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Temporal encephaloceles (TEs) are herniations of cerebral parenchyma through structural defects in the floor of the middle cranial fossa. They are a relatively common, but only relatively recently identified potential cause of drug-resistant epilepsy. Uncontrolled epilepsy is associated with many negative long term health consequences including a heightened risk of death. The most effective treatment for drug-resistant epilepsy is surgery. One of the most predictive factors associated with successful surgery is identification of an abnormality on imaging. However, TEs can be difficult to detect and are often overlooked on neuroimaging studies. Improving our ability to accurately detect TEs by MRI is an important step in improving surgical outcomes in patients with drug-resistant epilepsy. We performed a review on existing imaging modalities for detecting TEs and report on our attempt to use a voxel-based morphometry (VBM) algorithm to detect TEs in T1-weighted MRIs of 81 patients from a database comprised of 25 patients with confirmed encephaloceles and 56 controls. Our program's sensitivity and specificity were compared to those of two neuroradiologists and two epileptologists using visualization during surgery as the gold standard. On average, the neuroradiologists and epileptologists had sensitivities of 41 % and 58 % and specificities of 81 % and 60 % while our VBM-based approach had sensitivities and specificities ranging from 11 % to 50 % and 0.2 % to 17 %, respectively. This work provides an overview of the different imaging modalities utilized in the detection of TEs and highlights the difficulties associated with their detection for both experienced physicians and cutting-edge computational methods. Our findings suggest that VBM-based methods could potentially be used to enhance clinicians' ability to detect TEs thereby facilitating surgical planning, improving surgical outcomes by allowing for more specific targeting, and bettering the long-term health and well-being of patients with drug-resistant epilepsy secondary to TEs., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to disclose., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: γ-Aminobutyric acid (GABA) type A receptors (GABA A Rs) are ligand-gated Cl-channels that mediate the bulk of inhibitory neurotransmission in the mature CNS and are targets of many drugs. During cortical development, GABA A R-mediated signals are significantly modulated by changing subunit composition and expression of Cl-transporters as part of developmental processes and early network activity. To date, this developmental evolution has remained understudied, particularly at the level of cortical layer-specific changes. In this study, we characterized the expression of nine major GABA A R subunits and K-Cl transporter 2 (KCC2) in mouse somatosensory cortex from embryonic development to postweaning maturity., Methods: We evaluated expression of α1-5, β2-3, γ2, and δ GABA A R subunits using immunohistochemistry and Western blot techniques, and expression of KCC2 using immunohistochemistry in cortices from E13.5 to P25 mice., Results: We found that embryonic cortex expresses mainly α3, α5, β3, and γ2, while expression of α1, α2, α4, β2, δ, and KCC2 begins at later points in development; however, many patterns of nuanced expression can be found in specific lamina, cortical regions, and cells and structures., Discussion: While the general pattern of expression of each subunit and KCC2 is similar to previous studies, we found a number of unique temporal, regional, and laminar patterns that were previously unknown. These findings provide much needed knowledge of the intricate developmental evolution in GABA A R composition and KCC2 expression to accommodate developmental signals that transition to mature neurotransmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zavalin, Hassan, Zhang, Khera and Lagrange.)

K-Cl transporter KCC2 is an important regulator of neuronal development and neuronal function at maturity. Through its canonical transporter role, KCC2 maintains inhibitory responses mediated by γ-aminobutyric acid (GABA) type A receptors. During development, late onset of KCC2 transporter activity defines the period when depolarizing GABAergic signals promote a wealth of developmental processes. In addition to its transporter function, KCC2 directly interacts with a number of proteins to regulate dendritic spine formation, cell survival, synaptic plasticity, neuronal excitability, and other processes. Either overexpression or loss of KCC2 can lead to abnormal circuit formation, seizures, or even perinatal death. GABA has been reported to be especially important for driving migration and development of cortical interneurons (IN), and we hypothesized that properly timed onset of KCC2 expression is vital to this process. To test this hypothesis, we created a mouse with conditional knockout of KCC2 in Dlx5-lineage neurons (Dlx5 KCC2 cKO), which targets INs and other post-mitotic GABAergic neurons in the forebrain starting during embryonic development. While KCC2 was first expressed in the INs of layer 5 cortex, perinatal IN migrations and laminar localization appeared to be unaffected by the loss of KCC2. Nonetheless, the mice had early seizures, failure to thrive, and premature death in the second and third weeks of life. At this age, we found an underlying change in IN distribution, including an excess number of somatostatin neurons in layer 5 and a decrease in parvalbumin-expressing neurons in layer 2/3 and layer 6. Our research suggests that while KCC2 expression may not be entirely necessary for early IN migration, loss of KCC2 causes an imbalance in cortical interneuron subtypes, seizures, and early death. More work will be needed to define the specific cellular basis for these findings, including whether they are due to abnormal circuit formation versus the sequela of defective IN inhibition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zavalin, Hassan, Fu, Delpire and Lagrange.)

Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5β3γ2 and α1β3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Key Points: Physiologically relevant combinations of recombinant GABA A receptor (GABAR) subunits were expressed in HEK293 cells. Using whole-cell voltage clamp and rapid drug application, we measured the GABAR-subtype-specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4βδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5β2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1β2/3δ GABARs function well at prolonged, micromolar (>2 μm) GABA levels, but not to low tonic (<1 μm GABA) or synaptic/transient GABAergic signalling. These results help illuminate the context- and isoform-specific modes of GABAergic signalling in the brain., Abstract: GABA A receptors (GABARs) mediate a remarkable diversity of signalling modalities in vivo. Yet most published work characterizing responses to GABA has focused on the properties needed to convey fast, phasic synaptic inhibition. We therefore aimed to characterize the most prevalent (α4βδ, α5β3γ2L) and least prevalent (α1β2δ) non-synaptic GABAR currents, using whole-cell voltage clamp recordings of recombinant GABAR expressed in HEK293 cells and drug application protocols to recapitulate the GABA concentration profiles occurring during both fast synaptic and slow extrasynaptic signalling. We found that α4βδ GABARs were very sensitive to submicromolar GABA, with a rank order potency of α4β2δ ≥ α4β1δ ≈ α4β3δ GABARs. In comparison, the GABA EC 50 was up to 20 times higher for α1β2γ2L GABARs, with α1β2δ and α5β3γ2L GABARs having intermediate GABA potency. Both α4βδ and α5β3γ2L GABAR currents exhibited slow, but substantial, desensitization as well as prolonged rates of deactivation. These GABAR current properties defined distinct 'dynamic ranges' of responsiveness to changing GABA for α4β2δ (0.1-1 μm), α5β3γ2L (0.5-7 μm) and α1β2γ2L (0.6-9 μm) GABARs. Finally, α1β2δ GABARs were notable for their relative lack of desensitization and extremely quick deactivation. In summary, our results help delineate the roles that specific GABARs may play in mediating non-synaptic GABA signals. Since ambient GABA levels may be altered during development as well as by drugs and disease states, these findings may help future efforts to understand disrupted inhibition underlying a variety of neurological illnesses, such as epilepsy., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Myelin abnormalities are increasingly being recognized as an important component of a number of neurologic developmental disorders. The integration of many signaling pathways and cell types are critical for correct myelinogenesis. The PI3-K and mechanistic target of rapamycin (mTOR) pathways have been found to play key roles. mTOR is found within two distinct complexes, mTORC1 and mTORC2. mTORC1 activity has been shown to play a major role during myelination, while the role of mTORC2 is not yet well understood. To determine the role of mTORC2 signaling in myelinogenesis, we generated a mouse lacking the critical mTORC2 component Rictor in oligodendrocyte precursors (OPCs). Targeted deletion of Rictor in these cells decreases and delays the expression of myelin related proteins and reduces the size of cerebral white matter tracts. This is developmentally manifest as a transient reduction in myelinated axon density and g-ratio. OPC cell number is reduced at birth without detectable change in proliferation with proportional reductions in mature oligodendrocyte number at P15. The total number of oligodendrocytes as well as extent of myelination, does improve over time. Adult conditional knock-out (CKO) animals do not demonstrate a behavioral phenotype likely due in part to preserved axonal conduction velocities. These data support and extend prior studies demonstrating an important but transient contribution of mTORC2 signaling to myelin development.

Rationale: Hypermotor seizures are most often reported from the frontal lobe but may also have temporal, parietal, or insular origin. We noted a higher proportion of patients with temporal lobe epilepsy in our surgical cohort who had hypermotor seizures. We evaluated the anatomic localization and surgical outcome in patient with refractory hypermotor seizures who had epilepsy surgery in our center., Methods: We identified twenty three patients with refractory hypermotor seizures from our epilepsy surgery database. We analyzed demographics, presurgical evaluation including semiology, MRI, PET scan, interictal/ictal scalp video-EEG, intracranial recording, and surgical outcomes. We evaluated preoperative variables as predictors of outcome., Results: Most patients (65%) had normal brain MRI. Intracranial EEG was required in 20 patients (86.9%). Based on the presurgical evaluation, the resection was anterior temporal in fourteen patients, orbitofrontal in four patients, cingulate in four patients, and temporoparietal in one patient. The median duration of follow-up after surgery was 76.4months. Fourteen patients (60%) had been seizure free at the last follow up while 3 patients had rare disabling seizures., Conclusions: Hypermotor seizures often originated from the temporal lobe in this series of patients who had epilepsy surgery. This large proportion of temporal lobe epilepsy may be the result of a selection bias, due to easier localization and expected better outcome in temporal lobe epilepsy. With extensive presurgical evaluation, including intracranial EEG when needed, seizure freedom can be expected in the majority of patients., (Copyright © 2016. Published by Elsevier Inc.)

The subunit stoichiometry and arrangement of synaptic αβγ GABAA receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rationale: Epilepsy and psychogenic nonepileptic spells (PNES) can coexist, often posing diagnostic and therapeutic challenges. We sought to identify clinical and historical characteristics of two groups of patients, those with coexisting epilepsy and PNES and those with PNES alone, and determine the prevalence of coexisting epilepsy/PNES with strict diagnostic criteria in a large group of epilepsy monitoring unit (EMU) patients., Methods: We reviewed the medical records of all consecutive patients admitted to the Vanderbilt University Medical Center Adult EMU between July 1, 2007 and June 30, 2012. We identified patients with recorded PNES and classified them as having coexisting epilepsy/PNES or PNES alone and then systematically compared the clinical characteristics of these two groups., Results: A total of 1567 patient medical records were reviewed. The prevalence rate of coexisting epilepsy/PNES was 5.2% among all EMU admissions (12.3% of all patients with epilepsy and 14.8% of all patients with PNES). These rates were lower when patients with interictal epileptiform activity (IEA) alone and no recorded ictal discharges were not included in the group with epilepsy (2.6%, 6.2%, and 7.4%, respectively). The accuracy of pre-EMU clinical suspicion was significantly higher in the group with PNES-only. Patients with epilepsy/PNES were significantly more likely to require more than one EMU admission for definitive diagnosis. The first PNES event preceded an epileptic seizure (ES) in 94.4% of patients with epilepsy/PNES. The group with PNES-only had significantly higher suggestibility, and the group with epilepsy/PNES had a significantly higher presence of epilepsy risk factors. Abnormal neurological examination and abnormal brain MRI were also significantly more common in the group with epilepsy/PNES., Conclusions: Our study defined the prevalence of coexisting epilepsy/PNES in a large cohort with strict diagnostic criteria and outlined specific clinical and historical characteristics differentiating the two groups of patients with coexisting epilepsy/PNES and PNES-only. These findings should help guide clinicians to reach the correct diagnosis faster and provide appropriate treatment earlier., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: We report clinical and electrographic features of generalized onset seizures with focal evolution (GOFE) and present arguments for the inclusion of this seizure type in the seizure classification., Methods: The adult and pediatric Epilepsy Monitoring Unit databases at Vanderbilt Medical Center and Children's Hospital were screened to identify generalized onset seizures with focal evolution. We reviewed medical records for epilepsy characteristics, epilepsy risk factors, MRI abnormalities, neurologic examination, antiepileptic medications before and after diagnosis, and response to medications. We also reviewed ictal and interictal EEG tracings, as well as video-recorded semiology., Results: Ten patients were identified, 7 males and 3 females. All of the patients developed generalized epilepsy in childhood or adolescence (ages 3-15years). Generalized onset seizures with focal evolution developed years after onset in 9 patients, with a semiology concerning for focal seizures or nonepileptic events. Ictal discharges had a generalized onset on EEG, described as either generalized spike-and-wave and/or polyspike-and-wave discharges, or generalized fast activity. This electrographic activity then evolved to focal rhythmic activity most commonly localized to one temporal or frontal region; five patients had multiple seizures evolving to focal activity in different regions of both hemispheres. The predominant interictal epileptiform activity included generalized spike-and-wave and/or polyspike-and-wave discharges in all patients. Taking into consideration all clinical and EEG data, six patients were classified with genetic (idiopathic) generalized epilepsy, and four were classified with structural/metabolic (symptomatic) generalized epilepsy. All of the patients had modifications to their medications following discharge, with three becoming seizure-free and five responding with >50% reduction in seizure frequency., Conclusion: Generalized onset seizures may occasionally have focal evolution with semiology suggestive of focal seizures, leading to a misdiagnosis of focal onset. This unique seizure type may occur with genetic as well as structural/metabolic forms of epilepsy. The identification of this seizure type may help clinicians choose appropriate medications, avoiding narrow spectrum agents known to aggravate generalized onset seizures., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by a number of neurological problems, including developmental delay, movement disorders, and epilepsy. AS results from the loss of UBE3A (an imprinted gene) expressed from the maternal chromosome in neurons. Given the ubiquitous expression of Ube3a and the devastating nature of AS, the role of environmental and maternal effects has been largely ignored. Severe ataxia, anxiety-like behaviors and learning deficits are well-documented in patients and AS mice. More recently, clinical imaging studies of AS patients suggest myelination may be delayed or reduced. Utilizing a mouse model of AS, we found disrupted expression of cortical myelin proteins, the magnitude of which is influenced by maternal status, in that the aberrant myelination in the AS pups of AS affected mothers were more pronounced than those seen in AS pups raised by unaffected (Ube3a (m+/p-)) Carrier mothers. Furthermore, feeding the breeding mothers a higher fat (11% vs 5%) diet normalizes these myelin defects. These effects are not limited to myelin proteins. Since AS mice have abnormal stress responses, including altered glucocorticoid receptor (GR) expression, we measured GR expression in pups from Carrier and affected AS mothers. AS pups had higher GR expression than their WT littermates. However, we also found an effect of maternal status, with reduced GR levels in pups from affected mothers compared to genotypically identical pups raised by unaffected Carrier mothers. Taken together, our findings suggest that the phenotypes observed in AS mice may be modulated by factors independent of Ube3a genotype., (Published by Elsevier B.V.)

Angelman Syndrome (AS) is a devastating neurodevelopmental disorder characterized by developmental delay, speech impairment, movement disorder, sleep disorders and refractory epilepsy. AS is caused by loss of the Ube3a protein encoded for by the imprinted Ube3a gene. Ube3a is expressed nearly exclusively from the maternal chromosome in mature neurons. While imprinting in neurons of the brain has been well described, the imprinting and expression of Ube3a in other neural tissues remains relatively unexplored. Moreover, given the overwhelming deficits in brain function in AS patients, the possibility of disrupted Ube3a expression in the infratentorial nervous system and its consequent disability have been largely ignored. We evaluated the imprinting status of Ube3a in the spinal cord and sciatic nerve and show that it is also imprinted in these neural tissues. Furthermore, a growing body of clinical and radiological evidence has suggested that myelin dysfunction may contribute to morbidity in many neurodevelopmental syndromes. However, findings regarding Ube3a expression in non-neuronal cells of the brain have varied. Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types. Unlike many other neurodevelopmental disorders, AS symptoms do not become apparent until roughly 6 to 12 months of age. To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points. We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete. This furthers the hypothesis that the apparently normal window of development in AS patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this crucial epoch of early development.

GABAA receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABAA receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABAA receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2(+/-)) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABAA receptors.

Objective: To share our experience on clinical presentation and management of patients diagnosed with Hashimoto's Encephalopathy (HE) at Vanderbilt Medical Center between 1999 and 2012., Background: HE is a rare disorder characterized by encephalopathy and central nervous system (CNS) dysfunction, elevated antithyroid antibodies, the absence of infection or structural abnormalities in the CNS, and a response to treatment with steroids. The relationship between thyroid antibodies and encephalopathy has remained unresolved., Design/methods: Retrospective chart review., Results: We identified 13 patients who met the criteria for the diagnosis of HE. The median age was 49 years (range, 2-66) and all except one were women. Encephalopathy in the form of altered mental status, stroke-like symptoms or seizures, with prompt resolution of symptoms upon receiving steroids, was the commonest presentation, seen in 7 patients. The second commonest presentation was subacute progressive decrease in cognitive function, which reversed within days to weeks after steroid therapy, seen in 4 patients. Electroencephalogram (EEG) was available in 12 patients and was abnormal in 8, showing nonspecific cerebral dysfunction in all 8 and epileptiform activity in 3. Treatment consisted of steroids in the acute phase for 12 of 13 patients with rapid improvement in symptoms. Maintenance therapy was rituximab in 7 patients, intravenous immunoglobulin (IVIg) in 7, azathioprine in 4, mycophenolate mofetil in 3, and methotrexate in 1 (some patients received sequential therapy with different agents). There was complete or near complete resolution of symptoms in 12 of the 13 patients., Conclusions: We present a cohort of patients in whom CNS dysfunction was associated with elevated antithyroid antibodies and reversal of disease followed immunomodulatory therapies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Object: Traumatic brain injury (TBI) often causes an encephalopathic state, corresponding amplitude suppression, and disorganization of electroencephalographic activity. Clinical recovery in patients who have suffered TBI varies, and identification of patients with a poor likelihood of functional recovery is not always straightforward. The authors sought to investigate temporal patterns of electrophysiological recovery of neuronal networks in an animal model of TBI. Because thalamocortical circuit function is a critical determinant of arousal state, as well as electroencephalography organization, these studies were performed using a thalamocortical brain slice preparation., Methods: Adult rats received a moderate parietal fluid-percussion injury and were allowed to survive for 1 hour, 2 days, 7 days, or 15 days prior to in vitro electrophysiological recording. Thalamocortical brain slices, 450-μm thick, were prepared using a cutting angle that preserved reciprocal connections between the somatosensory cortex and the ventrobasal thalamic complex., Results: Extracellular recordings in the cortex of uninjured control brain slices revealed spontaneous slow cortical oscillations (SCOs) that are blocked by (2R)-amino-5-phosphonovaleric acid (50 μM) and augmented in low [Mg2+]o. These oscillations have been shown to involve simultaneous bursts of activity in both the cortex and thalamus and are used here as a metric of thalamocortical circuit integrity. They were absent in 84% of slices recorded at 1 hour postinjury, and activity slowly recovered to approximate control levels by Day 15. The authors next used electrically evoked SCO-like potentials to determine neuronal excitability and found that the maximum depression occurred slightly later, on Day 2 following TBI, with only 28% of slices showing evoked activity. In addition, stimulus intensities needed to create evoked SCO activity were elevated at 1 hour, 2 days, and 7 days following TBI, and eventually returned to control levels by Day 15. The SCO frequency remained low throughout the 15 days following TBI (40% of control by Day 15)., Conclusions: The suppression of cortical oscillatory activity following TBI observed in the rat model suggests an injury-induced functional disruption of thalamocortical networks that gradually recovers to baseline at approximately 15 days postinjury. The authors speculate that understanding the processes underlying disrupted thalamocortical circuit function may provide important insights into the biological basis of altered consciousness following severe head injury. Moreover, understanding the physiological basis for this process may allow us to develop new therapies to enhance the rate and extent of neurological recovery following TBI.

γ-aminobutyric acid type A (GABA(A)) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA(A) receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABA(A) receptors by glycosylation.

Purpose: A drug holiday seems to produce seizure interval prolongation (SIP) after reinstitution of antiepileptic drugs (AEDs). This effect was demonstrated mainly with carbamazepine. We evaluated SIP with newer AEDs and tested the relationship of SIP to history of AED tolerance., Methods: We prospectively studied patients with refractory epilepsy admitted to the Vanderbilt epilepsy monitoring unit (EMU) over a period of 12 months. We included only patients on levetiracetam, lamotrigine, or oxcarbazepine who had their AEDs withdrawn on admission and reinstituted without change upon discharge. We defined SIP as the interval from EMU discharge to first seizure minus the interval between the last two seizures before EMU admission., Results: A total of 43 patients completed the study; 15 were on monotherapy. SIP was greater than zero in this patient group (p < 0.0001), with a mean prolongation of 19.4 +/- 28.0 days. The average SIP was higher (p = 0.01) in patients on monotherapy (29.7 +/- 23.8 days) than patients on polytherapy (13.9 +/- 29.0 days). SIP tended to be greater in patients with a prior history of AED tolerance (25.7 +/- 36.8 days) compared to patient with no prior history of AED tolerance (14.0 +/- 16.3 days)., Discussion: SIP does occur after brief AED withdrawal. This effect is greater in patients on monotherapy and tends to be larger in patients with a history of AED tolerance. The SIP effect may be related to the phenomenon of tolerance, clinically seen as resistance to AED therapeutic effect.

Benzodiazepines (BDZs) are GABA(A) receptor modulators with anxiolytic, hypnotic, and anticonvulsant properties. BDZs are understood to potentiate GABA(A) receptor function by increasing channel opening frequency, in contrast to barbiturates, which increase channel open duration. However, the in vitro evidence demonstrating increased opening frequency involved prolonged exposure to sub-saturating GABA concentrations, conditions most similar to those found in extrasynaptic areas. In contrast, synaptic GABA(A) receptors are transiently activated by high GABA concentrations. To determine if BDZ modulation of single-channel opening frequency would be different for BDZ-sensitive receptors activated under synaptic versus extrasynaptic conditions, a combination of patch clamp recording and kinetic modeling was used. Consistent with the original experimental findings, BDZs were found to increase receptor affinity for GABA by decreasing the unbinding rate. While this mechanism was predicted to increase opening frequency under extrasynaptic conditions, simulations predicted that the same mechanism under synaptic conditions would increase the number, but not the frequency, of single-channel openings. Thus, a single mechanism (slower GABA unbinding) can produce differential changes in opening frequency under synaptic versus extrasynaptic conditions. The functional impact of BDZs on GABA(A) receptors therefore depends upon the physiological context of receptor activation.

The international seizure classification recognizes that partial-onset seizures can become secondarily generalized, but generalized-onset seizures are expected to remain generalized. We report six patients who had recorded seizures with generalized onset, but subsequent evolution into a focal discharge. The clinical seizure onset was generalized absence or myoclonic, and the most common subsequent clinical pattern was prolonged behavioral arrest with mild automatisms, and then postictal confusion. The ictal discharge started with generalized spike-and-wave activity and then acquired a focal predominance. Interictal epileptiform activity was generalized. There were no focal magnetic resonance imaging abnormalities. Four patients were misdiagnosed with complex partial seizures. All patients were initially refractory, but three became seizure-free and three improved after treatment with antiepileptic medications appropriate for absence or myoclonic seizures. Generalized-onset seizures that acquire focal features are easily misdiagnosed as complex partial. These seizures have a more favorable response to medications effective against generalized absence and myoclonic seizures.

Objective: Some seizures are characterized by a transitional sharp wave (TShW) at ictal onset. We evaluated the clinical significance and localizing value of TShW in partial-onset seizures., Methods: We identified and analyzed all scalp ictal recordings with a TShW at ictal onset in the Vanderbilt Epilepsy Monitoring Unit over a period of 12 months., Results: A total of 52 ictal discharges in 13 patients started with a TShW. The center of TShW field was concordant with the final localization/lateralization, while that of the subsequent ictal discharge was concordant in only 6 patients. The subsequent rhythmic ictal discharge was non-localizing in 4 patients and misleading in the remaining 3 patients. The final localization was neocortical temporal or frontal in 11 patients, occipito-parietal in one, and undetermined in 1 patient. None of 61 ictal discharges in 15 patients with mesial temporal lobe epilepsy studied in the same time period started with a TShW., Conclusion: In this patient series, the TShW was a marker of neocortical seizure onset. The TShW field provided more accurate localization or lateralization of the ictal focus than the following rhythmic ictal discharge., Significance: TShW at seizure onset should suggest a neocortical rather than hippocampal seizure onset.

Fast synaptic transmission is mediated by post-synaptic ligand-gated ion channels (LGICs) transiently activated by neurotransmitter released from pre-synaptic vesicles. Although disruption of synaptic transmission has been implicated in numerous neurological and psychiatric disorders, effective and practical methods for studying LGICs in vitro under synaptically relevant conditions are unavailable. Here, we describe a novel microfluidic approach to solution switching that allows for precise temporal control over the neurotransmitter transient while substantially increasing experimental throughput, flexibility, reproducibility, and cost-effectiveness. When this system was used to apply ultra-brief ( approximately 400micros) GABA pulses to recombinant GABA(A) receptors, members of the cys-loop family of LGICs, the resulting currents resembled hippocampal inhibitory post-synaptic currents (IPSCs) and differed from currents evoked by longer, conventional pulses, illustrating the importance of evaluating LGICs on a synaptic timescale. This methodology should therefore allow the effects of disease-causing mutations and allosteric modulators to be evaluated in vitro under physiologically relevant conditions.

Low folate levels are associated with an increased risk of spontaneous abortion and congenital malformations. Up to half of all pregnancies are unplanned, so official guidelines in a number of countries, including the US, recommend folic acid supplementation for all women who might become pregnant, before they actually start planning to have children. Children born to women who are taking antiepileptic medications, many of which antagonize folate action, are at an increased risk of birth defects. This article highlights a study by Pittschieler et al. that has suggested that supplementation with folic acid might be particularly important in women with epilepsy who are taking antiepileptic medications--especially valproate. However, the sample size in the study was too small to determine the optimum dose or to detect whether folic acid protects against birth defects. Nonetheless, it is clear that all women of reproductive age, especially those taking certain antiepileptic medications, should take folic acid supplements.

Adenosine-to-inosine (A-to-I) editing of RNA transcripts is an increasingly recognized cellular strategy to modulate the function of proteins involved in neuronal excitability. We have characterized the editing of transcripts encoding the alpha3 subunit of heteromeric GABA(A) receptors (Gabra3), in which a genomically encoded isoleucine codon (ATA) is converted to a methionine codon (ATI) in a region encoding the predicted third transmembrane domain of this subunit. Editing at this position (I/M site) was regulated in a spatiotemporal manner with approximately 90% of the Gabra3 transcripts edited in most regions of adult mouse brain, but with lower levels of editing in the hippocampus. Editing was low in whole-mouse brain at embryonic day 15 and increased during development, reaching maximal levels by postnatal day 7. GABA-evoked current in transfected cells expressing nonedited alpha3(I)beta3gamma2L GABA(A) receptors activated more rapidly and deactivated much more slowly than edited alpha3(M)beta3gamma2L receptors. Furthermore, currents from nonedited alpha3(I)beta3gamma2L receptors were strongly outwardly rectifying (corresponding to chloride ion influx), whereas currents from edited alpha3(M)beta3gamma2L receptors had a more linear current/voltage relationship. These studies suggest that increased expression of the nonedited alpha3(I) subunit during brain development, when GABA is depolarizing, may allow the robust excitatory responses that are critical for normal synapse formation. However, the strong chloride ion influx conducted by receptors containing the nonedited alpha3(I) subunit could act as a shunt to prevent excessive excitation, providing the delicate balance necessary for normal neuronal development.

Up-regulation of the GABAA receptor alpha4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of alpha4 subtype-containing GABAA receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABAA receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of alpha4beta3gamma2L receptors to those of the more widely distributed alpha1beta3gamma2L receptors. These receptor currents had similar peak current amplitudes and GABA EC50 values. However, alpha4beta3gamma2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (tau = 15-100 ms), and had less residual current during long GABA applications. In addition, alpha4beta3gamma2L currents deactivated more slowly than alpha1beta3gamma2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for alpha4beta3gamma2L currents than alpha1beta3gamma2L currents. Moreover, the time required to recover from desensitization was prolonged in alpha4beta3gamma2L currents compared to alpha1beta3gamma2L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of alpha4beta3gamma2L currents to higher concentrations of GABA, while alpha1beta3gamma2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that alpha4beta3gamma2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to alpha1beta3gamma2L receptors in their ability to respond to brief and low frequency synaptic inputs, alpha4beta3gamma2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures.

Juvenile myoclonic epilepsy (JME), accounting for approximately 25% of idiopathic generalized epilepsies, is genetically heterogeneous. Mutations in the alpha-1 subunit of the GABAA receptor (GABRA1) and EFHC1 genes have been reported in a few families with autosomal dominant (AD) JME. We have investigated the contribution of these two genes to familial JME in our cohort of 54 JME Caucasian families. Syndromic classification of JME was based on previously published criteria. We considered kindreds with at least one affected first-degree relative and the evidence of a vertical transmission as definite AD JME, and families with at least one affected second-degree relative as probable AD JME. We included 33 families meeting criteria for definitive AD JME and 21 that were classified as probable AD JME. None of these families were considered informative enough to analyze candidate loci for JME using linkage analysis. We have systematically screened coding exons of these two genes using temperature gradient capillary electrophoresis. Every heteroduplex with an abnormal mobility was sequenced. No disease-causing mutations in the GABRA1 gene were identified. Analysis of EFHC1 gene found one putative disease-causing mutation R221H that was previously reported as a tandem mutation. Several synonymous and non-synonymous coding polymorphisms were identified but the allelic frequency did not differ between controls and affected individuals. Our data suggests that the majority of familial AD JME is not caused by mutations in the GABRA1 and EFHC1 genes.

We report a case of new-onset seizures and narcolepsy in a previously healthy 40-year-old man. He developed severe daytime somnolence and cataplexy over the course of a few months. Brain MRI was normal, and polysomnography with multiple sleep latency testing confirmed a diagnosis of narcolepsy. His HLA haplotype is DQB1*0602 and cerebrospinal fluid analysis showed no detectable hypocretin. Approximately 18 months later, he developed complex partial seizures. Further MRI showed a progressively enlarging lesion involving the left frontotemporal and insular areas. Pathology from a partial resection was consistent with Rasmussen's syndrome. Evaluation for tumor, infectious, and paraneoplastic etiologies was negative. There was no further progression of the residual lesion on serial MRI. Although the pathophysiologic bases of narcolepsy and Rasmussen's syndrome are unknown, they may have an autoimmune basis. This unique case of both disorders in a single patient suggests the possibility of a common underlying disease process.

Here we report on the progress we have made in elucidating the mechanisms through which estrogen alters synaptic responses in hypothalamic neurons. We examined the modulation by estrogen of the coupling of various receptor systems to inwardly rectifying and small conductance, Ca(2+)-activated K(+) (SK) channels. We used intracellular sharp-electrode and whole-cell recordings in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples mu-opioid receptors from G protein-gated inwardly rectifying K(+) (GIRK) channels in beta-endorphin neurons, manifest by a reduction in the potency of mu-opioid receptor agonists to hyperpolarize these cells. This effect is blocked by inhibitors of protein kinase A and protein kinase C. Estrogen also uncouples gamma-aminobutyric acid (GABA)(B) receptors from the same population of GIRK channels coupled to mu-opioid receptors. At 24 h after steroid administration, the GABA(B)/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area (POA) is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of alpha(1)-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these POA GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of both arcuate and POA neurons, among which gonadotropin-releasing hormone (GnRH) neurons are particularly sensitive. These findings indicate a richly complex yet coordinated steroid modulation of K(+) channel activity that serves to control the excitability of hypothalamic neurons involved in regulating the reproductive axis.

17Beta-estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic neurons. We have used intracellular recordings from female guinea pig hypothalamic slices to characterize the receptor and intracellular pathway(s) mediating E2's rapid effects. E2 acts stereospecifically with physiologically relevant concentration-dependence (EC50 = 8 nM) to cause a fourfold reduction in the potency of the mu-opioid agonist (D-Ala2-N-Me-Phe4-Gly5-ol)-enkephalin and the GABA(B) agonist baclofen to activate an inwardly rectifying K+ conductance in hypothalamic neurons. Both the nonsteroidal estrogen diethylstilbestrol and the anti-estrogen ICI 164,384 blocked E2 actions to uncouple mu-opioid receptors. Using a pharmacological Schild analysis, we found that ICI 164,384 competed for this E2 receptor with a Ke of approximately 0.3 nM. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a rapid, nongenomic mechanism of E2 action. The effects of E2 were mimicked by the bath application of the protein kinase A (PKA) activators, forskolin and Sp-cAMP, and the protein kinase C (PKC) activator phorbol-12,13-dibutyrate. Furthermore, the selective PKA antagonists Rp-cAMP and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. In addition, the actions of E2 were blocked by the selective PKC inhibitor Calphostin C. Therefore, it appears that E2 can activate both PKA and PKC to cause a heterologous desensitization of both mu-opioid and GABA(B) receptors, which has the potential to alter synaptic transmission in many regions of the CNS.

17beta-Estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic (beta-endorphin) neurons. In the current study, we used intracellular recordings from guinea pig hypothalamic slices to characterize the receptor and intracellular effector system mediating the rapid effects of E2. E2 acted stereospecifically with physiologically relevant concentration dependence (EC50 = 8 nM) to cause a 4-fold reduction in the potency of a mu-opioid agonist to activate an inwardly rectifying K+ conductance. Using Schild analysis to estimate the affinity of the mu-opioid receptor for an antagonist (naloxone), we found that estrogen did not compete for the mu-opioid receptor or alter the affinity of the mu receptor. Both the nonsteroidal estrogen diethylstilbestrol and the "pure" antiestrogen ICI 164,384 blocked the actions of E2, the latter with a subnanomolar affinity. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a nongenomic mechanism of action by E2. The actions of E2 were mimicked by the protein kinase A (PKA) activators forskolin and cAMP, Sp-isomer triethylammonium salt. Furthermore, the selective PKA antagonists cAMP, Rp-isomer triethylammonium salt and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. Thus, estrogen binds to a specific receptor that activates PKA to rapidly uncouple the mu-opioid receptor from its K+ channel. Because we have previously shown that gamma-aminobutyric acidB receptors are also uncoupled by estrogen, this mechanism of action has the potential to alter synaptic transmission via G protein-coupled receptors throughout the brain.

The present study examined the potential for cross-tolerance development between mu-opioid and gamma-aminobutyric acidB receptor agonists, in hypothalamic arcuate neurons, resulting from chronic morphine treatment. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized female guinea pigs. The mu-opioid receptor agonist D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin and the gamma-aminobutyric acidB receptor agonist baclofen produced dose-dependent membrane hyperpolarizations of arcuate neurons. The reversal potential for both agonist-induced hyperpolarizations was near -95 mV, indicative of the activation of an underlying K+ conductance. Coadministration of maximally effective concentrations of D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin and baclofen produced a response that was not additive, indicating a convergence onto a common K+ channel. In arcuate neurons, including a subset that was immunopositive for tyrosine hydroxylase, chronic morphine treatment for 4 to 7 days produced a 3.2-fold reduction in the potency, with no change in the efficacy, of D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin. In contrast, it affected neither the potency nor the efficacy of baclofen. Therefore, chronic morphine exposure does not produce cross-tolerance between mu-opioid and gamma-aminobutyric acidB receptor agonists in A12 dopamine neurons, suggesting that convergence upon a common effector is not a sufficient criterion for the development of cross-tolerance between receptor systems.

GABA is a predominant neurotransmitter in the hypothalamus and an important regulator of hypothalamic function. To elucidate the cellular basis for GABAergic action in this region, we used intracellular recordings from identified hypothalamic neurons. Ninety-three percent of the mediobasal hypothalamic neurons responded to GABAB receptor stimulation, and the presence of bicuculline-sensitive synaptic potentials indicated a tonic, GABAA receptor-mediated input. Stimulation of GABAB receptors hyperpolarized these cells by activating an inwardly rectifying potassium conductance. We characterized GABAB responses by generating concentration-response curves to the GABAB agonist baclofen. There was heterogeneity in the responses to baclofen, with one third of the cells having low baclofen potency (EC50 = 5.0 microM). Two thirds of the neurons had a 4-fold higher potency (EC50 = 1.2 microM), larger somas and a more lateral distribution. Previous work has shown that hypothalamic GABAB and mu-opioid receptors open the same K+ channels and that the response to mu-opioid agonists is rapidly attenuated by 17 beta-estradiol (E2). In order to test the hypothesis that the coupling of GABAB receptors to K+ channels is also altered, baclofen concentration-response curves were generated before and after an E2 challenge (100 nM, 20 min). Consistent with our hypothesis, the potency of baclofen was decreased nearly 4-fold in a subset of the cells that had a high potency response to baclofen. Furthermore, decreased baclofen potency only occurred in those cells in which E2 also altered the mu-opioid responses. Therefore, our findings suggest that a discrete subpopulation of hypothalamic neurons is sensitive to estrogen actions to alter inhibitory transmission. We propose that the alteration of GABAB and mu-opioid input is consistent with estrogen's rapid inhibition of the reproductive axis.

The mu-opioid receptor is an autoreceptor on hypothalamic beta-endorphin neurons that when activated inhibits cell firing via increasing an inwardly rectifying potassium conductance. The membrane hyperpolarization to DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin) in beta-endorphin and other arcuate (ARC) neurons was investigated in hypothalamic slices from control and morphine-treated, ovariectomized guinea pigs. Chronic morphine treatment caused both a decreased potency (EC50 220 +/- 10 nM vs. 64 +/- 3 nM in controls) and a decreased efficacy (Vmax: -7.1 +/- 1.1 mV vs. -10.7 +/- 0.6 mV in controls) of DAMGO in a population of ARC neurons including beta-endorphin neurons. In another population of ARC neurons from morphine-treated animals, DAMGO was less potent (EC50: 110 +/- 4 nm) than in controls (EC50: 64 +/- 3nM), but there was not a significant change in the efficacy of DAMGO. Twenty percent of ARC neurons did not exhibit any signs of tolerance. The density of mu-opioid receptors labeled with the antagonist radioligand [3H]diprenorphine was found to be significantly decreased in the ARC and surrounding mediobasal hypothalamus after morphine treatment (Bmax: 217 +/- 9 vs. 276 +/- 16 fmol/mg protein in controls), which is consistent with the altered response in beta-endorphin neurons. In summary, chronic morphine treatment decreases mu-opioid receptor density and the functional coupling of mu-opioid receptors to K+ channels in ARC neurons. This expression of morphine tolerance by beta-endorphin (ARC) neurons may serve as a homeostatic mechanism to maintain opioid control of a variety of systems ranging from reproduction to motivation and reward.

Control of the HPG axis involves a rapid (30 min) inhibition of LH (GnRH) release by E2. The time course of this effect is faster than expected for a purely transcriptional mechanism of E2 action. To elucidate the mechanism of E2 action, intracellular recordings in TTX were performed in guinea pig hypothalamic GnRH neurons. These neurons were directly hyperpolarized by both the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol, 9 mV) and the GABAB agonist, baclofen (18 mV) by opening K+ channels. Schild analysis with naloxone (Ke = 2.4 nM) confirmed that mu-opioid receptors mediated the effect of DAMGO. E2 also directly hyperpolarized GnRH neurons by opening K+ channels. Coupled with previous work showing a rapid effect of E2 to alter mu-opioid potency (1), a model is presented in which E2 rapidly inhibits GnRH neurons through parallel, possibly synergistic pathways.