Seizures exacerbate excitatory: inhibitory imbalance in Alzheimer's disease and 5XFAD mice.

Approximately 22% of Alzheimer's disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbates AD pathology and related cognitive deficits, suggesting that seizures may be a targetable component of AD progression. Given that alterations in neuronal excitatory:inhibitory (E:I) balance occur in epilepsy, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients. We similarly hypothesized that in 5XFAD mice, the E:I imbalance would progress from an early stage (prodromal) to later symptomatic stages and be further exacerbated by pentylenetetrazol (PTZ) kindling. Post-mortem AD temporal cortical tissues from patients with or without seizure history were examined for changes in several markers of E:I balance, including levels of the inhibitory GABAA receptor, the sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2) and the excitatory NMDA and AMPA type glutamate receptors. We performed patch-clamp electrophysiological recordings from CA1 neurons in hippocampal slices and examined the same markers of E:I balance in prodromal 5XFAD mice. We next examined 5XFAD mice at chronic stages, after PTZ or control protocols, and in response to chronic mTORC1 inhibitor rapamycin, administered following kindled seizures, for markers of E:I balance. We found that AD patients with comorbid seizures had worsened cognitive and functional scores and decreased GABAA receptor subunit expression, as well as increased NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. Patch clamp recordings of prodromal 5XFAD CA1 neurons showed increased intrinsic excitability, along with decreased GABAergic inhibitory transmission and altered glutamatergic neurotransmission, indicating that E:I imbalance may occur in early disease stages. Furthermore, seizure induction in prodromal 5XFAD mice led to later dysregulation of NKCC1/KCC2 and a reduction in GluA2 AMPA glutamate receptor subunit expression, indicative of depolarizing GABA receptors and calcium permeable AMPA receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced amyloid-ß pathology and cognitive deficits, could also reverse elevations of the NKCC1/KCC2 ratio in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that targeting E:I balance, potentially with US Food and Drug Administration-approved mTOR inhibitors, hold therapeutic promise for AD patients with a seizure history.

Cannabidiol attenuates seizure susceptibility and behavioural deficits in adult CDKL5R59X knock-in mice.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is caused by a loss-of-function mutation in CDKL5 gene, encoding a serine-threonine kinase highly expressed in the brain. CDD manifests with early-onset epilepsy, autism, motor impairment and severe intellectual disability. While there are no known treatments for CDD, the use of cannabidiol has recently been introduced into clinical practice for neurodevelopmental disorders. Given the increased clinical utilization of cannabidiol, we examined its efficacy in the CDKL5R59X knock-in (R59X) mice, a CDD model based on a human mutation that exhibits both lifelong seizure susceptibility and behavioural deficits. We found that cannabidiol pre-treatment rescued the increased seizure susceptibility in response to the chemoconvulsant pentylenetetrazol (PTZ), attenuated working memory and long-term memory impairments, and rescued social deficits in adult R59X mice. To elucidate a potential mechanism, we compared the developmental hippocampal and cortical expression of common endocannabinoid (eCB) targets in R59X mice and their wild-type littermates, including cannabinoid type 1 receptor (CB1R), transient receptor potential vanilloid type 1 (TRPV1) and 2 (TRPV2), G-coupled protein receptor 55 (GPR55) and adenosine receptor 1 (A1R). Many of these eCB targets were developmentally regulated in both R59X and wild-type mice. In addition, adult R59X mice demonstrated significantly decreased expression of CB1R and TRPV1 in the hippocampus, and TRPV2 in the cortex, while TRPV1 was increased in the cortex. These findings support the potential for dysregulation of eCB signalling as a plausible mechanism and therapeutic target in CDD, given the efficacy of cannabidiol to attenuate hyperexcitability and behavioural deficits in this disorder.

Recognizing and Responding to the Needs of Future Child and Adult Neurology Care Through the Evolution of Residency Training.

Recent insights into the frequency of occurrence and the genetic and mechanistic basis of nervous system disease have demonstrated that neurologic disorders occur as a spectrum across all ages. To meet future needs of patients with neurologic disease of all ages and prepare for increasing implementaton of precision therapies, greater integration of child and adult neurology residency training is needed. ANN NEUROL 2023;94:1005-1007.

The role of mTORC1 activation in seizure-induced exacerbation of Alzheimer's disease.

The risk of seizures is 10-fold higher in patients with Alzheimer's disease than the general population, yet the mechanisms underlying this susceptibility and the effects of these seizures are poorly understood. To elucidate the proposed bidirectional relationship between Alzheimer's disease and seizures, we studied human brain samples (n = 34) from patients with Alzheimer's disease and found that those with a history of seizures (n = 14) had increased amyloid-ß and tau pathology, with upregulation of the mechanistic target of rapamycin (mTOR) pathway, compared with patients without a known history of seizures (n = 20). To establish whether seizures accelerate the progression of Alzheimer's disease, we induced chronic hyperexcitability in the five times familial Alzheimer's disease mouse model by kindling with the chemoconvulsant pentylenetetrazol and observed that the mouse model exhibited more severe seizures than the wild-type. Furthermore, kindled seizures exacerbated later cognitive impairment, Alzheimer's disease neuropathology and mTOR complex 1 activation. Finally, we demonstrated that the administration of the mTOR inhibitor rapamycin following kindled seizures rescued enhanced remote and long-term memory deficits associated with earlier kindling and prevented seizure-induced increases in Alzheimer's disease neuropathology. These data demonstrated an important link between chronic hyperexcitability and progressive Alzheimer's disease pathology and suggest a mechanism whereby rapamycin may serve as an adjunct therapy to attenuate progression of the disease.

Anti-seizure efficacy of perampanel in two established rodent models of early-life epilepsy.

Early-life seizures can be refractory to conventional antiseizure medications (ASMs) and can also result in chronic epilepsy and long-term behavioral and cognitive deficits. Treatments targeting age-specific mechanisms contributing to epilepsy would be of clinical benefit. One such target is the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subtype of excitatory glutamate receptor, which is upregulated in the developing brain. Perampanel is a non-competitive, selective AMPAR antagonist that is FDA-approved for focal onset seizures (FOS) or primary generalized tonic-clonic seizures (PGTC) in children and adults. However, the efficacy of perampanel treatment in epilepsy patients younger than 4 years has been less documented. We thus tested the efficacy of perampanel in two early-life seizure models: (1) a rat model of hypoxia-induced neonatal seizures and (2) a mouse model of Dravet syndrome with hyperthermia-induced seizures. Pretreatment with perampanel conferred dose-dependent protection against early-life seizures in both experimental models. These findings suggest that AMPAR-mediated hyperexcitability could be involved in the pathophysiology of early-life seizures, which may be amenable to treatment with perampanel.

Opportunities and limitations of genetically modified nonhuman primate models for neuroscience research.

The recently developed new genome-editing technologies, such as the CRISPR/Cas system, have opened the door for generating genetically modified nonhuman primate (NHP) models for basic neuroscience and brain disorders research. The complex circuit formation and experience-dependent refinement of the human brain are very difficult to model in vitro, and thus require use of in vivo whole-animal models. For many neurodevelopmental and psychiatric disorders, abnormal circuit formation and refinement might be at the center of their pathophysiology. Importantly, many of the critical circuits and regional cell populations implicated in higher human cognitive function and in many psychiatric disorders are not present in lower mammalian brains, while these analogous areas are replicated in NHP brains. Indeed, neuropsychiatric disorders represent a tremendous health and economic burden globally. The emerging field of genetically modified NHP models has the potential to transform our study of higher brain function and dramatically facilitate the development of effective treatment for human brain disorders. In this paper, we discuss the importance of developing such models, the infrastructure and training needed to maximize the impact of such models, and ethical standards required for using these models.

Adults with Cerebral Palsy Require Ongoing Neurologic Care: A Systematic Review.

Cerebral palsy (CP) neurologic care and research efforts typically focus on children. However, most people with CP are adults. Adults with CP are at increased risk of new neurologic conditions, such as stroke and myelopathy, that require ongoing neurologic surveillance to distinguish them from baseline motor impairments. Neurologic factors could also contribute to the motor function decline, chronic pain, and chronic fatigue that are commonly experienced by adults with CP. Based on a systematic literature review, we suggest (1) guidelines for neurologic surveillance and neurologist referral and (2) clinical research questions regarding the evolving neurologic risks for adults with CP. ANN NEUROL 2021;89:860-871.

A Pilot Randomized, Controlled, Double-Blind Trial of Bumetanide to Treat Neonatal Seizures.

OBJECTIVE: In the absence of controlled trials, treatment of neonatal seizures has changed minimally despite poor drug efficacy. We tested bumetanide added to phenobarbital to treat neonatal seizures in the first trial to include a standard-therapy control group. METHODS: A randomized, double-blind, dose-escalation design was employed. Neonates with postmenstrual age 33 to 44 weeks at risk of or with seizures were eligible. Subjects with electroencephalography (EEG)-confirmed seizures after ≥20 and <40mg/kg phenobarbital were randomized to receive additional phenobarbital with either placebo (control) or 0.1, 0.2, or 0.3mg/kg bumetanide (treatment). Continuous EEG monitoring data from ≥2 hours before to ≥48 hours after study drug administration (SDA) were analyzed for seizures. RESULTS: Subjects were randomized to treatment (n = 27) and control (n = 16) groups. Pharmacokinetics were highly variable among subjects and altered by hypothermia. The only statistically significant adverse event was diuresis in treated subjects (48% vs 13%, p = 0.02). One treated (4%) and 3 control subjects died (19%, p = 0.14). Among survivors, 2 of 26 treated subjects (8%) and 0 of 13 control subjects had hearing impairment, as did 1 nonrandomized subject. Total seizure burden varied widely, with much higher seizure burden in treatment versus control groups (median = 3.1 vs 1.2 min/h, p = 0.006). There was significantly greater reduction in seizure burden 0 to 4 hours and 2 to 4 hours post-SDA (both p < 0.01) compared with 2-hour baseline in treatment versus control groups with adjustment for seizure burden. INTERPRETATION: Although definitive proof of efficacy awaits an appropriately powered phase 3 trial, this randomized, controlled, multicenter trial demonstrated an additional reduction in seizure burden attributable to bumetanide over phenobarbital without increased serious adverse effects. Future trials of bumetanide and other drugs should include a control group and balance seizure severity. ANN NEUROL 2021;89:327-340.

Dysregulation of GABAA Receptor-Mediated Neurotransmission during the Auditory Cortex Critical Period in the Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is the leading monogenic form of intellectual disability and autism, with patients exhibiting numerous auditory-related phenotypes during their developmental period, including communication, language development, and auditory processing deficits. Despite FXS studies describing excitatory-inhibitory (E-I) imbalance in the auditory circuit and an impaired auditory critical period, evaluation of E-I circuitry maturation in the auditory cortex of FXS models remains limited. Here, we examined GABAA receptor (GABAAR)-mediated inhibitory synaptic transmission within the auditory cortex, characterizing normal intracortical circuit development patterns in wild-type (WT) mice and examining their dysregulation in developing Fmr1 knock-out (KO) mice. Electrophysiological recordings revealed gradual developmental shifts in WT L4-L2/3 connectivity, where circuit excitability significantly increased after critical period onset. KO mice exhibited accelerated developmental shifts related to aberrant GABAergic signaling. Specifically, Fmr1 KO L2/3 pyramidal neurons have enhanced developmental sensitivity to pharmacological GABAAR modulators, altered maturation of GABAAR voltage-dependent conductance, with additional presynaptic GABA release alterations. These differences are further accompanied by alterations in developmental long-term potentiation. Together, our results suggest that altered GABAergic signaling within developing Fmr1 KOs impairs the normal patterning of E-I circuit and synaptic plasticity maturation to contribute to the impaired auditory cortex critical period and functional auditory deficits in FXS.

Altered hippocampal dendritic spine maturation after hypoxia-induced seizures in neonatal rats.

Cognitive comorbidities often follow early-life seizures (ELS), especially in the setting of autism and other neurodevelopmental syndromes. However, there is an incomplete understanding of whether neuronal and synaptic development are concomitantly dysregulated. We have previously shown that hypoxia-induced seizures (HS) in postnatal day (P)10 rats increase acute and later-life hippocampal glutamatergic neurotransmission and spontaneous recurrent seizures, and impair cognition and behavior. As dendritic spines critically regulate synaptic function, we hypothesized that ELS can induce developmentally specific changes in dendritic spine maturation. At intervals during one month following HS in P10 rats, we assessed dendritic spine development on pyramidal neurons in the stratum radiatum of hippocampal area CA1. Compared to control rats in which spine density significantly decreased from P10 to early adulthood (P38), post-seizure rats failed to show a developmental decrease in spine density, and spines from P38 post-seizure rats appeared more immature-shaped (long, thin). In addition, compared to P38 control rats, post-seizure P38 rats expressed significantly more synaptic PSD-95, a marker of mature synapses. These changes were preceded by a transient increase in hippocampal expression of cofilin phosphorylated at Ser3, representing a decrease in cofilin activity. These results suggest that early-life seizures may impair normal dendritic spine maturation and pruning in CA1 during development, resulting in an excess of less efficient synapses, via activity-dependent modification of actin-regulating proteins such as cofilin. Given that multiple neurodevelopmental disorders show similar failures in developmental spine pruning, the current findings may represent a deficit in structural plasticity that could be a component of a mechanism leading to later-life cognitive consequences associated with early-life seizures.

Alzheimer-like amyloid and tau alterations associated with cognitive deficit in temporal lobe epilepsy.

Temporal lobe epilepsy represents a major cause of drug-resistant epilepsy. Cognitive impairment is a frequent comorbidity, but the mechanisms are not fully elucidated. We hypothesized that the cognitive impairment in drug-resistant temporal lobe epilepsy could be due to perturbations of amyloid and tau signalling pathways related to activation of stress kinases, similar to those observed in Alzheimer's disease. We examined these pathways, as well as amyloid-ß and tau pathologies in the hippocampus and temporal lobe cortex of drug-resistant temporal lobe epilepsy patients who underwent temporal lobe resection (n = 19), in comparison with age- and region-matched samples from neurologically normal autopsy cases (n = 22). Post-mortem temporal cortex samples from Alzheimer's disease patients (n = 9) were used as positive controls to validate many of the neurodegeneration-related antibodies. Western blot and immunohistochemical analysis of tissue from temporal lobe epilepsy cases revealed increased phosphorylation of full-length amyloid precursor protein and its associated neurotoxic cleavage product amyloid-ß*56. Pathological phosphorylation of two distinct tau species was also increased in both regions, but increases in amyloid-ß1-42 peptide, the main component of amyloid plaques, were restricted to the hippocampus. Furthermore, several major stress kinases involved in the development of Alzheimer's disease pathology were significantly activated in temporal lobe epilepsy brain samples, including the c-Jun N-terminal kinase and the protein kinase R-like endoplasmic reticulum kinase. In temporal lobe epilepsy cases, hippocampal levels of phosphorylated amyloid precursor protein, its pro-amyloidogenic processing enzyme beta-site amyloid precursor protein cleaving enzyme 1, and both total and hyperphosphorylated tau expression, correlated with impaired preoperative executive function. Our study suggests that neurodegenerative and stress-related processes common to those observed in Alzheimer's disease may contribute to cognitive impairment in drug-resistant temporal lobe epilepsy. In particular, we identified several stress pathways that may represent potential novel therapeutic targets.

AMPA Receptor Dysregulation and Therapeutic Interventions in a Mouse Model of CDKL5 Deficiency Disorder.

Pathogenic mutations in cyclin-dependent kinase-like 5 (CDKL5) result in CDKL5 deficiency disorder (CDD), a rare disease marked by early-life seizures, autistic behaviors, and intellectual disability. Although mouse models of CDD exhibit dendritic instability and alterations in synaptic scaffolding proteins, studies of glutamate receptor levels and function are limited. Here we used a novel mouse model of CDD, the Cdkl5R59X knock-in mouse (R59X), to investigate changes in synaptic glutamate receptor subunits and functional consequences. Male mice were used for all experiments to avoid the confounding effects of X-inactivation that would be present in female heterozygous mice. We showed that adult male R59X mice recapitulated the behavioral outcomes observed in other mouse models of CDD, including social deficits and memory and learning impairments, and exhibited decreased latency to seizure upon pentylenetetrazol administration. Furthermore, we observed a specific increase in GluA2-lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (AMPARs) in the adult R59X hippocampus, which is accompanied electrophysiologically by increased rectification ratio of AMPAR EPSCs and elevated early-phase long term potentiation (LTP). Finally, we showed that acute treatment with the GluA2-lacking AMPAR blocker IEM-1460 decreased AMPAR currents, and rescued social deficits, working memory impairments, and seizure behavior latency in R59X mice.SIGNIFICANCE STATEMENT CDKL5 deficiency disorder (CDD) is a rare disease marked by autistic-like behaviors, intellectual disability, and seizures. While synaptic dysfunction has been observed in mouse models of CDD, there is limited information on how synaptic alterations contribute to behavioral and functional changes in CDD. Here we reveal elevated hippocampal GluA2-lacking AMPAR expression in a novel mouse model of CDD that is accompanied by changes in synaptic AMPAR function and plasticity. We also show, for the first time, that acutely targeting GluA2-lacking AMPAR dysregulation rescues core synaptic and neurobehavioral deficits in CDD.

Mechanistic target of rapamycin complex 1 and 2 in human temporal lobe epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is a chronic epilepsy syndrome defined by seizures and progressive neurological disabilities, including cognitive impairments, anxiety, and depression. Here, human TLE specimens were investigated focusing on the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and complex 2 (mTORC2) activities in the brain, given that both pathways may represent unique targets for treatment. METHODS: Surgically resected hippocampal and temporal lobe samples from therapy-resistant TLE patients were analyzed by western blotting to quantify the expression of established mTORC1 and mTORC2 activity markers and upstream or downstream signaling pathways involving the two complexes. Histological and immunohistochemical techniques were used to assess hippocampal and neocortical structural abnormalities and cell-specific expression of individual biomarkers. Samples from patients with focal cortical dysplasia (FCD) type II served as positive controls. RESULTS: We found significantly increased expression of phospho-mTOR (Ser2448), phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), and phospho-Akt (Ser473) in TLE samples compared to controls, consistent with activation of both mTORC1 and mTORC2. Our work identified the phosphoinositide 3-kinase and Ras/extracellular signal-regulated kinase signaling pathways as potential mTORC1 and mTORC2 upstream activators. In addition, we found that overactive mTORC2 signaling was accompanied by induction of two protein kinase B-dependent prosurvival pathways, as evidenced by increased inhibitory phosphorylation of forkhead box class O3a (Ser253) and glycogen synthase kinase 3 beta (Ser9). INTERPRETATION: Our data demonstrate that mTOR signaling is significantly dysregulated in human TLE, offering new targets for pharmacological interventions. Specifically, clinically available drugs that suppress mTORC1 without compromising mTOR2 signaling, such as rapamycin and its analogs, may represent a new group of antiepileptogenic agents in TLE patients. Ann Neurol 2018;83:311-327.

Regulation of seizure-induced MeCP2 Ser421 phosphorylation in the developing brain.

Neonatal seizures disrupt normal synaptic maturation and often lead to later-life epilepsy and cognitive deficits. During early life, the brain exhibits heightened synaptic plasticity, in part due to a developmental overabundance of CaV1.2 L-type voltage gated calcium (Ca2+) channels (LT-VGCCs) and Ca2+-permeable AMPARs (CP-AMPARs) lacking GluA2 subunits. We hypothesized that early-life seizures overactivate these channels, in turn dysregulating Ca2+-dependent signaling pathways including that of methyl CPG binding protein 2 (MeCP2), a transcription factor implicated in the autism spectrum disorder (ASD) Rett Syndrome. Here, we show that in vivo hypoxia-induced seizures (HS) in postnatal day (P)10 rats acutely induced phosphorylation of the neuronal-specific target of activity-dependent MeCP2 phosphorylation, S421, as well as its upstream activator CaMKII T286. We next identified mechanisms by which activity-dependent Ca2+ influx induced MeCP2 phosphorylation using in vitro cortical and hippocampal neuronal cultures at embryonic day (E)18 + 10 days in vitro (DIV). In contrast to the prevalent role of NMDARs in the adult brain, we found that both CP-AMPARs and LT-VGCCs mediated MeCP2 S421 and CaMKII T286 phosphorylation induced by kainic acid (KA) or high potassium chloride (KCl) stimulation. Furthermore, in vivo post-seizure treatment with the broad-spectrum AMPAR antagonist NBQX, the CP-AMPAR blocker IEM-1460, or the LT-VGCC antagonist nimodipine blocked seizure-induced MeCP2 phosphorylation. Collectively, these results demonstrate that early-life seizures dysregulate critical activity-dependent developmental signaling pathways, in part via CP-AMPAR and LT-VGCC activation, providing novel age-specific therapeutic targets for convergent pathways underlying epilepsy and ASDs.

Bioresorbable silicon electronics for transient spatiotemporal mapping of electrical activity from the cerebral cortex.

Bioresorbable silicon electronics technology offers unprecedented opportunities to deploy advanced implantable monitoring systems that eliminate risks, cost and discomfort associated with surgical extraction. Applications include postoperative monitoring and transient physiologic recording after percutaneous or minimally invasive placement of vascular, cardiac, orthopaedic, neural or other devices. We present an embodiment of these materials in both passive and actively addressed arrays of bioresorbable silicon electrodes with multiplexing capabilities, which record in vivo electrophysiological signals from the cortical surface and the subgaleal space. The devices detect normal physiologic and epileptiform activity, both in acute and chronic recordings. Comparative studies show sensor performance comparable to standard clinical systems and reduced tissue reactivity relative to conventional clinical electrocorticography (ECoG) electrodes. This technology offers general applicability in neural interfaces, with additional potential utility in treatment of disorders where transient monitoring and modulation of physiologic function, implant integrity and tissue recovery or regeneration are required.

Early-life seizures alter synaptic calcium-permeable AMPA receptor function and plasticity.

Calcium (Ca2+)-mediated4 signaling pathways are critical to synaptic plasticity. In adults, the NMDA glutamate receptor (NMDAR) represents a major route for activity-dependent synaptic Ca2+ entry. However, during neonatal development, when synaptic plasticity is particularly high, many AMPA glutamate receptors (AMPARs) are also permeable to Ca2+ (CP-AMPAR) due to low GluA2 subunit expression, providing an additional route for activity- and glutamate-dependent Ca2+ influx and subsequent signaling. Therefore, altered hippocampal Ca2+ signaling may represent an age-specific pathogenic mechanism. We thus aimed to assess Ca2+ responses 48h after hypoxia-induced neonatal seizures (HS) in postnatal day (P)10 rats, a post-seizure time point at which we previously reported LTP attenuation. We found that Ca2+ responses were higher in brain slices from post-HS rats than in controls and that this increase was CP-AMPAR-dependent. To determine whether synaptic CP-AMPAR expression was also altered post-HS, we assessed the expression of GluA2 at hippocampal synapses and the expression of long-term depression (LTD), which has been linked to the presence of synaptic GluA2. Here we report a decrease 48h after HS in synaptic GluA2 expression at synapses and LTD in hippocampal CA1. Given the potentially critical role of AMPAR trafficking in disease progression, we aimed to establish whether post-seizure in vivo AMPAR antagonist treatment prevented the enhanced Ca2+ responses, changes in GluA2 synaptic expression, and diminished LTD. We found that NBQX treatment prevents all three of these post-seizure consequences, further supporting a critical role for AMPARs as an age-specific therapeutic target.

Developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits in human white and gray matter: potential mechanism of increased vulnerability in the immature brain.

The pathophysiology of perinatal brain injury is multifactorial and involves hypoxia-ischemia (HI) and inflammation. N-methyl-d-aspartate receptors (NMDAR) are present on neurons and glia in immature rodents, and NMDAR antagonists are protective in HI models. To enhance clinical translation of rodent data, we examined protein expression of 6 NMDAR subunits in postmortem human brains without injury from 20 postconceptional weeks through adulthood and in cases of periventricular leukomalacia (PVL). We hypothesized that the developing brain is intrinsically vulnerable to excitotoxicity via maturation-specific NMDAR levels and subunit composition. In normal white matter, NR1 and NR2B levels were highest in the preterm period compared with adult. In gray matter, NR2A and NR3A expression were highest near term. NR2A was significantly elevated in PVL white matter, with reduced NR1 and NR3A in gray matter compared with uninjured controls. These data suggest increased NMDAR-mediated vulnerability during early brain development due to an overall upregulation of individual receptors subunits, in particular, the presence of highly calcium permeable NR2B-containing and magnesium-insensitive NR3A NMDARs. These data improve understanding of molecular diversity and heterogeneity of NMDAR subunit expression in human brain development and supports an intrinsic prenatal vulnerability to glutamate-mediated injury; validating NMDAR subunit-specific targeted therapies for PVL.

Mammalian target of rapamycin complex 1 activation negatively regulates Polo-like kinase 2-mediated homeostatic compensation following neonatal seizures.

Homeostatic plasticity is characterized by compensatory changes in synaptic strength and intrinsic membrane properties in response to chronic changes in neuronal activity. Neonatal seizures are a naturally occurring source of neuronal overactivation and can lead to long-term epilepsy and cognitive deficits. Using a rodent model of hypoxia-induced neonatal seizures that results in a persistent increase in AMPA receptor (AMPAR) function in hippocampal CA1 pyramidal neurons, we aimed to determine whether there was any evidence of an opposing endogenous homeostatic antiepileptic response. Given that this model results in long-term epilepsy, we also examined mechanisms whereby this homeostasis fails. Whole-cell patch-clamp recordings from neurons in slices removed at intervals following seizure onset revealed an initial up-regulation of AMPAR function that was followed by a transient dynamic attenuation of this enhancement by 48-72 h, although AMPAR function was still increased compared with nonseizure control baseline. This secondary down-regulation of enhanced AMPAR function was coincident with a marked transient increase in expression and function of the Polo-like kinase 2 (PLK2), which has previously been implicated in homeostatic down-regulation of neuronal excitability in cell/slice culture models. The effects were transient and at 1 wk AMPAR function once again became up-regulated, simultaneous with a decrease in PLK2 expression and function. This negative regulation was mediated by subacute postseizure increases in mammalian target of rapamycin (mTOR). Application of the mTOR inhibitor rapamycin prevented post-hypoxic seizure impairment of homeostasis, suggesting that homeostatic plasticity mechanisms may be potentially modifiable therapeutic targets in epileptogenesis.

Chloride cotransporter NKCC1 inhibitor bumetanide protects against white matter injury in a rodent model of periventricular leukomalacia.

BACKGROUND: Periventricular leukomalacia (PVL) is a major form of preterm brain injury. Na(+)-K(+)-Cl(-) 1 cotransporter (NKCC1) expression on neurons and astrocytes is developmentally regulated and mediates Cl(-) reversal potential. We hypothesized that NKCC1 is highly expressed on oligodendrocytes (OLs) and increases vulnerability to hypoxia-ischemia (HI) mediated white matter injury, and that the NKCC1 inhibitor bumetanide would be protective in a rodent PVL model. METHODS: Immunohistochemistry in Long-Evans rats and PLP-EGFP transgenic mice was used to establish cell-specific expression of NKCC1 in the immature rodent brain. HI was induced on postnatal day 6 (P6) in rats and the protective efficacy of bumetanide (0.3 mg/kg/i.p. q12h × 60 h) established. RESULTS: NKCC1 was expressed on OLs and subplate neurons through the first 2 postnatal weeks, peaking in white matter and the subplate between P3-7. Following HI, NKCC1 is expressed on OLs and neurons. Bumetanide treatment significantly attenuates myelin basic protein loss and neuronal degeneration 7 d post-HI. CONCLUSION: Presence and relative overexpression of NKCC1 in rodent cerebral cortex coincides with a period of developmental vulnerability to HI white matter injury in the immature prenatal brain. The protective efficacy of bumetanide in this model of preterm brain injury suggests that Cl(-) transport is a factor in PVL and that its inhibition may have clinical application in premature human infants.