Progesterone Receptor Activation Regulates Sensory Sensitivity and Migraine Susceptibility.

Women develop chronic pain during their reproductive years more often than men, and estrogen and progesterone regulate this susceptibility. We tested whether brain progesterone receptor (PR) signaling regulates pain susceptibility. During the estrous cycle, animals were more sensitive to mechanical stimulus during the estrus stage than in the diestrus stage, suggesting a role for reproductive hormones, estrogen, and progesterone. Progesterone treatment of ovariectomized and estrogen-primed mice caused a delayed reduction in the mechanical threshold. Segesterone, a specific agonist of PRs replicated this effect, whereas, the segesterone-induced reduction in mechanical threshold was blocked in the mice lacking PRs in the nervous system. Segesterone treatment also did not alter mechanical threshold in adult male and juvenile female mice. PR activation increased the cold sensitivity but did not affect the heat and light sensitivity. We evaluated whether PR activation altered experimental migraine. Segesterone and nitroglycerin when administered sequentially, reduced the pain threshold but not when given separately. PRs were expressed in several components of the migraine ascending pain pathway, and their deletion blocked the painful effects of nitroglycerin. PR activation also increased the number of active neurons in the components of the migraine ascending pain pathway. These studies have uncovered a pain-regulating function of PRs. Targeting PRs may provide a novel therapeutic avenue to treat chronic pain and migraine in women. PERSPECTIVE: This article provides evidence for the role of progesterone receptors in regulating pain sensitivity and migraine susceptibility in females. Progesterone receptors may be a therapeutic target to treat chronic pain conditions more prevalent in women than men.

Progesterone receptor activation regulates sensory sensitivity.

Women develop chronic pain during their reproductive years more often than men, and estrogen and progesterone regulate this susceptibility. We tested whether brain progesterone receptor (PR) signaling regulates pain susceptibility. During the estrous cycle, animals were more sensitive to pain during the estrus stage than in the diestrus stage, suggesting a role for reproductive hormones, estrogen, and progesterone. We measured the pain threshold daily for four days in ovariectomized, estrogen-primed animals treated with progesterone. The pain threshold was lower 2 days later and stayed that way for the duration of the testing. A specific progesterone-receptor (PR) agonist, segesterone, promoted pain, and mice lacking PR in the brain (PRKO) did not experience lowered pain threshold when treated with progesterone or segesterone. PR activation increased the cold sensitivity but did not affect the heat sensitivity and had a small effect on light sensitivity. Finally, we evaluated whether PR activation altered experimental migraine. Segesterone and nitroglycerin (NTG) when administered sequentially, reduced pain threshold but not separately. These studies have uncovered a pain-regulating function of PRs. Targeting PRs may provide a novel therapeutic avenue to treat chronic pain in women.

Limbic progesterone receptors regulate spatial memory.

Progesterone and its receptors (PRs) participate in mating and reproduction, but their role in spatial declarative memory is not understood. Male mice expressed PRs, predominately in excitatory neurons, in brain regions that support spatial memory, such as the hippocampus and entorhinal cortex (EC). Furthermore, segesterone, a specific PR agonist, activates neurons in both the EC and hippocampus. We assessed the contribution of PRs in promoting spatial and non-spatial cognitive learning in male mice by examining the performance of mice lacking this receptor (PRKO), in novel object recognition, object placement, Y-maze alternation, and Morris-Water Maze (MWM) tasks. In the recognition test, the PRKO mice preferred the familiar object over the novel object. A similar preference for the familiar object was also seen following the EC-specific deletion of PRs. PRKO mice were also unable to recognize the change in object position. We confirmed deficits in spatial memory of PRKO mice by testing them on the Y-maze forced alternation and MWM tasks; PR deletion affected animal's performance in both these tasks. In contrast to spatial tasks, PR removal did not alter the response to fear conditioning. These studies provide novel insights into the role of PRs in facilitating spatial, declarative memory in males, which may help with finding reproductive partners.

Increased glutamatergic synaptic transmission during development in layer II/III mouse motor cortex pyramidal neurons.

Postnatal maturation of the motor cortex is vital to developing a variety of functions, including the capacity for motor learning. The first postnatal weeks involve many neuronal and synaptic changes, which differ by region and layer, likely due to different functions and needs during development. Motor cortex layer II/III is critical to receiving and integrating inputs from somatosensory cortex and generating attentional signals that are important in motor learning and planning. Here, we examined the neuronal and synaptic changes occurring in layer II/III pyramidal neurons of the mouse motor cortex from the neonatal (postnatal day 10) to young adult (postnatal day 30) period, using a combination of electrophysiology and biochemical measures of glutamatergic receptor subunits. There are several changes between p10 and p30 in these neurons, including increased dendritic branching, neuronal excitability, glutamatergic synapse number and synaptic transmission. These changes are critical to ongoing plasticity and capacity for motor learning during development. Understanding these changes will help inform future studies examining the impact of early-life injury and experiences on motor learning and development capacity.

Limbic progesterone receptor activity enhances neuronal excitability and seizures.

OBJECTIVE: Emerging evidence raises the possibility that progesterone receptor (PR) signaling may contribute to the reproductive hormone fluctuation-linked seizure precipitation, called catamenial epilepsy. Therefore, we studied PR isoform expression in limbic regions involved in temporal lobe epilepsy and the effect of PR activation on neuronal activity and seizures. METHODS: We evaluated PR expression in the limbic regions, entorhinal cortex (EC), hippocampus, and amygdala in female rats using quantitative real-time polymerase chain reaction (qRT-PCR). A selective agonist, Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) activated PRs, and the effect on excitability and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission of EC neurons was studied using electrophysiology. Finally, we assessed PR regulation of epileptic seizures and status epilepticus (SE) induced by lithium-pilocarpine in female rats with the global deletion of PRs (PR knockout; PRKO) using video electroencephalography (-EEG). RESULTS: Limbic regions EC, hippocampus, and amygdala robustly expressed PR messenger RNA (mRNA). Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) treatment reduced the action potential threshold of layer II/III EC neurons and increased the frequency of AMPA receptor-mediated synaptic currents of ovariectomized and estrogen-primed female rats. Female rats lacking PRs (PRKO) experienced a shorter duration, less intense, and less fatal SE than wild-type (WT) animals. Furthermore, Nestorone treatment caused seizure exacerbation in the WT epileptic animals, but not in the PRKO epileptic animals. SIGNIFICANCE: Activation of PRs expressed in the EC and hippocampus increased neuronal excitability and worsened seizures. These receptors may play a role in catamenial epilepsy.

Difluoroboron ß-diketonate polylactic acid oxygen nanosensors for intracellular neuronal imaging.

Critical for metabolism, oxygen plays an essential role in maintaining the structure and function of neurons. Oxygen sensing is important in common neurological disorders such as strokes, seizures, or neonatal hypoxic-ischemic injuries, which result from an imbalance between metabolic demand and oxygen supply. Phosphorescence quenching by oxygen provides a non-invasive optical method to measure oxygen levels within cells and tissues. Difluoroboron ß-diketonates are a family of luminophores with high quantum yields and tunable fluorescence and phosphorescence when embedded in certain rigid matrices such as poly (lactic acid) (PLA). Boron nanoparticles (BNPs) can be fabricated from dye-PLA materials for oxygen mapping in a variety of biological milieu. These dual-emissive nanoparticles have oxygen-insensitive fluorescence, oxygen-sensitive phosphorescence, and rigid matrix all in one, enabling real-time ratiometric oxygen sensing at micron-level spatial and millisecond-level temporal resolution. In this study, BNPs are applied in mouse brain slices to investigate oxygen distributions and neuronal activity. The optical properties and physical stability of BNPs in a biologically relevant buffer were stable. Primary neuronal cultures were labeled by BNPs and the mitochondria membrane probe MitoTracker Red FM. BNPs were taken up by neuronal cell bodies, at dendrites, and at synapses, and the localization of BNPs was consistent with that of MitoTracker Red FM. The brain slices were stained with the BNPs, and the BNPs did not significantly affect the electrophysiological properties of neurons. Oxygen maps were generated in living brain slices where oxygen is found to be mostly consumed by mitochondria near synapses. Finally, the BNPs exhibited excellent response when the conditions varied from normoxic to hypoxic and when the neuronal activity was increased by increasing K+ concentration. This work demonstrates the capability of BNPs as a non-invasive tool in oxygen sensing and could provide fundamental insight into neuronal mechanisms and excitability research.

Progesterone modulates neuronal excitability bidirectionally.

Progesterone acts on neurons directly by activating its receptor and through metabolic conversion to neurosteroids. There is emerging evidence that progesterone exerts excitatory effects by activating its cognate receptors (progesterone receptors, PRs) through enhanced expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Progesterone metabolite 5α,3α-tetrahydro-progesterone (allopregnanolone, THP) mediates its anxiolytic and sedative actions through the potentiation of synaptic and extrasynaptic γ-aminobutyric acid type-A receptors (GABAARs). Here, we review progesterone's neuromodulatory actions exerted through PRs and THP and their opposing role in regulating seizures, catamenial epilepsy, and seizure exacerbation associated with progesterone withdrawal.

Neocortical injury-induced status epilepticus.

OBJECTIVE: To characterize neocortical onset status epilepticus (SE) in the C57BL/6J mouse. METHODS: We induced SE by administering homocysteine 16-18 hours after cobalt (Co) implantation. SE was monitored by video and electroencephalography (EEG). We evaluated brain structure with magnetic resonance imaging (MRI). Neurodegeneration was evaluated 72 hours after SE using Fluoro-Jade C staining. RESULTS: Cobalt triggered seizures in a dose-dependent manner (median effective dose, ED50  = 0.78 mg) and the latency to peak seizure frequency shortened with increased dose. Animals developed SE after homocysteine administration. SE began with early intermittent focal seizures, consisting of frontal onset rhythmic spike-wave discharges manifested as focal dystonia with clonus. These focal seizures then evolved into generalized continuous convulsive activity. Behavioral manifestations of SE included tonic stiffening, bilateral limb clonus, and bilateral tonic-clonic movements, which were accompanied by generalized rhythmic spike-wave discharges on EEG. After prolonged seizures, animals became comatose with intermittent bilateral myoclonic seizures or jerks. During this period, EEG showed seizures interspersed with generalized periodic discharges on a suppressed background. MRI obtained when animals were in a coma revealed edema, midline shift in frontal lobe around the Co implantation site, and ventricular effacement. Fluoro-Jade C staining revealed neurodegeneration in the cortex, amygdala, and thalamus. SIGNIFICANCE: We have developed a mouse model of severe, refractory cortical-onset SE, consisting of convulsions merging into a coma, EEG patterns of cortical seizures, and injury, with evidence of widespread neocortical edema and damage. This model replicates many features of acute seizures and SE resulting from traumatic brain injury, subarachnoid, and lobar hemorrhage.

Characterization of kindled VGAT-Cre mice as a new animal model of temporal lobe epilepsy.

OBJECTIVE: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism. METHODS: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology. RESULTS: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus. SIGNIFICANCE: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics.

The Need to Intervene Before Time Point 2: Evidence From Clinical and Animal Data That Status Epilepticus Damages the Brain.

Status epilepticus, a condition characterized by abnormally prolonged seizures, has the potential to cause irreversible, structural or functional, injury to the brain. Unfavorable consequences of these seizures include mortality, the risk of developing epilepsy, and cognitive impairment. We highlight key findings of clinical and laboratory studies that have provided insights into aspects of cell death, and anatomical and functional alterations triggered by status epilepticus that support the need to intervene before time point 2, the time after which the risk of these long-term consequences increases.

The Frail Male: Early Life Inflammation Promotes Chloride Loading in Adult Male Mice.

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Reduced neurosteroid potentiation of GABAA receptors in epilepsy and depolarized hippocampal neurons.

OBJECTIVE: Neurosteroids regulate neuronal excitability by potentiating γ-aminobutyric acid type-A receptors (GABARs). In animal models of temporal lobe epilepsy, the neurosteroid sensitivity of GABARs is diminished and GABAR subunit composition is altered. We tested whether similar changes occur in patients with epilepsy and if depolarization-induced increases in neuronal activity can replicate this effect. METHODS: We determined GABAR α4 subunit expression in cortical tissue resected from pediatric epilepsy patients. Modulation of human GABARs by allopregnanolone and Ro15-4513 was measured in Xenopus oocytes using whole-cell patch clamp. To extend the findings obtained using tissue from epilepsy patients, we evaluated GABAR expression and modulation by allopregnanolone and Ro15-4513 in cultured rat hippocampal neurons exposed to high extracellular potassium (HK) to increase neuronal activity. RESULTS: Expression of α4 subunits was increased in pediatric cortical epilepsy specimens encompassing multiple pathologies. The potentiation of GABA-evoked currents by the neurosteroid allopregnanolone was decreased in Xenopus oocytes expressing GABARs isolated from epilepsy patients. Furthermore, receptors isolated from epilepsy but not control tissue were sensitive to potentiation by Ro15-4513, indicating higher expression of α4 ßx γ2 subunit-containing receptors. Correspondingly, increasing the activity of cultured rat hippocampal neurons reduced allopregnanolone potentiation of miniature inhibitory postsynaptic currents (mIPSCs), increased modulation of tonic GABAR current by Ro15-4513, upregulated the surface expression of α4 and γ2 subunits, and increased the colocalization of α4 and γ2 subunit immunoreactivity. INTERPRETATION: These findings suggest that seizure activity-induced upregulation of α4 ßx γ2 subunit-containing GABARs could affect the anticonvulsant actions of neurosteroids.

A deletion in Eml1 leads to bilateral subcortical heterotopia in the tish rat.

Children with malformations of cortical development (MCD) are at risk for epilepsy, developmental delays, behavioral disorders, and intellectual disabilities. For a subset of these children, antiseizure medications or epilepsy surgery may result in seizure freedom. However, there are limited options for treating or curing the other conditions, and epilepsy surgery is not an option in all cases of pharmacoresistant epilepsy. Understanding the genetic and neurobiological mechanisms underlying MCD is a necessary step in elucidating novel therapeutic targets. The tish (telencephalic internal structural heterotopia) rat is a unique model of MCD with spontaneous seizures, but the underlying genetic mutation(s) have remained unknown. DNA and RNA-sequencing revealed that a deletion encompassing a previously unannotated first exon markedly diminished Eml1 transcript and protein abundance in the tish brain. Developmental electrographic characterization of the tish rat revealed early-onset of spontaneous spike-wave discharge (SWD) bursts beginning at postnatal day (P) 17. A dihybrid cross demonstrated that the mutant Eml1 allele segregates with the observed dysplastic cortex and the early-onset SWD bursts in monogenic autosomal recessive frequencies. Our data link the development of the bilateral, heterotopic dysplastic cortex of the tish rat to a deletion in Eml1.

α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Plasticity Sustains Severe, Fatal Status Epilepticus.

OBJECTIVE: Generalized convulsive status epilepticus is associated with high mortality. We tested whether α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor plasticity plays a role in sustaining seizures, seizure generalization, and mortality observed during focal onset status epilepticus. We also determined whether modified AMPA receptors generated during status epilepticus could be targeted with a drug. METHODS: Electrically induced status epilepticus was characterized by electroencephalogram and behavior in GluA1 knockout mice and in transgenic mice with selective knockdown of the GluA1 subunit in hippocampal principal neurons. Excitatory and inhibitory synaptic transmission in CA1 neurons was studied using patch clamp electrophysiology. The dose response of N,N,H,-trimethyl-5-([tricyclo(3.3.1.13,7)dec-1-ylmethyl]amino)-1-pentanaminiumbromide hydrobromide (IEM-1460), a calcium-permeable AMPA receptor antagonist, was determined. RESULTS: Global removal of the GluA1 subunit did not affect seizure susceptibility; however, it reduced susceptibility to status epilepticus. GluA1 subunit knockout also reduced mortality, severity, and duration of status epilepticus. Absence of the GluA1 subunit prevented enhancement of glutamatergic synaptic transmission associated with status epilepticus; however, γ-aminobutyric acidergic synaptic inhibition was compromised. Selective removal of the GluA1 subunit from hippocampal principal neurons also reduced mortality, severity, and duration of status epilepticus. IEM-1460 rapidly terminated status epilepticus in a dose-dependent manner. INTERPRETATION: AMPA receptor plasticity mediated by the GluA1 subunit plays a critical role in sustaining and amplifying seizure activity and contributes to mortality. Calcium-permeable AMPA receptors modified during status epilepticus can be inhibited to terminate status epilepticus. ANN NEUROL 2020;87:84-96.

Progesterone receptor activation regulates seizure susceptibility.

OBJECTIVE: Progesterone is a potent neuromodulator that exerts effects on the brain through neurosteroids, progesterone receptors (PRs), and other molecules. Whether PR activation regulates seizures is not known. We determined whether PR activation increased seizure susceptibility. METHODS: Adult female rats that developed epilepsy following lithium-pilocarpine-induced status epilepticus (SE) were used. Seizures were recorded by continuous-video EEG and read by an individual blinded to the treatment of the animals. The animals were treated for a week with progesterone (50 mg/kg per day), and the effect of progesterone withdrawal on seizure frequency was assessed during the subsequent week. During the week of progesterone treatment, the animals were treated with PR antagonist RU-486 (10 mg/kg per day) or a vehicle control, which was administered 30 min before progesterone. In another set of animals, we determined the effect of the PR agonist Nestorone (3 mg/kg per day) on seizure frequency. The animals were treated with Nestorone or vehicle for a week, and seizure frequencies at baseline and during the treatment week were compared. RESULTS: Progesterone withdrawal induced twofold increase in seizures in 57% of animals (n = 14). RU-486 treatment in combination with progesterone, prevented this increase, and a smaller fraction of animals (17%) experienced withdrawal seizures (n = 13). The specific activation of PRs by Nestorone also increased the seizure frequency. Forty-six percent (n = 14) of Nestorone-treated animals experienced at least a 50% increase in seizures compared to only 9% of the vehicle-treated animals (n = 11). INTERPRETATION: PR activation increased seizure frequency in epileptic animals. Thus, PRs may be novel targets for treating catamenial epilepsy.

Parallel pathways of seizure generalization.

Generalized convulsive status epilepticus is a life-threatening emergency, because recurrent convulsions can cause death or injury. A common form of generalized convulsive status epilepticus is of focal onset. The neuronal circuits activated during seizure spread from the hippocampus, a frequent site of seizure origin, to the bilateral motor cortex, which mediates convulsive seizures, have not been delineated. Status epilepticus was initiated by electrical stimulation of the hippocampus. Neurons transiently activated during seizures were labelled with tdTomato and then imaged following brain slice clearing. Hippocampus was active throughout the episode of status epilepticus. Neuronal activation was observed in hippocampus parahippocampal structures: subiculum, entorhinal cortex and perirhinal cortex, septum, and olfactory system in the initial phase status epilepticus. The tdTomato-labelled neurons occupied larger volumes of the brain as seizures progressed and at the peak of status epilepticus, motor and somatosensory cortex, retrosplenial cortex, and insular cortex also contained tdTomato-labelled neurons. In addition, motor thalamic nuclei such as anterior and ventromedial, midline, reticular, and posterior thalamic nuclei were also activated. Furthermore, circuits proposed to be crucial for systems consolidation of memory: entorhinal cortex, retrosplenial cortex, cingulate gyrus, midline thalamic nuclei and prefrontal cortex were intensely active during periods of generalized tonic-clonic seizures. As the episode of status epilepticus waned, smaller volume of brain was activated. These studies suggested that seizure spread could have occurred via canonical thalamocortical pathway and many cortical structures involved in memory consolidation. These studies may help explain retrograde amnesia following seizures.

Neurosteroid regulation of GABAA receptors: A role in catamenial epilepsy.

The female reproductive hormones progesterone and estrogen regulate network excitability. Fluctuations in the circulating levels of these hormones during the menstrual cycle cause frequent seizures during certain phases of the cycle in women with epilepsy. This seizure exacerbation, called catamenial epilepsy, is a dominant form of drug-refractory epilepsy in women of reproductive age. Progesterone, through its neurosteroid derivative allopregnanolone, increases γ-aminobutyric acid type-A receptor (GABAR)-mediated inhibition in the brain and keeps seizures under control. Catamenial seizures are believed to be a neurosteroid withdrawal symptom, and it was hypothesized that exogenous administration of progesterone to maintain its levels high during luteal phase will treat catamenial seizures. However, in a multicenter, double-blind, phase III clinical trial, progesterone treatment did not suppress catamenial seizures. The expression of GABARs with reduced neurosteroid sensitivity in epileptic animals may explain the failure of the progesterone clinical trial. The expression of neurosteroid-sensitive δ subunit-containing GABARs is reduced, and the expression of α4γ2 subunit-containing GABARs is upregulated, which alters the inhibition of dentate granule cells in epilepsy. These changes reduce the endogenous neurosteroid control of seizures and contribute to catamenial seizures.

Mechanisms of status epilepticus: α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor hypothesis.

Prolonged seizures of status epilepticus (SE) result from failure of mechanisms of seizure termination or activation of mechanisms that sustain seizures. Reduced γ-aminobutyric acid type A receptor-mediated synaptic transmission contributes to impairment of seizure termination. However, mechanisms that sustain prolonged seizures are not known. We propose that insertion of GluA1 subunits at the glutamatergic synapses causes potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR)-mediated neurotransmission, which helps to spread and sustain seizures. The AMPAR-mediated neurotransmission of CA1 pyramidal neurons was increased in animals in SE induced by pilocarpine. The surface membrane expression of GluA1 subunit-containing AMPARs on CA1 pyramidal neurons was also increased. Blockade of N-methyl-d-aspartate receptors 10 minutes after the onset of continuous electrographic seizure activity prevented the increase in the surface expression of GluA1 subunits. N-methyl-d-aspartate receptor antagonist MK-801 in conjunction with diazepam also terminated seizures that were refractory to MK-801 or diazepam alone. Future studies using mice lacking the GluA1 subunit expression will provide further insights into the role of GluA1 subunit-containing AMPAR plasticity in sustaining seizures of SE.

Status epilepticus: Role for etiology in determining response to benzodiazepines.

OBJECTIVE: Clinical factors contributing to benzodiazepine failure in treating status epilepticus (SE) include suboptimal dosing and seizure duration. As many benzodiazepine-refractory episodes of SE arise from acute etiologies, we sought to determine whether etiology impacts SE treatment. METHODS: The potency of diazepam to terminate SE induced by lithium-pilocarpine (LiPilo-SE) or kainic acid (KA-SE) in 3-week-old rats was studied by video-electroencephalography. Synaptic γ-aminobutyric acid type A receptor (GABAR)-mediated currents were recorded from dentate granule cells using voltage-clamp electrophysiology. Surface expression of γ2 subunit-containing GABARs and Kv4.2 potassium channels in hippocampal slices was determined using a biotinylation assay. Expression of phosphorylated forms of ß2/3 and γ2 subunits was determined using phosphospecific antibodies and Western blotting. RESULTS: Diazepam failed to terminate late SE in LiPilo-SE animals but was successful in terminating KA-SE of 1- and 3-hour duration. One hour after SE onset, GABAR-mediated synaptic inhibition and γ2 subunit-containing GABAR surface expression were reduced in LiPilo-SE animals. These were unchanged in KA-SE animals at 1 and 3 hours. Phosphorylation of γ2 subunit residue S327 was unchanged in both models, although GABAR ß3 subunit S408/409 residues were dephosphorylated in the LiPilo-SE animals. Kv4.2 potassium channel surface expression was increased in LiPilo-SE animals but reduced in KA-SE animals. INTERPRETATION: SE-model-dependent differences support a novel hypothesis that the development of benzodiazepine pharmacoresistance may be etiologically predetermined. Further studies are required to investigate the mechanisms that underlie such etiological differences during SE and whether etiology-dependent protocols for the treatment of SE need to be developed. Ann Neurol 2018;83:830-841.

Electroencephalography and behavior patterns during experimental status epilepticus.

OBJECTIVE: To characterize the evolution of behavioral and electrographic seizures in an experimental electrical stimulation-based model of status epilepticus (SE) in C57Bl/6 mice, and to relate SE to various outcomes, including death and epileptogenesis. METHODS: SE was induced by continuous hippocampal stimulation and was evaluated by review of electroencephalographic recordings, spectral display, and behavior. RESULTS: Seizures were initially locked to the electrical trains but later became independent of them. Following the end of stimulation, autonomous seizures continued for >5 minutes in 85% of the animals. There was ongoing 2-3-Hz rhythmic, high-amplitude, slow spike-wave discharges (HASDs) associated with purposeless, repetitive, continuously circling and exploratory behavior. There were high-amplitude fast discharges (HAFDs) associated with worsening of behavioral seizures that were interspersed with the ongoing HASDs. Death during SE occurred in 23% of the animals, and it was preceded by a stage 5 behavioral seizure. In the waning stage of SE, severe seizures and HAFDs dissipated, HASDs slowed down, and normal behavior was restored in most animals. Epilepsy developed in 33% of the animals monitored after SE. SIGNIFICANCE: The electrical stimulation model of SE can be used to study mechanisms of SE and its adverse consequences, including death and epileptogenesis.