TRPV1 promotes repetitive febrile seizures by pro-inflammatory cytokines in immature brain.

Febrile seizure (FS) is the most common seizure disorder in children, and children with FS are regarded as a high risk for the eventual development of epilepsy. Brain inflammation may be implicated in the mechanism of FS. Transient receptor potential vanilloid 1 (TRPV1) is believed to act as a monitor and regulator of body temperature. The role of inflammation in synaptic plasticity mediation indicates that TRPV1 is relevant to several nervous system diseases, such as epilepsy. Here, we report a critical role for TRPV1 in a febrile seizure mouse model and reveal increased levels of pro-inflammatory factors in the immature brain. Animals were subjected to hyperthermia for 30 min, which generates seizures lasting approximately 20 min, and then were used for experiments. To invoke frequently repetitive febrile seizures, mice are exposed to hyperthermia for three times daily at an interval of 4h between every time induced seizure, and a total of 4 days to induce. Behavioral testing for febrile seizures revealed that a TRPV1 knock-out mouse model demonstrated a prolonged onset latency and a shortened duration and seizure grade of febrile seizure when compared with wild type (WT) mice. The expression levels of both TRPV1 mRNA and protein increased after a hyperthermia-induced febrile seizure in WT mice. Notably, TRPV1 activation resulted in a significant elevation in the expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and HMGB1) in the hippocampus and cortex. These data indicate that the reduction of TRPV1 expression parallels a decreased susceptibility to febrile seizures. Thus, preventative strategies might be developed for use during febrile seizures.

Decreased A-currents in hippocampal dentate granule cells after seizure-inducing hypoxia in the immature rat.

PURPOSE: Cerebral hypoxia is a major cause of neonatal seizures, and can lead to epilepsy. Pathologic anatomic and physiologic changes in the dentate gyrus have been associated with epileptogenesis in many experimental models, as this region is widely believed to gate the propagation of limbic seizures. However, the consequences of hypoxia-induced seizures for the immature dentate gyrus have not been extensively examined. METHODS: Seizures were induced by global hypoxia (5-7% O2 for 15 min) in rat pups on postnatal day 10. Whole-cell voltage-clamp recordings were used to examine A-type potassium currents (IA ) in dentate granule cells in hippocampal slices obtained 1-17 days after hypoxia treatment. KEY FINDINGS: Seizure-inducing hypoxia resulted in decreased maximum IA amplitude in dentate granule cells recorded within the first week but not at later times after hypoxia treatment. The decreased IA amplitude was not associated with changes in the voltage-dependence of activation or inactivation removal, or in sensitivity to inhibition by 4-aminopyridine (4-AP). However, consistent with the role of IA in shaping firing patterns, we observed in the hypoxia group a significantly decreased latency to first spike with depolarizing current injection from hyperpolarized potentials. These differences were not associated with changes in resting membrane potential or input resistance, and were eliminated by application of 10 m 4-AP. SIGNIFICANCE: Given the role of IA to slow action potential firing, decreased IA could contribute to long-term hippocampal pathology after neonatal seizure-inducing hypoxia by increasing dentate granule cell excitability during a critical window of activity-dependent hippocampal maturation.

Synaptic connections of hilar basal dendrites of dentate granule cells in a neonatal hypoxia model of epilepsy.

Numerous animal models of epileptogenesis demonstrate neuroplastic changes in the hippocampus. These changes occur not only for the mature neurons and glia, but also for the newly generated granule cells in the dentate gyrus. One of these changes, the sprouting of mossy fiber axons, is derived predominantly from newborn granule cells in adult rats with pilocarpine-induced temporal lobe epilepsy. Newborn granule cells also mainly contribute to another neuroplastic change, hilar basal dendrites (HBDs), which are synaptically targeted by mossy fibers in the hilus. Both sprouted mossy fibers and HBDs contribute to recurrent excitatory circuitry that is hypothesized to be involved in increased seizure susceptibility and the development of spontaneous recurrent seizures (SRS) that occur following the initial pilocarpine-induced status epilepticus. Considering the putative role of these neuroplastic changes in epileptogenesis, a critical question is whether similar anatomic phenomena occur after epileptogenic insults to the immature brain, where the proportion of recently born granule cells is higher due to ongoing maturation. The current study aimed to determine if such neuroplastic changes could be observed in a standardized model of neonatal seizure-inducing hypoxia that results in development of SRS. We used immunoelectron microscopy for the immature neuronal marker doublecortin to label newborn neurons and their HBDs following neonatal hypoxia. Our goal was to determine whether synapses form on HBDs from neurons born after neonatal hypoxia. Our results show a robust synapse formation on HBDs from animals that experienced neonatal hypoxia, regardless of whether the animals experienced tonic-clonic seizures during the hypoxic event. In both cases, the axon terminals that synapse onto HBDs were identified as mossy fiber terminals, based on the appearance of dense core vesicles. No such synapses were observed on HBDs from newborn granule cells obtained from sham animals analyzed at the same time points. This aberrant circuit formation may provide an anatomic substrate for increased seizure susceptibility and the development of epilepsy.

Dexamethasone after early-life seizures attenuates increased susceptibility to seizures, seizure-induced microglia activation and neuronal injury later in life.

Chronic epilepsy can begin with isolated early-life prolonged seizures followed by remission and the re-emergence of seizures later in life. Seizures are known to trigger a neuroinflammatory response to promote neuronal damage and increase the risk of epilepsy. We examined whether post-seizure anti-inflammatory treatment with dexamethasone after early-life seizures could decrease future seizure susceptibility and ameliorate heightened microglia activation and cell injury in response to later-life seizures. Using a "two-hit" model, early-life seizures (SZ) were induced in rats on postnatal day (P) 25 by systemic kainic acid (KA) injection followed by later-life KA at P39. P25 animals were administered anti-inflammatory drugs for 2 or 7 days after first KA exposure to inhibit seizure-induced inflammation. Hippocampal microglial activation was measured after first or second KA treatments to assay neuroinflammation, and the latency and severity of seizures to the second KA treatment were measured to determine seizure susceptibility. In situ end labeling for DNA fragmentation was used to compare KA-induced neuronal injury between treatment groups after the second KA administration. KA-SZ at P25 caused marked microglia activation within 48 hours. At P39, KA-SZ in rats without prior seizures caused a modest (2-fold) increase in microglia assayed 72 hours after KA. In contrast, microglia were markedly activated (5-fold) in response to a second KA-SZ at P39. Short-course (2 days) dexamethasone significantly decreased seizure-induced microglia activation at P25, and ameliorated the exaggerated microglia activation, cell injury, and heightened susceptibility to second-hit seizures. Although short-course dexamethasone was effective, longer term (7 days) administration of dexamethasone resulted in decreased weight gain and increased mortality in animals with or without KA-induced seizures. These data indicated that acute short-term steroid therapy after SZ could inhibit seizure-induced microglia activation and decrease the long-term damaging effects of early-life SZ. These results further implicate seizure-induced inflammation and activation of innate immunity mediated by microglia in the pathogenesis of childhood epilepsy.

Chemokine CCL2 and its receptor CCR2 are increased in the hippocampus following pilocarpine-induced status epilepticus.

BACKGROUND: Neuroinflammation occurs after seizures and is implicated in epileptogenesis. CCR2 is a chemokine receptor for CCL2 and their interaction mediates monocyte infiltration in the neuroinflammatory cascade triggered in different brain pathologies. In this work CCR2 and CCL2 expression were examined following status epilepticus (SE) induced by pilocarpine injection. METHODS: SE was induced by pilocarpine injection. Control rats were injected with saline instead of pilocarpine. Five days after SE, CCR2 staining in neurons and glial cells was examined using imunohistochemical analyses. The number of CCR2 positive cells was determined using stereology probes in the hippocampus. CCL2 expression in the hippocampus was examined by molecular assay. RESULTS: Increased CCR2 was observed in the hippocampus after SE. Seizures also resulted in alterations to the cell types expressing CCR2. Increased numbers of neurons that expressed CCR2 was observed following SE. Microglial cells were more closely apposed to the CCR2-labeled cells in SE rats. In addition, rats that experienced SE exhibited CCR2-labeling in populations of hypertrophied astrocytes, especially in CA1 and dentate gyrus. These CCR2+ astroctytes were not observed in control rats. Examination of CCL2 expression showed that it was elevated in the hippocampus following SE. CONCLUSION: The data show that CCR2 and CCL2 are up-regulated in the hippocampus after pilocarpine-induced SE. Seizures also result in changes to CCR2 receptor expression in neurons and astrocytes. These changes might be involved in detrimental neuroplasticity and neuroinflammatory changes that occur following seizures.

A Rat Model of Perinatal Seizures Provoked by Global Hypoxia.

Hypoxic-ischemic encephalopathy (HIE) refers to acute brain injury that results from perinatal asphyxia. HIE is a major cause of neonatal seizures, and outcomes can range from apparent recovery to severe cognitive impairment, cerebral palsy, and epilepsy. Acute partial seizures frequently aid in indicating the severity and localization of brain injury. However, evidence also suggests that the occurrence of seizures further increases the likelihood of epilepsy in later life regardless of the severity of the initial injury. Here, we describe a neonatal rat model of seizure-provoking mild hypoxia without overt brain injury that has been used to investigate potential epileptogenic effects of hypoxia-associated seizures alone on neonatal brain development. Clinically, HIE is defined by brain injury, and thus, this model is not intended to mimic clinical HIE. Rather, its utility is in providing a model to understand the dynamic and long-term regulation of brain function and how this can be perturbed by early life seizures that are provoked by a commonly encountered pathophysiological trigger. Additionally, the model allows the study of brain pathophysiology without the potential confound of variable neuroanatomical changes that are reactive to widespread cell death.

Vitexin protects against hypoxic-ischemic injury via inhibiting Ca2+/Calmodulin-dependent protein kinase II and apoptosis signaling in the neonatal mouse brain.

Neonatal hypoxic-ischemic is a major cause of death and disability in neonates. In this study, we suggest for the first time that pretreatment with vitexin may suppress a pro-apoptotic signaling pathway in hypoxic-ischemic neuronal injury in neonates by inhibition of the phosphorylation of Ca2+/Calmodulin-dependent protein kinase II. Here we found that vitexin pretreatment reduced brain infarct volume in a dose-dependent manner. In addition, vitexin decreased the number of TUNEL-positive cells and brain atrophy. Furthermore, vitexin improved neurobehavioral outcomes. Vitexin also reduced oxygen glucose deprivation-induced neuronal injury and calcium entry. Vitexin pretreatment increased the Bcl-2/Bax protein ratio and decreased phosphorylation of Ca2+/Calmodulin-dependent protein kinase II and NF-κB, cleaved caspase-3 protein expression 24 hours after injury. Our data indicate that pretreatment with vitexin protects against neonatal hypoxic-ischemic brain injury and thus has potential as a treatment for hypoxic-ischemic brain injury.

AMPA/kainate receptor-mediated downregulation of GABAergic synaptic transmission by calcineurin after seizures in the developing rat brain.

Hypoxia is the most common cause of perinatal seizures and can be refractory to conventional anticonvulsant drugs, suggesting an age-specific form of epileptogenesis. A model of hypoxia-induced seizures in immature rats reveals that seizures result in immediate activation of the phosphatase calcineurin (CaN) in area CA1 of hippocampus. After seizures, CA1 pyramidal neurons exhibit a downregulation of GABA(A) receptor (GABA(A)R)-mediated inhibition that was reversed by CaN inhibitors. CaN activation appears to be dependent on seizure-induced activation of Ca2+-permeable AMPA receptors (AMPARs), because the upregulation of CaN activation and GABA(A)R inhibition were attenuated by GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] or Joro spider toxin. GABA(A)R beta2/3 subunit protein was dephosphorylated at 1 h after seizures, suggesting this subunit as a possible substrate of CaN in this model. Finally, in vivo administration of the CaN inhibitor FK-506 significantly suppressed hypoxic seizures, and posttreatment with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) or FK-506 blocked the hypoxic seizure-induced increase in CaN expression. These data suggest that Ca2+-permeable AMPARs and CaN regulate inhibitory synaptic transmission in a novel plasticity pathway that may play a role in epileptogenesis in the immature brain.

Vitexin reduces hypoxia-ischemia neonatal brain injury by the inhibition of HIF-1alpha in a rat pup model.

Previous studies have demonstrated that the early suppression of HIF-1α after hypoxia-ischemia (HI) injury provides neuroprotection. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside), an HIF-1α inhibitor, is a c-glycosylated flavone that has been identified in medicinal plants. Therefore, we hypothesized that treatment with vitexin would protect against HI brain injury. Newborn rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O2 at 37 °C). Vitexin (30, 45 or 60 mg/kg) was administered intraperitoneally at 5 min or 3 h after HI. Vitexin, administered 5 min after HI, was neuroprotective as seen by decreased infarct volume evaluated at 48 h post-HI. This neuroprotection was removed when vitexin was administered 3 h after HI. Neuronal cell death, blood-brain barrier (BBB) integrity, brain edema, HIF-1α and VEGF protein levels were evaluated using a combination of Nissl staining, IgG staining, brain water content, immunohistochemistry and Western blot at 24 and 48 h after HI. The long-term effects of vitexin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests. Vitexin (45 mg/kg) ameliorated brain edema, BBB disruption and neuronal cell death; Upregulation of HIF-1α by dimethyloxalylglycine (DMOG) increased the BBB permeability and brain edema compared to HI alone. Vitexin attenuated the increase in HIF-1α and VEGF. Vitexin also had long-term effects of protecting against the loss of ipsilateral brain and improveing neurobehavioral outcomes. In conclusion, our data indicate early HIF-1α inhibition with vitexin provides both acute and long-term neuroprotection in the developing brain after neonatal HI injury.

Molecular biology and ontogeny of glutamate receptors in the mammalian central nervous system.

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system. After release from presynaptic terminals, glutamate binds to both ionotropic and metabotropic receptors to mediate fast, slow, and persistent effects on synaptic transmission and integrity. There are three types of ionotropic glutamate receptors. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate receptors are principally activated by the agonist bearing its name and are permeable to cationic flux; hence, their activation results in membrane depolarization. All ionotropic glutamate receptors are believed to be composed of four distinct subunits, each of which is topologically arranged with three transmembrane-spanning and one pore-lining (hairpin loop) domain. In contrast, metabotropic glutamate receptors are G protein (guanine nucleotide-binding protein) -coupled receptors linked to second-messenger systems. Group I metabotropic glutamate receptors are linked to phospholipase C, which results in phosphoinositide hydrolysis and release of calcium from intracellular stores. Group II and group III metabotropic glutamate receptors are negatively linked to adenylate cyclase, which catalyzes the production of cyclic adenosine monophosphate. Each metabotropic glutamate receptor is composed of seven transmembrane-spanning domains, similar to other members of the superfamily of metabotropic receptors, which includes noradrenergic, muscarinic acetylcholinergic, dopaminergic, serotonergic (except type 3 receptors), and gamma-aminobutyric acid (GABA) type B receptors. This review summarizes the relevant molecular biology and ontogeny of glutamate receptors in the central nervous system and highlights some of the roles that they can play during brain development and in certain disease states.

Regulation of Kv4.2 A-Type Potassium Channels in HEK-293 Cells by Hypoxia.

We previously observed that A-type potassium currents were decreased and membrane excitability increased in hippocampal dentate granule cells after neonatal global hypoxia associated with seizures. Here, we studied the effects of hypoxia on the function and expression of Kv4.2 and Kv4.3 α subunit channels, which encode rapidly inactivating A-type K currents, in transfected HEK-293 cells to determine if hypoxia alone could regulate IA in vitro. Global hypoxia in neonatal rat pups resulted in early decreased hippocampal expression of Kv4.2 mRNA and protein with 6 or 12 h post-hypoxia. Whole-cell voltage-clamp recordings revealed that similar times after hypoxia (1%) in vitro decreased peak currents mediated by recombinant Kv4.2 but not Kv4.3 channels. Hypoxia had no significant effect on the voltage-dependencies of activation and inactivation of Kv4.2 channels, but increased the time constant of activation. The same result was observed when Kv4.2 and Kv4.3 channels were co-expressed in a 1:1 ratio. These data suggested that hypoxia directly modulates A-type potassium channels of the subfamily typically expressed in principal hippocampal neurons, and does so in a manner to decrease function. Given the role of IA to slow action potential firing, these data are consistent with a direct effect of hypoxia to decrease IA as a mechanism of increased neuronal excitability and promotion of seizures.

Chronic Cellular Hyperexcitability in Elderly Epileptic Rats with Spontaneous Seizures Induced by Kainic Acid Status Epilepticus while Young Adults.

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.

Gabapentin promotes inhibition by enhancing hyperpolarization-activated cation currents and spontaneous firing in hippocampal CA1 interneurons.

The H-current (I(H)) regulates membrane electrical activity in many excitable cells. The antiepileptic drug gabapentin (GBP) has been shown to increase I(H) in hippocampal area CA1 pyramidal neurons, and this has been proposed as an anticonvulsant mechanism of action. I(H) also regulates excitability in some types of hippocampal interneuron that provide synaptic inhibition to CA1 pyramidal neurons, suggesting that global pharmacological I(H) enhancement could have more complex effects on the local synaptic network. However, whether I(H) in CA1 interneurons is modulated by GBP has not been examined. In this study, we tested the effects of GBP on I(H) on hippocampal area CA1 stratum oriens non-pyramidal neurons, and on spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons in immature rat brain slices. GBP (100µM) increased I(H) in approximately 67% of interneurons that exhibited I(H), with no apparent effect on cell types that did not exhibit I(H). GBP also increased the frequency of spontaneous (but not miniature) inhibitory postsynaptic currents in pyramidal neurons without altering amplitudes or rise and decay times. These data indicate that I(H) in a subset of CA1 interneuron types can be increased by GBP, similarly to its effect on I(H) in pyramidal neurons, and further, that indirectly increased spontaneous inhibition of pyramidal neurons could contribute to its anticonvulsant effects.

Increased basal synaptic inhibition of hippocampal area CA1 pyramidal neurons by an antiepileptic drug that enhances I(H).

The hyperpolarization-activated cation current (I(H)) regulates the electrical activity of many excitable cells, but its precise function varies across cell types. The antiepileptic drug lamotrigine (LTG) was recently shown to enhance I(H) in hippocampal CA1 pyramidal neurons, showing a potential anticonvulsant mechanism, as I(H) can dampen dendrito-somatic propagation of excitatory postsynaptic potentials in these cells. However, I(H) is also expressed in many hippocampal interneurons that provide synaptic inhibition to CA1 pyramidal neurons, and thus, I(H) modulation may indirectly regulate the inhibitory control of principal cells by direct modulation of interneuron activity. Whether I(H) in hippocampal interneurons is sensitive to modulation by LTG, and the manner by which this may affect the synaptic inhibition of pyramidal cells has not been investigated. In this study, we examined the effects of LTG on I(H) and spontaneous firing of area CA1 stratum oriens interneurons, as well as on spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons in immature rat brain slices. LTG (100 microM) significantly increased I(H) in the majority of interneurons, and depolarized interneurons from rest, promoting spontaneous firing. LTG also caused an increase in the frequency of spontaneous (but not miniature) IPSCs in pyramidal neurons without significantly altering amplitudes or rise and decay times. These data indicate that I(H) in CA1 interneurons can be increased by LTG, similarly to I(H) in pyramidal neurons, that I(H) enhancement increases interneuron excitability, and that these effects are associated with increased basal synaptic inhibition of CA1 pyramidal neurons.

Persistently decreased basal synaptic inhibition of hippocampal CA1 pyramidal neurons after neonatal hypoxia-induced seizures.

Hypoxia is the most common cause of neonatal seizures and can lead to epilepsy, but the epileptogenic mechanisms are not yet understood. We have previously shown that hypoxia-induced seizures in the neonatal rat result in acutely decreased amplitudes and frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in hippocampal CA1 pyramidal neurons. In the current study, we asked whether such changes persist for several days following hypoxia-induced seizures. Similar to the acute findings, we observed decreased frequency and amplitudes of sIPSCs and decreased mIPSC amplitudes in CA1 pyramidal neurons at 3-5 days after hypoxia. However, in contrast to the acute findings, we observed no differences between hypoxia-treated and control groups in mIPSC frequency. Additionally, by 7 days after hypoxia, sIPSC amplitudes in the hypoxia group had recovered to control levels, but sIPSC frequency remained decreased. These data indicate that the persistently decreased sIPSC frequency result from decreased firing of presynaptic inhibitory interneurons, with only transient possible changes in postsynaptic responses to GABA release.

Decreased IH in hippocampal area CA1 pyramidal neurons after perinatal seizure-inducing hypoxia.

PURPOSE: The hyperpolarization-activated cation current (IH) has been proposed to play a role in some forms of epileptogenesis, as it critically regulates synaptic integration and intrinsic excitability of principal limbic neurons and can be pathologically altered after experimentally induced seizures. In hippocampal CA1 pyramidal neurons, IH is functionally decreased after kainate-induced status epilepticus in adult rats but is increased after hyperthermia-induced seizures in immature rat pups. This study aimed to determine whether and how IH may be altered in CA1 pyramidal neurons after seizure-inducing global hypoxia in the neonatal brain. METHODS: Seizures were induced in rat pups on postnatal day 10 by 14- to 16-min exposure to 5-7% O2. Whole-cell patch-clamp recordings were obtained from hippocampal CA1 pyramidal neurons in slices 30 min to 3 days after hypoxia treatment, and from control age-matched littermates. IH was isolated under voltage-clamp by subtracting current responses to hyperpolarizing voltage steps before and during application of the IH blocker ZD 7288 (100 microM). RESULTS: IH was significantly decreased in pyramidal neurons from the hypoxia-treated group compared with controls (p<0.001; 19 controls; 15 hypoxia). Analyses of tail currents and activation kinetics indicated no statistically significant differences between groups in the voltage dependence or time constants of activation. CONCLUSIONS: These data indicate that a single episode of neonatal hypoxia that induces seizures can persistently decrease IH in CA1 pyramidal neurons, raising this as a potential contributing mechanism to epileptogenesis in this setting. Our findings further indicate that the consequences of seizures for IH may depend more on seizure etiology than on maturational stage.

Mature myelin basic protein-expressing oligodendrocytes are insensitive to kainate toxicity.

We examined the vulnerability to excitotoxicity of rat oligodendrocytes in dissociated cell culture at different developmental stages. Mature oligodendrocytes that express myelin basic protein were resistant to excitotoxic injury produced by kainate, whereas earlier stages in the oligodendrocyte lineage were vulnerable to this insult. To test the hypothesis that the sensitivity of immature oligodendrocytes and the resistance of mature oligodendrocytes to kainate toxicity were due to differences in membrane responsiveness to kainate, we used whole-cell patch-clamp recording. Oligodendrocyte precursors in cultures vulnerable to kainate toxicity responded to 500 microM kainate with large inward currents, whereas mature myelin basic protein-expressing oligodendrocytes in cultures resistant to kainate toxicity showed no clear response to application of this agonist. We assayed expression of glutamate receptor subunits (GluR) -2, -4, -6, -7, and KA2 using immunoblot analysis and found that expression of all of these glutamate receptors was significantly down-regulated in mature oligodendrocytes. These results suggest a striking developmental regulation of glutamate receptors in oligodendrocytes and suggest that the vulnerability of oligodendrocytes to non- N-methyl-D-aspartate receptor-mediated excitotoxicity might be much greater in developing oligodendrocytes than after the completion of myelination.