within the white matter of the corpus callosum of both hemispheres. Immunohistochemical techniques were used to determine that this heterotopia primarily consists of neurons born between gestational d 17-19 and exhibits a dose-dependent increase in size with decreases in thyroid hormone levels. Importantly, this structural abnormality is evident at modest levels of maternal thyroid hormone insufficiency ( approximately 45% reductions in T(4) with no change in T(3)), persists in adult offspring despite a return to normal hormonal status, and is dramatically reduced in size with prenatal thyroid hormone replacement. Developmental exposure to methimazole, another goitrogen, also induced formation of this heterotopia. Whereas the long-term consequence of this cortical malformation on brain function remains to be determined, the presence of the heterotopia underscores the critical role thyroid hormone plays in brain development during the prenatal period and provides a new model in which to study mechanisms of cortical development and cortical dysplasia.

Seizure susceptibility in intact and ovariectomized female rats treated with the convulsant pilocarpine.

Despite numerous neuroendocrinological studies of seizures, the influence of estrogen and progesterone on seizures and epilepsy remains unclear. This may be due to the fact that previous studies have not systematically compared distinct endocrine conditions and included all relevant controls. The goal of the present study was to conduct such a study using pilocarpine as chemoconvulsant. Thus, age and weight-matched, intact or ovariectomized rats were tested to determine incidence of status epilepticus and to study events leading to status. Intact female rats were sampled at each cycle stage (proestrus, estrus, metestrus, or diestrus 2). Convulsant was administered at the same time of day, 10:00-10:30 a.m. Statistical analysis showed that there was a significantly lower incidence of status on the morning of estrus, but differences were attenuated in older animals. Ovariectomized rats were distinct in their rapid progression to status. These results show that the incidence of status in female rats following pilocarpine injection, and the progression to pilocarpine-induced status, are influenced by reproductive state as well as age. The hormonal milieu present specifically on the morning of estrus appears to decrease susceptibility to pilocarpine-induced status, particularly at young ages. In contrast, the chronic absence of reproductive steroids that characterizes the ovariectomized rat leads to a more rapid progression to status. This dissociation between incidence vs. progression provides new insight into the influence of estrogen and progesterone on seizures.

Kynurenate and 7-chlorokynurenate formation in chronically epileptic rats.

PURPOSE: The tryptophan metabolite kynurenic acid (KYNA) and its synthetic derivative, 7-chlorokynurenic acid (7-Cl-KYNA), are antagonists of the glycine co-agonist ("glycine(B)") site of the N-methyl-D-aspartate (NMDA)-receptor. Both compounds have neuroprotective and anticonvulsive properties but do not readily penetrate the blood-brain barrier. However, KYNA and 7-Cl-KYNA can be formed in, and released from, astrocytes after the peripheral administration of their transportable precursors kynurenine and 4-chlorokynurenine, respectively. The present study was designed to examine these biosynthetic processes, as well as astrogliosis, in animals with spontaneously recurring seizures. METHODS: The fate and formation of KYNA and 7-Cl-KYNA was studied in vivo (microdialysis) and in vitro (tissue slices) in rats exhibiting chronic seizure activity (pilocarpine model) and in appropriate controls. Neuronal loss and gliosis in these animals were examined immunohistochemically. RESULTS: In vivo microdialysis revealed higher ambient extracellular KYNA levels and enhanced de novo formation of 7-Cl-KYNA in the entorhinal cortex and hippocampus in epileptic rats. Complementary studies in tissue slices showed increased neosynthesis of KYNA and 7-Cl-KYNA in the same two brain areas. Microscopic analysis revealed pronounced astrocytic reactions in entorhinal cortex and hippocampus in epileptic animals. CONCLUSIONS: These results demonstrate that the epileptic brain can synthesize glycine(B) receptor antagonists in situ. Astrogliosis probably accounts for their enhanced production in chronically epileptic rats. These results bode well for the use of 4-chlorokynurenine in the treatment of chronic seizure disorders.

Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus.

Adult dentate neurogenesis is important for certain types of hippocampal-dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety-related behaviour and learning and memory. We report that adult NPY receptor knock-out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin-positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1-receptor mediated and involves extracellular signal-regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY-releasing interneurons may modulate dentate neurogenesis.

Preemptive low-frequency stimulation decreases the incidence of amygdala-kindled seizures.

PURPOSE: The use of electrical stimulation as a therapy for epilepsy is currently being studied in experimental animals and in patients with epilepsy. This study examined the effect of preemptive, low-frequency, 1-Hz sine wave stimulation (LFS) on the incidence of amygdala-kindled seizures in the rat. METHODS: Electrodes were implanted into the basolateral amygdalae of adult male rats. All animals received a kindling stimulus of 60-Hz, 400-microA, sine wave for 1 s twice a day. Experimental animals received an additional LFS consisting of 1 Hz, 50 microA for 30 s immediately before the kindling stimulus. Afterdischarge (AD) duration, behavioral seizure score, the number of stimulations required to elicit the first stage five seizure and to become fully kindled were measured. After 20 stimulations, a crossover procedure was performed. Fully kindled rats from each group were switched, so that the original controls received LFS plus the kindling stimulus, and the original experimental rats received only the kindling stimulus. RESULTS: During kindling acquisition, LFS induced a significant decrease in AD duration. A significant increase in the number of times the kindling stimulus failed to elicit an AD was noted. Control rats exhibited an AD 99% of the time compared with 70% in experimental rats (p < 0.0001; Fisher's Exact test). In fully kindled animals, the incidence of stage five seizures in the original controls significantly decreased from 98% to 42% (p < 0.0001) when the LFS was added to the kindling paradigm. CONCLUSIONS: The dramatic decrease in the incidence of stage 5 seizures in fully kindled animals after preemptive LFS strongly suggests that LFS may be an effective therapy for the prevention of seizures in patients with epilepsy.

Vascular endothelial growth factor (VEGF) in seizures: a double-edged sword.

Vascular endothelial growth factor (VEGF) is a vascular growth factor which induces angiogenesis (the development of new blood vessels), vascular permeability, and inflammation. In brain, receptors for VEGF have been localized to vascular endothelium, neurons, and glia. VEGF is upregulated after hypoxic injury to the brain, which can occur during cerebral ischemia or high-altitude edema, and has been implicated in the blood-brain barrier breakdown associated with these conditions. Given its recently-described role as an inflammatory mediator, VEGF could also contribute to the inflammatory responses observed in cerebral ischemia. After seizures, blood-brain barrier breakdown and inflammation is also observed in brain, albeit on a lower scale than that observed after stroke. Recent evidence has suggested a role for inflammation in seizure disorders. We have described striking increases in VEGF protein in both neurons and glia after pilocarpine-induced status epilepticus in the brain. Increases in VEGF could contribute to the blood-brain barrier breakdown and inflammation observed after seizures. However, VEGF has also been shown to be neuroprotective across several experimental paradigms, and hence could potentially protect vulnerable cells from damage associated with seizures. Therefore, the role of VEGF after seizures could be either protective or destructive. Although only further research will determine the exact nature of VEGF's role after seizures, preliminary data indicate that VEGF plays a protective role after seizures.

Brain stimulation as a therapy for epilepsy.

The failure of current antiepileptic therapies to adequately treat a significant number of epileptic patients highlights the need for the development of new treatments for the disorder. A new strategy that is currently being developed is to deliver electrical stimulation directly to the brain to decrease or prevent seizure activity. Clinical evidence that electrical stimulation could interfere with seizure activity was initially reported in the 1930's. However, many of these early studies consisted of case reports or were poorly controlled. In addition, there were a number of studies that failed to observe any beneficial effect of brain stimulation on seizures. More recently, deep brain stimulation has been used successfully to treat patients with movement disorders and vagus nerve stimulation has been shown to effectively decrease seizure activity in a select population of epilepsy patients. These advances have led to a reexamination of the potential therapeutic benefits of deep brain stimulation for the treatment of epilepsy. There is now experimental and clinical evidence that direct electrical stimulation of the brain can prevent or decrease seizure activity. However, several fundamental questions remain to be resolved. They include where in the brain the stimulus should be delivered and what type of stimulation would be most effective. One goal of this research is to combine the beneficial aspects of electrical stimulation with seizure detection technology in an implantable responsive stimulator. The device will detect the onset of a seizure and deliver an electrical stimulus that will safely block seizure activity without interfering with normal brain function.

Hippocampal excitability increases during the estrous cycle in the rat: a potential role for brain-derived neurotrophic factor.

To test the hypothesis that induction of BDNF may contribute to changes in hippocampal excitability occurring during the female reproductive cycle, we examined the distribution of BDNF immunoreactivity and changes in CA1 and CA3 electrophysiology across the estrous cycle in rats. Hippocampal BDNF immunoreactivity increased on the day of proestrus as well as on the following morning (estrus), relative to metestrus or ovariectomized animals. Changes in immunoreactivity were clearest in mossy fiber axons of dentate gyrus granule cells, which contain the highest concentration of BDNF. Increased immunoreactivity was also apparent in the neuropil-containing dendrites of CA1 and CA3 neurons. Electrophysiological recordings in hippocampal slices showed robust cycle-dependent differences. Evoked responses of CA1 neurons to Schaffer collateral stimulation changed over the cycle, with larger maximum responses at both proestrus and estrus relative to metestrus. In area CA3, repetitive hilar stimuli frequently evoked multiple population spikes at proestrus and estrus but only rarely at other cycle stages, and never in slices of ovariectomized rats. Hyperexcitability in area CA3 at proestrus was blocked by exposure to the high-affinity neurotrophin receptor antagonist K252a, or an antagonist of the alpha7 nicotinic cholinergic receptor, whereas it was induced at metestrus by the addition of BDNF to hippocampal slices. These studies suggest that hippocampal BDNF levels change across the estrous cycle, accompanied by neurophysiological responses that resemble the effects of BDNF treatment. An estrogen-induced interaction of BDNF and alpha7 nicotinic receptors on mossy fibers seems responsible for estrous cycle changes in area CA3. Periovulatory changes in hippocampal function may, thus, involve estrogen-induced increases in BDNF expression.