Bachelor of Science, Bates College (2008)
Doctor of Philosophy, University of Virginia (2018)
Maintenance of a low intracellular Cl- concentration ([Cl-]i) is critical for enabling inhibitory neuronal responses to GABAA receptor-mediated signaling. Cl- transporters, including KCC2, and extracellular impermeant anions ([A]o) of the extracellular matrix are both proposed to be important regulators of [Cl-]i Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl-]i remains relatively low in RT neurons. Although we identify a small contribution of [A]o to a low [Cl-]i in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl- Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl- following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl- extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy.SIGNIFICANCE STATEMENT Precise regulation of intracellular Cl- levels ([Cl-]i) preserves appropriate, often inhibitory, GABAergic signaling within the brain. However, there is disagreement over the relative contribution of various mechanisms that maintain low [Cl-]i We found that the Cl- transporter KCC2 is an important Cl- extruder in the reticular thalamic (RT) nucleus, despite this nucleus having remarkably low KCC2 immunoreactivity relative to other regions of the adult brain. We also identified a smaller contribution of fixed, impermeant anions ([A]o) to lowering [Cl-]i in RT neurons. Inhibitory signaling among RT neurons is important for preventing excessive activation of RT neurons, which can be responsible for generating seizures. Our work suggests that KCC2 critically restricts the spread of activity within the RT nucleus.
View details for DOI 10.1523/JNEUROSCI.1345-17.2017
View details for PubMedID 29273603
View details for PubMedCentralID PMC5792478
Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca2+ levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage-dependent L- and T-type Ca2+ channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately -85 mV and has been considered electrically silent, low voltage-activated T-type Ca2+ channels are assumed to provide the primary Ca2+ signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain-of-function mutations in L-type CaV 1.3 channels in aldosterone-producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca2+ signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV 3.2 channels can generate a significant and recurring Ca2+ signal, and how CaV 1.3 channels may contribute to the Ca2+ signal that drives aldosterone production.
View details for DOI 10.1113/JP271896
View details for Web of Science ID 000385399100013
View details for PubMedID 26845064
View details for PubMedCentralID PMC5063946
Neonatal seizures are commonly caused by hypoxic and/or ischemic injury during birth and can lead to long-term epilepsy and cognitive deficits. In a rodent hypoxic seizure (HS) model, we have previously demonstrated a critical role for seizure-induced enhancement of the AMPA subtype of glutamate receptor (GluA) in epileptogenesis and cognitive consequences, in part due to GluA maturational upregulation of expression. Similarly, as the expression and function of the N-Methyl-D-aspartate (NMDA) subtype of glutamate receptor (GluN) is also developmentally controlled, we examined how early life seizures during the critical period of synaptogenesis could modify GluN development and function. In a postnatal day (P)10 rat model of neonatal seizures, we found that seizures could alter GluN2/3 subunit composition of GluNs and physiological function of synaptic GluNs. In hippocampal slices removed from rats within 48-96 h following seizures, the amplitudes of synaptic GluN-mediated evoked excitatory postsynaptic currents (eEPSCs) were elevated in CA1 pyramidal neurons. Moreover, GluN eEPSCs showed a decreased sensitivity to GluN2B selective antagonists and decreased Mg(2+) sensitivity at negative holding potentials, indicating a higher proportion of GluN2A and GluN3A subunit function, respectively. These physiological findings were accompanied by a concurrent increase in GluN2A phosphorylation and GluN3A protein. These results suggest that altered GluN function and expression could potentially contribute to future epileptogenesis following neonatal seizures, and may represent potential therapeutic targets for the blockade of future epileptogenesis in the developing brain.
View details for DOI 10.3389/fncel.2015.00362
View details for Web of Science ID 000361627000001
View details for PubMedID 26441533
View details for PubMedCentralID PMC4585040
Tropomyosin-related kinase receptor B (TrkB) activation has been implicated in epileptogenesis. We investigated hippocampal levels of phosphorylated TrkB (p-TrkB) and potential antiepileptogenic actions of the tyrosine kinase inhibitor, lestaurtinib (CEP-701) in postnatal day 10 (P10) rat pups following hypoxic seizures (HS). Hippocampal expression of p-TrkB over total TrkB protein levels were assessed by immunoblot at 6, 12, or 24 h post-HS, and revealed a statistically significant and transient 1.5-fold increase in hippocampal p-TrkB 12 h post-HS compared to littermate normoxic controls. To investigate the effects of CEP-701, pups were treated with 2 doses of CEP-701 intraperitoneally (i.p.), 3 mg/kg/dose, immediately after and 12 h post-HS. P-TrkB levels and susceptibility to kainic acid (KA)-induced seizures at P14 were compared between post-HS CEP-701-treated pups, post-HS vehicle-treated pups and normoxic littermates. Post-treatment with CEP-701 reversed the increased TrkB phosphorylation to baseline normoxic levels and attenuated the HS-related enhanced susceptibility to KA-induced seizures at P14. Given its known clinical safety profile, CEP-701 is a promising clinically translatable therapy to prevent epileptogenesis in the immature brain.
View details for DOI 10.1016/j.eplepsyres.2014.01.019
View details for Web of Science ID 000335623100024
View details for PubMedID 24582454
To determine whether AMPA receptor (AMPAR) antagonist NBQX can prevent early mammalian target of rapamycin (mTOR) pathway activation and long-term sequelae following neonatal seizures in rats, including later-life spontaneous recurrent seizures, CA3 mossy fiber sprouting, and autistic-like social deficits.Long-Evans rats experienced hypoxia-induced neonatal seizures (HS) at postnatal day (P)10. NBQX (20 mg/kg) was administered immediately following HS (every 12 h × 4 doses). Twelve hours post-HS, we assessed mTOR activation marker phosphorylated p70-S6 kinase (p-p70S6K) in hippocampus and cortex of vehicle (HS + V) or NBQX-treated post-HS rats (HS + N) versus littermate controls (C + V). Spontaneous seizure activity was compared between groups by epidural cortical electroencephalography (EEG) at P70-100. Aberrant mossy fiber sprouting was measured using Timm staining. Finally, we assessed behavior between P30 and P38.Postseizure NBQX treatment significantly attenuated seizure-induced increases in p-p70S6K in the hippocampus (p < 0.01) and cortex (p < 0.001). Although spontaneous recurrent seizures increased in adulthood in HS + V rats compared to controls (3.22 ± 1 seizures/h; p = 0.03), NBQX significantly attenuated later-life seizures (0.14 ± 0.1 seizures/h; p = 0.046). HS + N rats showed less aberrant mossy fiber sprouting (115 ± 8.0%) than vehicle-treated post-HS rats (174 ± 10%, p = 0.004), compared to controls (normalized to 100%). Finally, NBQX treatment prevented alterations in later-life social behavior; post-HS rats showed significantly decreased preference for a novel over a familiar rat (71.0 ± 12 s) compared to controls (99.0 ± 15.6 s; p < 0.01), whereas HS + N rats showed social novelty preference similar to controls (114.3 ± 14.1 s).Brief NBQX administration during the 48 h postseizure in P10 Long-Evans rats suppresses transient mTOR pathway activation and attenuates spontaneous recurrent seizures, social preference deficits, and mossy fiber sprouting observed in vehicle-treated adult rats after early life seizures. These results suggest that acute AMPAR antagonist treatment during the latent period immediately following neonatal HS can modify seizure-induced activation of mTOR, reduce the frequency of later-life seizures, and protect against CA3 mossy fiber sprouting and autistic-like social deficits.
View details for DOI 10.1111/epi.12378
View details for Web of Science ID 000326551300013
View details for PubMedID 24117347
View details for PubMedCentralID PMC4262152
Microarray profiling was used to investigate gene expression in the hypoxic seizure model of acquired epilepsy in the rat, with the aim of characterizing functional pathways which are persistently activated or repressed during epileptogenesis. Hippocampal and cortical tissues were transcriptionally profiled over a one week period following an initial series of seizures induced by mild hypoxia at post-natal day 10 (P10), and the gene expression data was then analyzed with a focus on gene set enrichment analysis, an approach which emphasizes regulation of entire pathways rather than of individual genes. Animals were subjected to one of three conditions: a control with no hypoxia, hypoxic seizures, and hypoxic seizures followed by treatment with the AMPAR antagonist NBQX, a compound currently proposed to be a modulator of epileptogenesis. While temporal gene expression in the control samples was found to be consistent with known processes of neuronal maturation in the rat for the given time window, the hypoxic seizure response was found to be enriched for components of the PI3K/mTOR and Wnt signaling pathways, alongside gene sets representative of glutamatergic, synaptic and axonal processes, perhaps regulated as a downstream consequence of activation of these pathways. Wnt signaling components were also found enriched in the more specifically epileptogenic NBQX-responsive gene set. While activation of the mTOR pathway is consistent with its known role in epileptogenesis and strengthens the case for mTOR or PI3K pathway inhibitors as potential anti-epileptogenic drugs, investigation of the role of Wnt signaling and the effect of appropriate inhibitors might offer a parallel avenue of research toward anti-epileptogenic treatment of epilepsy.
View details for DOI 10.1371/journal.pone.0074428
View details for Web of Science ID 000325223900026
View details for PubMedID 24086344
View details for PubMedCentralID PMC3785482
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.
View details for DOI 10.1073/pnas.1208010110
View details for Web of Science ID 000318031900072
View details for PubMedID 23479645
View details for PubMedCentralID PMC3612683
Neonatal seizures can lead to later life epilepsy and neurobehavioral deficits, and there are no treatments to prevent these sequelae. We showed previously that hypoxia-induced seizures in a neonatal rat model induce rapid phosphorylation of serine-831 (S831) and Serine 845 (S845) sites of the AMPA receptor GluR1 subunit and later neuronal hyperexcitability and epilepsy, suggesting that seizure-induced posttranslational modifications may represent a novel therapeutic target. To unambiguously assess the contribution of these sites, we examined seizure susceptibility in wild-type mice versus transgenic knock-in mice with deficits in GluR1 S831 and S845 phosphorylation [GluR1 double-phosphomutant (GluR1 DPM) mice]. Phosphorylation of the GluR1 S831 and S845 sites was significantly increased in the hippocampus and cortex after a single episode of pentyleneterazol-induced seizures in postnatal day 7 (P7) wild-type mouse pups and that transgenic knock-in mice have a higher threshold and longer latencies to seizures. Like the rat, hypoxic seizures in P9 C57BL/6N wild-type mice resulted in transient increases in GluR1 S831 and GluR1 S845 phosphorylation in cortex and were associated with enhanced seizure susceptibility to later-life kainic-acid-induced seizures. In contrast, later-life seizure susceptibility after hypoxia-induced seizures was attenuated in GluR1 DPM mice, supporting a role for posttranslational modifications in seizure-induced network excitability. Finally, human hippocampal samples from neonatal seizure autopsy cases also showed an increase in GluR1 S831 and S845, supporting the validation of this potential therapeutic target in human tissue.
View details for DOI 10.1523/JNEUROSCI.6121-11.2012
View details for Web of Science ID 000312402200024
View details for PubMedID 23223299
View details for PubMedCentralID PMC3574823
Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.
View details for DOI 10.1371/journal.pone.0035885
View details for Web of Science ID 000305341500033
View details for PubMedID 22567115
View details for PubMedCentralID PMC3342334
Tuberous sclerosis complex (TSC) is a multiorgan genetic disease in which brain involvement causes epilepsy, intellectual disability, and autism. The hallmark pathological finding in TSC is the cerebral cortical tuber and its unique constituent, giant cells. However, an animal model that replicates giant cells has not yet been described. Here, we report that mosaic induction of Tsc1 loss in neural progenitor cells in Tsc1(cc) Nestin-rtTA(+) TetOp-cre(+) embryos by doxycycline leads to multiple neurological symptoms, including severe epilepsy and premature death. Strikingly, Tsc1-null neural progenitor cells develop into highly enlarged giant cells with enlarged vacuoles. We found that the vacuolated giant cells had multiple signs of organelle dysfunction, including markedly increased mitochondria, aberrant lysosomes, and elevated cellular stress. We found similar vacuolated giant cells in human tuber specimens. Postnatal rapamycin treatment completely reversed these phenotypes and rescued the mutants from epilepsy and premature death, despite prenatal onset of Tsc1 loss and mTOR complex 1 activation in the developing brain. This TSC brain model provides insights into the pathogenesis and organelle dysfunction of giant cells, as well as epilepsy control in patients with TSC.
View details for DOI 10.1073/pnas.1106454108
View details for Web of Science ID 000296700000010
View details for PubMedID 22025691
View details for PubMedCentralID PMC3214999
To study the development of epilepsy following hypoxia-induced neonatal seizures in Long-Evans rats and to establish the presence of spontaneous seizures in this model of early life seizures.Long-Evans rat pups were subjected to hypoxia-induced neonatal seizures at postnatal day 10 (P10). Epidural cortical electroencephalography (EEG) and hippocampal depth electrodes were used to detect the presence of seizures in later adulthood (> P60). In addition, subdermal wire electrode recordings were used to monitor age at onset and progression of seizures in the juvenile period, at intervals between P10 and P60. Timm staining was performed to evaluate mossy fiber sprouting in the hippocampi of P100 adult rats that had experienced neonatal seizures.In recordings made from adult rats (P60-180), the prevalence of epilepsy in cortical and hippocampal EEG recordings was 94.4% following early life hypoxic seizures. These spontaneous seizures were identified by characteristic spike and wave activity on EEG accompanied by behavioral arrest and facial automatisms (electroclinical seizures). Phenobarbital injection transiently abolished spontaneous seizures. EEG in the juvenile period (P10-60) showed that spontaneous seizures first occurred approximately 2 weeks after the initial episode of hypoxic seizures. Following this period, spontaneous seizure frequency and duration increased progressively with time. Furthermore, significantly increased sprouting of mossy fibers was observed in the CA3 pyramidal cell layer of the hippocampus in adult animals following hypoxia-induced neonatal seizures. Notably, Fluoro-Jade B staining confirmed that hypoxic seizures at P10 did not induce acute neuronal death.The rodent model of hypoxia-induced neonatal seizures leads to the development of epilepsy in later life, accompanied by increased mossy fiber sprouting. In addition, this model appears to exhibit a seizure-free latent period, following which there is a progressive increase in the frequency of electroclinical seizures.
View details for DOI 10.1111/j.1528-1167.2011.02992.x
View details for Web of Science ID 000289156700016
View details for PubMedID 21366558
View details for PubMedCentralID PMC3071424
Chronic hyperoxia during the first 1-4 postnatal weeks attenuates the hypoxic ventilatory response (HVR) subsequently measured in adult rats. Rather than focusing on this long-lasting plasticity, the present study considered the influence of hyperoxia on respiratory control during the neonatal period. Sprague-Dawley rats were born and raised in 60% O2 until studied at postnatal ages (P) of 4, 6-7, or 13-14 days. Ventilation and metabolism were measured in normoxia (21% O2) and acute hypoxia (12% O2) using head-body plethysmography and respirometry, respectively. Compared with age-matched rats raised in room air, the major findings were 1) diminished pulmonary ventilation and metabolic O2 consumption in normoxia at P4 and P6-7; 2) decreased breathing stability during normoxia; 3) attenuation of the early phase of the HVR at P6-7 and P13-14; and 4) a sustained increase in ventilation during hypoxia (vs. the normal biphasic HVR) at all ages studied. Attenuation of the early HVR likely reflects progressive impairment of peripheral arterial chemoreceptors while expression of a sustained HVR in neonates before P7 suggests that hyperoxia also induces plasticity within the central nervous system. Together, these results suggest a complex interaction between inhibitory and excitatory effects of hyperoxia on the developing respiratory control system.
View details for DOI 10.1152/japplphysiol.00510.2010
View details for Web of Science ID 000281958800023
View details for PubMedID 20576840
View details for PubMedCentralID PMC2944635
Gyrodactylus pisculentus sp. n. is described from the head, body and fins of the northern pipefish, Syngnathus fuscus Storer (Syngnathidae) from the environs of Woods Hole, Massachusetts, USA. The new species is compared with Gyrodactylus shorti and Gyrodactylus syngnathi, species previously recorded from pipefish in the United States and Norway, respectively. Gyrodactylus pisculentus was frequently associated with mortality of northern pipefish held in quarantine at the Woods Hole Science Aquarium during 2002-2005. The clinical account of the infections and treatment protocols are presented.
View details for Web of Science ID 000262527700003
View details for PubMedID 19175204