Sex differences in behavioral pathology induced by subconvulsive stimulation during early postnatal life are overcome by epileptic activity in the pre-juvenile weanling period.

Chronic subconvulsive activity in early life leads to sex-related autistic-like deficits in handling, object recognition, and social performance in pre-pubertal rats. Since autism and epilepsy are common neurodevelopmental disorders with high coincidence, we tested whether early-life chronic subconvulsive activity compared to convulsive activity alters handling, spatial memory, lateralization, coping strategy and the seizure threshold in a sex-dependent manner. A hypothesis is that convulsive seizures may alter sex differences induced by subconvulsive (SC) activity. Serial subconvulsive doses of kainic acid (KA) were administered postnatally (0.25-1 mg/kg) for 15 days to induce the chronic subconvulsive phenotype (SC group). Age-matched controls and a subset of SC pups were exposed to a convulsive dose of KA (KA and SC + KA groups; 7.5 mg/kg) or flurothyl vapors. In our open handling test, controls and the ASD groups escaped to a similar degree whereas after convulsive seizures, the pups exhibited freezing behavior; no escapes occurred. In the spontaneous alternating T-Maze control males and females entered the left arm with higher frequency. The SC males but not SC females entered left and right arms to a similar degree; alternation rates were reduced to chance revealing a sex difference. However, in KA and SC + KA groups, there was a sharp loss of spontaneous alternation rates. The rapid repetitive entries shifted to the right in both sexes possibly be due to hippocampal injury and changes in network activity induced by status epilepticus. In the forced swim test (FST), control and CS females were more active than corresponding males. After convulsions, immobility was reduced and vertical mobility was increased in SC and SC + KA males suggesting an elevated coping strategy compared to females. Onset and severity of KA induced status epilepticus was delayed in SC males and females possibly due to desensitization of KA receptors. Following flurothyl exposure, control males had faster onset of twitches and clonic seizures than control females which disappeared after the sub-convulsive pre-treatment. Data suggest that behavioral manifestations are more readily detectable between males and females when low levels of hyperexcitation are present chronically in early postnatal development but diminished after tonic-clonic convulsions persist. Therefore, therapeutic interventions may benefit patients if initiated upon the initial onset of sex-related autistic pathologies, particularly in males, which may reduce subsequent vulnerability to seizures.

Cannabidiol reduces lesion volume and restores vestibulomotor and cognitive function following moderately severe traumatic brain injury.

Despite the high incidence of traumatic brain injury (TBI), there is no universal treatment to safely treat patients. Blunt brain injuries destroy primary neural tissue that results in impaired perfusion, excessive release of glutamate, inflammation, excitotoxicity, and progressive secondary neuronal cell death. We hypothesized that administration of cannabidiol (CBD) directly to a brain contusion site, will optimize delivery to the injured tissue which will reduce local neural excitation and inflammation to spare neural tissue and improve neurological outcome following TBI. CBD was infused into a gelfoam matrix forming an implant (CBDi), then applied over the dura at the contusion site as well as delivered systemically by injection (CBD.IP). Post-injury administration of CBDi+IP greatly reduced defecation scores, lesion volume, the loss of neurons in the ipsilateral hippocampus, the number of injured neurons of the contralateral hippocampus, and reversed TBI-induced glial fibrillary acidic protein (GFAP) upregulation which was superior to either CBD.IP or CBDi treatment alone. Vestibulomotor performance on the beam-balance test was restored by 12 days post-TBI and sustained through 28 days. CBDi+IP treated rats exhibited preinjury levels of spontaneous alternation on the spontaneous alternation T-maze. In the object recognition test, they had greater mobility and exploration of novel objects compared to contusion or implant alone consistent with reduced anxiety and restored cognitive function. These results suggest that dual therapy by targeting the site of injury internally with a CBD-infused medical carrier followed by systemic supplementation may offer a more effective countermeasure than systemic or implant treatment alone for the deleterious effects of penetrating head wounds.

Chronic subconvulsive activity during early postnatal life produces autistic behavior in the absence of neurotoxicity in the juvenile weanling period.

The diagnosis of autism spectrum disorder (ASD) varies from very mild to severe social and cognitive impairments. We hypothesized that epigenetic subconvulsive activity in early postnatal life may contribute to the development of autistic behavior in a sex-related manner. Low doses of kainic acid (KA) (25-100 µg) were administered to rat pups for 15 days beginning on postnatal (P) day 6 to chronically elevate neuronal activity. A battery of classical and novel behavioral tests was used, and sex differences were observed. Our novel open handling test revealed that ASD males nose poked more often and ASD females climbed and escaped more frequently with age. In the social interaction test, ASD males were less social than ASD females who were more anxious in handling and elevated plus maze (EPM) tasks. To evaluate group dynamics, sibling and non-sibling control and experimental animals explored 3 different shaped novel social environments. Control pups huddled quickly and more frequently in all environments whether they socialized with littermates or non-siblings compared to ASD groups. Non-sibling ASD pups were erratic and huddled in smaller groups. In the object recognition test, only ASD males spent less time with the novel object compared to control pups. Data suggest that chronic subconvulsive activity in early postnatal life leads to an ASD phenotype in the absence of cell death. Males were more susceptible to developing asocial behaviors and cognitive pathologies, whereas females were prone to higher levels of hyperactivity and anxiety, validating our postnatal ASD model apparent in the pre-juvenile period.

Exposing immature hippocampal neurons to excitotoxins reveals distinct transcriptome and protein regulation with induction of common survival signaling pathways.

Early life traumas lead to neuroprotection by preconditioning mechanisms. To determine which genes and pathways are most likely involved in specific adaptive effects, immature hippocampal cultures were exposed to a single high dose of glutamate (250 µM), NMDA (100 µM), or KA (300 µM) for 48 h (5-7 DIV) based on our prior "two hit" in vitro model of preconditioning. Transcriptome profiling and immunocytochemistry of gene candidates were performed 7 days later when cultured neurons mature (14 DIV). Many genes were up- and down- regulated involving distinct Ca2+-binding protein families, G-coupled proteins, various growth factors, synaptic vesicle docking factors, certain neurotransmitter receptors, heat shock, oxidative stress, and certain anti-apoptotic Bcl-2 gene members that influence neuronal survival. Immunohistochemistry showed a marked decrease in the number of Calb1 and Calm2 positive neurons following NMDA but not after glutamate exposure whereas ryanodine and Cav1.2 voltage gated channel expression was less affected. Survivors had marked increases in Calm2 immunostaining; however, high-density neural clusters observed in controls, were depleted after NMDA and partly diminished after glutamate. While NR1 mRNA expression was decreased in the microarray, specific antibodies revealed selective loss of the NR1C1 splice variant. Calm2 which can inactivate NMDA receptors by binding to C1 but not C2 regions of its NR1 subunit suggests that loss of the C1 splice variant will reduce co-regulation with Calm2 and alter NR1 trafficking, phosphorylation, and NMDA currents following early life NMDA exposure. A dramatic reduction in the density of GABAAα5 and GABAB receptor expressing neurons was observed after NMDA exposure but immunodensity measurements were unchanged as was the expression of the GABA synthesizing enzyme, GAD, suggesting that fast inhibitory neurotransmission and response to benzodiazepines and GABAB-mediated IPSPs may be preserved in matured survivors. Selective upregulation of Chat and CNRIP was detected after glutamate treatment suggesting this condition would decrease cholinergic and excitatory neurotransmission by decreasing Ach content and CB1 interacting protein function. This decrease likely contributes to memory and attention tasks deficits that follow a single early neurological insult. Diverse changes that follow overactivation of excitatory networks of immature neurons appear long-lasting or permanent and are expected to have profound effects on network function and adaptive responses to further insult.

Selective inhibition of metabotropic glutamate type 1 alpha receptor (mGluR1α) reduces cell proliferation and migration following status epilepticus in early development.

The present study examined whether a single or multiple episode(s) of status epilepticus induced with kainic acid (KA) during the first 3 weeks of postnatal (P) development would aberrantly stimulate proliferation zones that alters migration to potentially injured areas and whether they would be blocked by selective Group I mGluR antagonists. mGluR1α (LY367385) and mGluR5 (MPEP) antagonists were administered 2h following KA-induced status epilepticus and animals were examined after 7days. Proliferating cells of the subventricular zone (SVZ), third ventricle, hippocampus, amygdala cortical complex were analyzed with the proliferative marker, Ki67; and two complementary retrograde dye tracers. Proliferation increased in extrahippocampal limbic structures when KA was administered on P13 or P20 which correlated with number of injured cells at the older age. LY367385 post-treatment caused striking decreases in proliferation in all limbic structures in the presence and absence of injury, whereas a reduction with MPEP was observed only within the amygdala cortical complex (Amg/ERcx) in the presence of multiple seizures (3×KA). After 3×KA and LY367385 post-treatments, diminished co-staining of dye tracers with Ki67 was observed within the Amg/ERcx despite high levels of progenitors marked by the retrograde tracers in this region. This indicates that not only was local proliferation within the SVZ and distant structures inhibited, but also that migration itself was reduced indirectly since there were less cells to migrate from the SVZ. Co-labeling with biomarkers provided evidence for neuronal differentiation suggesting potential aberrant integration may occur in distant locations, and that targeting of mGluR1α receptors may be a potential therapeutic strategy for future development.

Efficacy of Retigabine on Acute Limbic Seizures in Adult Rats.

BACKGROUND AND PURPOSE: The efficacy of retigabine (RGB), a positive allosteric modulator of K+ channels indicated for adjunct treatment of partial seizures, was studied in two adult models of kainic acid (KA)-induced status epilepticus to determine it's toleratbility. METHODS: Retigabine was administered systemiclly at high (5 mg/kg) and low (1-2 mg/kg) doses either 30 min prior to or 2 hr after KA-induced status epilepticus. High (1 µg/µL) and low (0.25 µg/µL) concentrations of RGB were also delivered by intrahippocampal microinjection in the presence of KA. RESULTS: Dose-dependent effects of RGB were observed with both models. Lower doses increased seizure behavior latency and reduced the number of single spikes and synchronized burst events in the electroencephalogram (EEG). Higher doses worsened seizure behavior, produced severe ataxia, and increased spiking activity. Animals treated with RGB that were resistant to seizures did not exhibit significant injury or loss in GluR1 expression; however if stage 5-6 seizures were reached, typical hippocampal injury and depletion of GluR1 subunit protein in vulernable pyramidal fields occurred. CONCLUSIONS: RGB was neuroprotective only if seizures were significantly attenuated. GluR1 was simultaneously suppressed in the resistant granule cell layer in presence of RGB which may weaken excitatory transmission. Biphasic effects observed herein suggest that the human dosage must be carefully scrutinized to produce the optimal clinical response.

Retigabine calms seizure-induced behavior following status epilepticus.

In adult rats, intraperitoneal injection of kainate (KA) results in sustained status epilepticus and persistent behavioral comorbidities such as hyperexcitability, anxiety, and altered response to environmental cues. Intrahippocampal KA also results in sustained status epilepticus and continuous high frequency oscillations in the electroencephalograph (EEG), although subsequent behavioral side effects are unknown. We hypothesized that retigabine, a recently discovered anticonvulsant and potent positive modulator of Kv7 channels, may attenuate seizure-induced behavioral abnormalities. Status epilepticus was induced by administration of KA either intraperitoneally (15 mg/kg) or by single intrahippocampal injection (1.0 µg/0.5 µL). After 24 h, half of systemically KA-treated animals that reached stage 6 seizures were injected once daily with retigabine (5 mg/kg) for 14 continuous days. All groups underwent three behavioral tests--capture and handling, open field, and elevated plus maze--24 h following the last retigabine treatment and were sacrificed at 25-28 days. In the capture and handling test, systemic KA treatment resulted in frisky behavior and resistance to capture with wild attempts to escape during the 1st, 2nd, and 3rd weeks of the observation period. In contrast, these behaviors were attenuated in KA+retigabine-treated animals. In the open-field test, KA-treated animals spent more time in the center zone, but KA+retigabine-treated rats had greater overall activity compared with those having vehicle, KA, or retigabine-only treatment. In the elevated plus maze, KA+retigabine-treated animals traveled greater distances in open and closed arms (proximal and distal) compared with controls, also signifying anxiety reduction. Retigabine-only-treated rats traveled more in the open proximal arms compared with controls, indicating increased hyperlocomotion in normotensive rats. Although treatment with KA+retigabine resulted in anxiolytic-like effects in all three behavioral tasks compared with vehicle, this group did not significantly differ from systemically KA-treated rats in most measurements in open-field and elevated plus maze tasks, suggesting that retigabine may also cause hyperlocomotion unrelated to anxiety level. Despite that intrahippocampal KA-treated rats displayed comparable seizure behavior, epileptiform activity, and hippocampal injury, their behavior resembled the controls, suggesting that molecular and subsequent cellular changes are also partially responsible for anxiolytic-like effects and that these results are likely independent of the hippocampus.

Transcriptome profiling of hippocampal CA1 after early-life seizure-induced preconditioning may elucidate new genetic therapies for epilepsy.

Injury of the CA1 subregion induced by a single injection of kainic acid (1 × KA) in juvenile animals (P20) is attenuated in animals with two prior sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early-life conditioning seizures we profiled and compared the transcriptomes of CA1 subregions from control, 1 × KA- and 3 × KA-treated animals. More genes were regulated following 3 × KA (9.6%) than after 1 × KA (7.1%). Following 1 × KA, genes supporting oxidative stress, growth, development, inflammation and neurotransmission were upregulated (e.g. Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12 and Grik1). After 3 × KA, protective genes were differentially over-expressed [e.g. Cat, Gpx7, Gad1, Hspa12A, Foxn1, adenosine A1 receptor, Ca(2+) adaptor and homeostasis proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp and suppressor of cytokine signaling (Socs3)]. Distinct anti-inflammatory interleukins (ILs) not observed in adult tissues [e.g. IL-6 transducer, IL-23 and IL-33 or their receptors (IL-F2 )] were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1 × KA but reduced after 3 × KA; the pro-inflammatory gene Cox1 was either upregulated or unchanged after 1 × KA but reduced by ~70% after 3 × KA. Enhanced GFAP immunostaining following 1 × KA was selectively attenuated in the CA1 subregion after 3 × KA. The observed differential transcriptional responses may contribute to early-life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca(2+) permeability of the hippocampus and redirecting inflammatory and apoptotic pathways. These changes could lead to new genetic therapies for epilepsy.

Lack of resveratrol neuroprotection in developing rats treated with kainic acid.

In adult rats, trans-resveratrol attenuates kainic acid (KA)-induced convulsions and the associated hippocampal neurotoxicity. Increased neuronal survival was correlated with reduced lipid peroxidation. Since free radical generation after KA is age dependent and does not correlate with the onset of seizure-induced injury, the present study investigated whether daily trans-resveratrol treatment in development could protect the juvenile hippocampus from seizures and onset of damage at postnatal (P) day 21. Rat pups were treated with daily injections of trans-resveratrol under three dosage regimens (1-15 mg/kg and 20-50mg/kg). Weight, electroencephalography (EEG), histology, and N-methyl-d-aspartate (NMDA) receptor expression were determined. Malondialdehyde (MDA) concentration was assessed from separate animals. trans-Resveratrol did not interfere with growth or attenuate KA-induced EEG seizures. However, modest protection was afforded in the CA1, the subregion most sensitive to injury at this age. The CA3 and entorhinal amygdala cortex (AMG/EC) were not spared. Changes in NR1 subunit or NR1 C2 splice variant expression were also not prevented. Baseline MDA concentrations of hippocampal subfields were low at P14, P21, and P60 and high in aged adults. Glutamate (100 µM) to stimulate peroxidation products was significant at young ages but was much greater at older ages. After KA, elevated MDA levels were observed at 24h but only in adult preparations. Thus, while antioxidant therapy with trans-resveratrol may be considered as an adjunctive therapy to hinder epileptic activity and neurodegeneration at adult ages, it had only modest effects at young ages when production of free radicals within limbic structures is limited in this experimental model of seizures.

Inverse relationship of cannabimimetic (R+)WIN 55, 212 on behavior and seizure threshold during the juvenile period.

Cannabinoids have anti-convulsant effects in both in vivo and in vitro models of status epilepticus. Since the development of spontaneous seizures and neuronal vulnerability are age-dependent, we hypothesized that the anti-convulsant effects of cannabimimetics are also age-dependent. We administered a single injection of varied doses of (R+)WIN 55,212 (0.5, 1, 5 mg/kg) to postnatal (P) day 20 rats 90 min prior to induction of kainate (KA)-induced status epilepticus. The highest dose of (R+)WIN 55,212 (5 mg/kg) resulted in rapid onset of behavioral stupor, loss of balance, stiffening and immobility while standing on hind legs or laying flat in prone position; lower doses had minimal or no behavioral effect. After KA administration, seizure scores and electroencephalography (EEG) recordings were inversely related to (R+)WIN 55,212 dosage whereby higher doses were associated with high seizures scores and synchronous epileptiform activity and low doses with low seizure scores and diminished spiking in the EEG. Immunohistochemistry revealed a dose-dependent reduction in CB1 receptor expression with increasing concentrations of (R+)WIN 55,212 in presence or absence of KA seizures. Nissl and NeuN staining showed hippocampal injury was attenuated only when seizures were mild following low doses of WIN 55,212 (0.5, 1 mg/kg), consistent with the level of CB1 expression. Since low doses abolished seizures without psychotropic side-effects further study may facilitate a groundbreaking cannabamimetic therapeutic strategy to treat early-life seizures. Higher doses had adverse effects on behavior and failed to prevent seizures and protect CA1 neurons possibly due to inactivation or loss of CB1 receptors.

Glutamatergic and morphological alterations associated with early life seizure-induced preconditioning in young rats.

Postnatal day (P)20 rats are sensitive to CA1 injury following a single injection of kainic acid (KA) but are resistant to this injury when animals have a history of two neonatal seizures. We hypothesized that the two earlier seizures led to neuroprotection by a preconditioning mechanism. Therefore, morphology, [Ca(2+)](i) and NMDA subunit proteins of the hippocampus were examined after KA was administered once (1 × KA, on P6, P9, P13 or P20), twice (2 × KA, on P6 and P9) or three times (3 × KA, on P6, P9, P13 or P20). After 1 × KA on P20, the Golgi method revealed marked decreases in spine densities and aborization of CA1 and CA3 apical dendrites. After 3 × KA, morphological alterations were attenuated in CA1 neurons and were similar to pruning observed after 1 × KA on P6 or 2 × KA. After 1 × KA at P13, baseline [Ca(2+)](i) was elevated within pyramidal and dentate granule cells. N-methyl-D-aspartate (NMDA) responses were simultaneously enhanced. After 3 × KA, Ca(2+) elevations were attenuated. Immunohistochemistry revealed selective depletion of the NR2A/B subunit modulator in the same areas. NR1 subunit expression was downregulated in the subiculum and increased in the CA3, causing a significant shift in the NR1:NR2A/B ratio throughout the hippocampus. After 1 × KA or 3 × KA at P20, reduced expression was only observed in areas of cell injury. Results indicate that different changes in morphology and excitatory responses occur depending upon when seizures begin. Partial pruning and persistent shift in the NR1:NR2A/B ratio among excitatory synapses of the hippocampus early in life may produce epileptic tolerance and protect against subsequent insults.

NR1 knockdown reveals CA1 injury during a developmental period of high seizure susceptibility despite reduced seizure activity.

NMDA receptors (NMDARs) are important for the propagation of seizures. To understand the role of NR1 subunits in the propagation of seizures we knocked down the NR1 subunit by intracranial injection of antisense deoxyoligonucleotides (NR1-AS-ODNs) into the right hippocampus during a window of maximal seizure susceptibility in development. Control missense and sense ODNs followed by focal injection of NMDA (2.5-25 nmoles) into the hippocampal CA1 and sensorimotor cortex of P15 rat pups resulted in behavioral and electrographic (EEG) seizures. After NR1 knockdown, low- and high-doses produced little or no spike activity in the hippocampus and overlying sensorimotor cortex as predicted. Despite reduced activity in the hippocampal and cortical EEG, intracranial NMDA or peripheral kainate (KA)-induced seizures led to paradoxical cell death of CA1 neurons, which is not typically observed in this age group. Histological changes were modest or absent in the cortex away from the infusion site. Signal specificity of the targeted CA1 or cortex was observed in autoradiograms, immunohistochemistry and Western blots. After knockdown, Ca2+ influx was suppressed as both NMDA and muscimol-stimulated Ca2+ permeability of the immature CA1 was blocked in ex-vivo slices measured with FURA-2AM optical dye imaging. Data suggest that certain constituent levels of NMDA receptors distributed on excitatory and/or inhibitory interneurons may be developmentally required for survival of CA1 pyramidal neurons during a critical period when ictal activity is present. Moreover, selective NR1 subunit downregulation simultaneously reduces NMDA and GABA A receptor Ca2+ ion permeability properties that may contribute to a premature cell death mechanism.

Maturational effects of single and multiple early-life seizures on AMPA receptors in prepubescent hippocampus.

The effects of single versus multiple episodes of status epilepticus on the expression of AMPA receptors during a critical growth spurt are unknown. To determine whether the pattern of hippocampal AMPA receptor subunit expression depends upon the age of the animal, timing and number of perinatal seizures, we characterized maturational changes in AMPA receptor protein levels of the hippocampus with immunohistochemistry and Western blotting in rats of juvenile ages with and without a history of neonatal seizures. Kainic acid (KA) was used to induce a single episode of status epilepticus (1 x KA) in rats on P20 or P30. Animals with a history of multiple seizures (3 x KA) were given KA on P6, P9, and then on P20 or P30. After 1 x KA, in P20 and P30 rats that are preferentially sensitive to CA1 damage, GluR1 immunoreactivity was depleted remarkably in CA1 stratum pyramidale and stratum lucidum and only morphologically healthy cells were faintly labeled. At P30, GluR2 subunit expression was nearly absent in the healthy cells and increased within the injured CA1 neuronal population. Western blot analysis confirmed that the GluR1/GluR2 ratio was decreased at P20 and further decreased at P30. A history of perinatal seizures (3 x KA) prevented the age-dependent alterations in the CA1. Except for areas of cell loss, NR1 and NR2A/B antibody labeling was relatively stable throughout the hippocampus at both ages and conditions examined. Data suggest that (i) Ca2+ permeable AMPA receptors may not be responsible for neuronal injury or irreversible cell loss and that (ii) the expression of AMPA receptors after status epilepticus depends upon the age of the animal, the timing of the first insult and subsequent formation of AMPA receptor subunit compositions within specific populations of hippocampal neurons.

Perinatal seizures preferentially protect CA1 neurons from seizure-induced damage in prepubescent rats.

Neonatal seizures may increase neuronal vulnerability later in life. Therefore, status epilepticus was induced with kainate (KA) during the first and second postnatal (P) weeks to determine whether early seizures shift the window of neuronal vulnerability to a younger age. KA was injected (i.p.) once (1x KA) on P13, P20 or P30 or three times (3 x KA), once on P6 and P9, and then either on P13, P20 or P30. After 1x KA, onset to behavioral seizures increased with age. Electroencephalography (EEG) showed interictal events appeared with maturation. After 3 x KA, spike number, frequency, spike amplitude, and high-frequency synchronous events and duration were increased at P13 when compared to age-matched controls. In contrast, P20 and P30 rats had decreases in EEG parameters relative to P20 and P30 rats with 1x KA despite that these animals had the same history of perinatal seizures on P6 and P9. In P13 rats with 1x KA, silver impregnation, hematoxylin/eosin and TUNEL methods showed no significant hippocampal injury and damage was minimal with 3 x KA. In contrast, P20 and P30 rats with 1x KA had robust eosinophilic or TUNEL positive labeling and preferential accumulation of silver ions within inner layer CA1 neurons. After 3 x KA, the CA1 but not CA3 of P20 and P30 rats was preferentially protected following 3 or 6 days. Although paradoxical changes occur in the EEG with maturation, the results indicate that early perinatal seizures do not significantly shift the window of hippocampal vulnerability to an earlier age but induce a tolerance that leads to long-term neuroprotection that differentially affects endogenous properties of CA1 versus CA3 neurons.

Suppression of hippocampal neurogenesis is associated with developmental stage, number of perinatal seizure episodes, and glucocorticosteroid level.

Seizures increase dentate granule cell proliferation in adult rats but decrease proliferation in young pups. The particular period and number of perinatal seizures required to cause newborn granule cell suppression in development are unknown. Therefore, we examined cell proliferation with bromodeoxyuridine (BrdU) immunohistochemistry during the peak of neurogenesis (e.g., P6 and P9) and at later postnatal ages (e.g., P13, P20, or P30) following single and multiple episodes of perinatal status epilepticus induced by kainate (KA). Because an inverse relationship exists between glucocorticosteroids (CORT) levels and granule cell proliferation, plasma CORT levels and electroencephalographic (EEG) activity were simultaneously monitored to elucidate underlying mechanisms that inhibit cell proliferation. In control animals, the number of BrdU-labeled cells increased then declined with maturation. After 1x KA or 2x KA administered on P6 and P9, the numbers of BrdU-labeled cells were not different from age-matched controls. However, rat pups with 3x KA (on P6, P9, and P13) had marked suppression of BrdU-labeled cells 48-72 h after the last seizure (43 +/- 6.5% of control). Cell proliferation was also significantly inhibited on P20 after 2x KA (to 56 +/- 6.9%) or 3x KA (to 54 +/- 7.9%) and on P30 with 3x KA (to 74.5 +/- 8.2% of age-matched controls). Cell death was not apparent as chromatin stains showed increased basophilia of only inner cells lining the granule cell layers, in the absence of eosinophilia, argyrophilia, or terminal deoxynucleotidyl dUTP nick endlabeling (TUNEL) labeling at times examined. In P13 pups with 3x KA, electron microscopy revealed an increased number of immature granule cells and putative stem cells with irregular shape, condensed cytoplasm, and electron dense nuclei, and they were also BrdU positive. The EEG showed no relationship between neurogenesis and duration of high-synchronous ictal activity. However, endocrine studies showed a correlation with BrdU number and age, sustained increases in circulating CORT levels following 1x KA on P6 (0.7 +/- 0.1 to 2.40 +/- 0.86 microg/dl), and cumulative increases that exceeded 10 microg/dl at 4-8 h after 3x KA on P13 or P20. In conclusion, a history of only one or two perinatal seizure(s) can suppress neurogenesis if a second or third seizure recurs after a critical developmental period associated with a marked surge in CORT. During the first 2 weeks of postnatal life sustained increases in postictal circulating CORT levels but not duration or intensity of ictal activity has long-term consequences on neurogenesis. The occurrence of an increased proportion of immature granule cells and putative stem cells with irregular morphology in the absence of neurodegeneration suggests that progenitors may not differentiate properly and remain in an immature state.

Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia.

Hypothermia may afford histological neuroprotection induced by ischemia by preventing aberrant Ca2+ influx through NMDA (N-methyl-D-aspartic acid) or Ca2+-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Expression of hippocampal GluR1A, GluR2B, GluR3C and NMDAR1 (NR1) subunits was investigated by in situ hybridization at 1 and 7 days after 10-min transient global ischemia in the presence and absence of intraischemic or postischemic brain hypothermia (30 degrees C). At 1 day, normothermic ischemia markedly suppressed the expression of GluR1A, GluR2B, and GluR3C receptor mRNAs to a similar degree in the vulnerable CA1. Less vulnerable CA3a-c subregions were also acutely downregulated. NR1 mRNA expression was reduced in CA1 but to a lesser extent than AMPA mRNAs. At 7 days after normothermic ischemia, a time of marked CA1 cell loss, all three AMPA transcripts were nearly absent in CA1 while a percentage (33.9+/-7.2%) of NR1 mRNA remained. Intraischemic hypothermia fully blocked the damage and non-selective mRNA downregulations at 1 and 7 days. By contrast, postischemic hypothermia postponed neurodegeneration but only partially rescued the expression of AMPA and NR1 mRNAs at 7 days and not at 1 day after the insult. Therefore, hippocampal AMPA receptor mRNAs decline at a relatively similar rate after normothermic global ischemia and cellular neuroprotection by intraischemic hypothermia occurred independently of altered subunit composition of AMPA receptors. Since decreases persist within resistant neurons under the postischemic condition, AMPA receptor-mediated Ca2+ currents probably do not contribute to selective vulnerability.

Distribution of glutamate and preproenkephalin messenger RNAs following transient focal cerebral ischemia.

Middle cerebral artery occlusion may result in increased activation of N-methyl-D-aspartate- or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type receptors by glutamate and lead to neuronal cell death. To characterize molecular events that precede cell death following transient focal ischemia, in situ hybridization histochemistry was used to measure levels of glutamate receptor subunit 1 (GluR1), GluR2, GluR3, N-methyl-D-aspartate receptor subunit 1 (NR1) and preproenkephalin messenger RNAs in adult rats at various recirculation times (1.5, 3 and 24 h) following a 90-min period of middle cerebral artery occlusion. At 1.5 and 3 h recirculation, autoradiography showed pronounced but differential decreases in AMPA, NR1 and preproenkephalin messenger RNA expression throughout the infarcted ipsilateral striatum. Non-uniform patterns of in situ hybridization grains emerged such that many striatal neurons were depleted of AMPA and preproenkephalin messenger RNAs, while others retained control levels. In cortical regions destined to undergo infarction, GluR2 and NR1 messenger RNAs were preferentially reduced relative to the contralateral side (to 75+/-8.5% and 66+/-4.5%, respectively); GluR1, GluR3 and preproenkephalin messenger RNAs were unaltered. At 24 h recirculation, depletion of striatal and cortical messenger RNAs became less selective. GluR3 and preproenkephalin messenger RNAs were up-regulated in ipsilateral spared regions of the striatum, and GluR1 and GluR2 messenger RNAs increased bilaterally in the cingulate cortex and in selective nuclei of the amygdala. Histological cell death or neurodegeneration was not detected in areas of reduced glutamate and preproenkephalin messenger RNA expression in either the ipsilateral striatum or cortex before 24 h. These findings suggest that complex and long-lasting decreases in messenger RNA expression occur prior to significant cell loss in regions destined to undergo infarction. Increased formation of Ca2+-permeable AMPA receptor assemblies may occur in "unspared" and "spared" regions via different mechanisms and contribute to alterations in post-ischemic synaptic activity. The possibility arises that there may be altered relationships between glutamatergic and enkephalin synapses, since the dorsolateral striatum, where preproenkephalin messenger RNA expression is acutely reduced, receives innervation by the affected ipsilateral cortical region.

Unilateral GluR2(B) hippocampal knockdown: a novel partial seizure model in the developing rat.

Kainic acid (KA) induces status epilepticus in both adult and young rats but with different consequences on pathology and gene expression. In adults, GluR2(B) AMPA subunit expression is markedly reduced in CA3 neurons before neurodegeneration. In pups, the GluR2(B) subunit is sustained, possibly contributing to neuronal survival. Mechanisms underlying the reduced vulnerability of developing neurons to seizures was investigated by examining the effects of unilateral microinfusions of GluR2(B) antisense oligodeoxynucleotides (AS-ODNs) into the hippocampus of young rats in the presence or absence of a subconvulsive dose of KA. GluR2(B) AS-ODN infusions resulted in spontaneous seizure-like behavior, high stimulus intensity population spikes in the absence of long-term potentiation, and neurodegeneration of CA3 neurons lateral to the infusion site. Electroencephalography revealed paroxysmal activity and high-frequency high-amplitude discharges associated with vigorous and continuous scratching, wild running, or bilateral jerking movements. Pups lacking phenotypic behavior exhibited high-rhythmic oscillations and status epilepticus by the dose of KA used. Radiolabeled AS-ODNs accumulated throughout the ipsilateral dorsal hippocampus. GluR2(B) but not GluR1(A) receptor protein was markedly reduced after GluR2(B) knockdown. In contrast, GluR1(A) knockdown reduced GluR1(A) but not GluR2(B) protein without change in behavior or morphology. Therefore, unilateral downregulation of hippocampal GluR2(B) but not GluR1(A) protein reduces the seizure threshold and survival of CA3 neurons in the immature hippocampus, possibly providing a novel partial seizure model in the developing rat.

Age-dependent vulnerability to seizures.

Seizure disorders frequently occur early in life. Seizures are classified as reactive, symptomatic, or idiopathic depending on whether their cause can be identified. Reactive seizures are the result of acute environmental perturbations. Early in life, many stressors can produce seizures and the ultimate outcome may depend on the particular precipitating factor and its intensity. Febrile convulsions are the most common reactive seizures, although they must be differentiated from symptomatic seizures precipitated by fever. Symptomatic seizures are often associated with varying degrees of central nervous system (CNS) insults, including congenital malformations and metabolic storage diseases of the gray matter. These seizures may have age-specific characteristics and may at times be difficult to treat with conventional antiepileptic treatments. To develop a better understanding of the pathophysiology of seizures early in life, we have extensively used animal models of epilepsy. In this chapter, we report our findings with a rat model of developmental cortical dysplasias produced by intrauterine injections of methylazoxymethanol acetate. These rats are more susceptible to kainic acid, flurothyl, and hyperthermic seizures than normal rats. Rats with severe cortical dysplasia are most susceptible to seizures. We have also studied the mechanisms involved in the control of seizures during development because status epilepticus is more prevalent in infants than in adults. Our data suggest that the substantia nigra may play a crucial role in status epilepticus as a function of age. In the adult substantia nigra two regions mediate opposing effects on seizures following infusions of gamma-aminobutyric acid type A (GABAA) agents. One region is located in the anterior substantia nigra, and muscimol infusions in this region mediate anticonvulsant effects. The second region is in the posterior substantia nigra, and here muscimol infusions produce proconvulsant effects. In situ hybridization data demonstrate that, at the cellular level, neurons in the two substantia nigra regions differ in the amount of hybridization grains for GABAA receptor alpha 1 and gamma 2L subunit mRNAs. In developing male rats, only the "proconvulsant" region is present up to the age of 21 days. The transition from the immature to mature substantia nigra mediated seizure control occurs between age 25 and 30 days. The identification of age-dependent functional networks involved in the containment of seizures may lead to possible new pharmacologic strategies to control seizures, thus aiding the development of age-appropriate treatments of seizure disorders.