Biology subjects

Staley, K. J.

Publications and source records attributed to Staley, K. J..

12 recordsLinked to original sources

bioRxiv2026

Acute ClC-2 blockade increases neuronal intracellular chloride and depolarizes EGABA

Neuronal intracellular chloride concentration shapes the polarity and strength of GABAA receptor-mediated inhibition. ClC-2 is a hyperpolarization-activated chloride channel proposed to support chloride extrusion, but its acute contribution to EGABA and neuronal chloride homeostasis remains incompletely understood. Here, we used the selective ClC-2 inhibitor AK-42 to test how acute ClC-2 inhibition affects GABAergic chloride signaling in organotypic hippocampal slice cultures. We combined targeted single-interneuron optogenetic activation of stCoChR-expressing GABAergic interneurons with gramicidin-perforated patch recordings to measure optogenetically evoked inhibitory postsynaptic currents and calculate EGABA and we used SuperClomeleon imaging to measure neuronal intracellular chloride. AK-42 depolarized EGABA, with a larger effect when stimulation was delivered 2 s rather than 100 ms after voltage-step onset. This delay-dependent effect is consistent with greater engagement of voltage- and time-dependent ClC-2 activity during prolonged voltage steps. AK-42 also abolished the more hyperpolarized EGABA normally measured at the 2-s stimulation delay and shifted baseline holding currents at hyperpolarized command potentials without measurably changing estimated resting membrane potential. SuperClomeleon imaging showed that AK-42 increased neuronal intracellular chloride in the presence of TTX, supporting a role for ClC-2 in basal chloride regulation. Together, these findings show that acute ClC-2 inhibition depolarizes EGABA, increases neuronal intracellular chloride, and alters the temporal dynamics of GABAergic chloride signaling, identifying ClC-2 as an acute regulator of synaptic GABAergic chloride signaling.

neuroscience↗

bioRxiv2026

Robust Induction of Spontaneous Recurrent Seizures and Hippocampal Sclerosis with Synthetic Kainic Acid in the Intrahippocampal Kainate Model

Intrahippocampal kainate (IHK) is a well-established mouse model of temporal lobe epilepsy, in which focal hippocampal injection of kainic acid induces excitotoxic injury followed by the development of chronic spontaneous seizures. Commercial sources of kainic acid (KA) have increasingly transitioned from naturally derived to synthetic KA, which has introduced a potential source of variability to the implementation of the IHK model. Here, we evaluated injection parameters for synthetic KA, with the goal of identifying conditions that reliably produce spontaneous seizures while minimizing mortality. We found that intrahippocampal injection of 200nL of 5mM synthetic KA resulted in low mortality compared to 50-100nL of 20mM synthetic KA (a dosing range commonly used with naturally derived KA), while robustly inducing TLE-like hippocampal sclerosis and recurrent spontaneous electroclinical seizures. We further demonstrated high inter-animal variability in both seizure frequency and temporal clustering of seizures across animals receiving either injection protocol (200nL of 5mM or 50nL of 20mM). Together, these findings provide a framework for implementing the IHK model with synthetic KA and support its continued use to advance our understanding of the cellular and network mechanisms underlying temporal lobe epilepsy.

neuroscience↗

bioRxiv2026

Individualized and stereotypical seizure semiology in a porcine model of post-traumatic epilepsy.

Large-animal models of traumatic brain injury may yield translatable data on epileptogenesis, given their similarities in anatomy, brain size, and immune systems to humans. Adult male and female swine received bilateral cortical impact (N=16) or sham surgery (N=6) and were screened for convulsions via video-EEG for up to one year. Post-traumatic epilepsy (PTE) was defined as 2 seizures after 1-week post-injury. Nine out of sixteen pigs (56%) receiving bilateral cortical impact developed PTE, with an average 4.6 months ({+/-}3.4,SD) latent period. Seizures (N = 199) began focally, sometimes with motor onset including automatisms, before becoming generalized, with tonic-clonic or tonic convulsions. We defined a library differentiating peri-ictal behaviors (N = 31) from rhythmic/odd behaviors typical in healthy pigs (N = 12). Seizures had an average of 7.3 behaviors per seizure (max 26) lasting an average of 1.8 minutes (max 7.9). For seizures comprised of multiple convulsive episodes, the first convulsion had a greater number of peri-ictal behaviors than subsequent convulsions (P < 0.001). The array of peri-ictal behaviors displayed was pig-specific, with many behaviors consistently observed across seizures. The seizure frequency detected was 0.38/day. This large-brain model of PTE exhibits a variable period of epileptogenesis, a substantial rate of PTE, and an expansive repertoire of ictal behaviors. This first description of semiology in this species will serve as a guide for other porcine epilepsy models. Biofidelic models of PTE are expected to increase our understanding of the pathophysiology, enabling the identification and testing of therapeutics that translate into human patients. HighlightsO_LIThe average time from bilateral cortical impact to post-traumatic epilepsy in swine is 6 months, and is highly variable, ranging from 2 to 47 weeks post-TBI. C_LIO_LISwine with post-traumatic epilepsy display an array of specific behaviors around convulsions, distinct from pigs without post-traumatic epilepsy. C_LIO_LIThough the duration of convulsion was typically a few seconds, the entire seizure, with the associated peri-ictal behaviors, lasts up to 7.9 minutes. C_LIO_LIThe complexity of behaviors around convulsions tended to increase from early traumatic seizures to post-traumatic seizures. C_LIO_LIPeri-ictal behaviors observed around convulsions in an individual were often displayed prior to the first convulsion. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/708000v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@9523aborg.highwire.dtl.DTLVardef@158cfa6org.highwire.dtl.DTLVardef@1a8b499org.highwire.dtl.DTLVardef@e8c86f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Ongoing loss of viable neurons for weeks after mild perinatal hypoxia-ischemia

Mild hypoxic-ischemic encephalopathy is common in neonates with no evidence-based therapies, and 30-40% of patients experience adverse outcomes. The nature and progression of mild injury is poorly understood. Thus, we studied the evolution of mild perinatal brain injury using longitudinal two-photon imaging of transgenic fluorescent proteins as a novel readout of neuronal viability and activity at cellular resolution. In vitro, perinatal murine organotypic hippocampal cultures underwent 15-20 minutes of oxygen-glucose deprivation. In vivo, mild hypoxia-ischemia was completed in post-natal day 10 mouse pups of both sexes with carotid ligation and 15 minutes of hypoxia. Consistent with a mild injury, minimal immediate neuronal death was seen and there was no volumetric evidence of injury by ex vivo MRI 2.5 weeks after injury. In both the hippocampus and neocortex, these mild injuries resulted in a significantly delayed and progressive neuronal loss in the second week after injury, measured by fluorophore quenching. Mild hypoxia-ischemia transiently suppressed cortical network activity followed by normal maturation. No post-injury seizures were seen. The participation in network activity of individual neurons destined to die was indistinguishable from those that survived for 4 days post-injury. In conclusion, our results showed that mild perinatal brain injury resulted in a prolonged increase of neuronal death. Neurons that died late were functioning normally for days after injury, suggesting a new pathophysiology of neuronal death. Critically, the neurons destined to die late demonstrated multiple biomarkers of viability long after mild injury, suggesting their later death may be modified with neuroprotective interventions. SIGNIFICANCE STATEMENTNeonatal encephalopathy due to peripartum hypoxia-ischemia (HI) is a major cause of neonatal mortality and morbidity worldwide. Of these infants, most are categorized as having mild HI. Infants with mild HI have significant long-term disabilities. There are currently no evidence-based therapies, largely because the progression and pathophysiology of mild injury is poorly understood. We have identified, for the first time, that mild perinatal HI results in a delayed and prolonged increase in neuronal death. The cortical and hippocampal neurons that die over a week after injury participate normally in neural network activity and exhibit robust viability for many days after injury, indicating a novel pathophysiology of neuronal death. Clinically, these data suggest an extended therapeutic window for mild perinatal HI.

neuroscience↗

bioRxiv2023

Brief Apnea and Hypoventilation Reduces Seizure Duration and Shifts Seizure Location for Several Hours in a Model of Severe Traumatic Brain Injury.

ObjectiveSeizures are difficult to control in infants and toddlers. Seizures with periods of apnea and hypoventilation are common following severe traumatic brain injury (TBI). In our multifactorial, severe TBI model (cortical impact, mass effect, subdural hematoma, subarachnoid hemorrhage, seizures induced with kainic acid, and brief apnea and hypoventilation), we observed that brief apnea with hypoventilation (A&H) after induced seizure acutely interrupted seizures, leading us to hypothesize that brief A&H might reduce seizure duration beyond the brief hypoxia and hypercapnia for several hours thereafter. The effects of the timing of A&H on seizure duration and location might inform the pathophysiology of this hypoxic-ischemic injury as well as potential treatments. MethodsPiglets (1 week or 1 month old) received multi-factorial injuries. Apnea and hypoventilation (1 min apnea, 10 min hypoventilation; A&H) was induced either before or after seizure induction, or as a control piglets received subdural/subarachnoid hematoma and seizure without A&H. In an intensive care unit, piglets were sedated, intubated, mechanically ventilated, and epidural EEG was recorded for an average of 18 hours after seizure induction. ResultsIn our severe TBI model, A&H after seizure reduced ipsilateral seizure burden by 80% compared to the same injuries without A&H. In the A&H before seizure induction group, more piglets had exclusively contralateral seizures though most piglets in all groups had seizures that shifted location throughout the several hours of seizure. After 8-10 hours, seizures transitioned to interictal epileptiform discharges regardless of timing of A&H. SignificanceEven brief A&H may alter traumatic seizures We will address the possibility of induced spreading depolarization prior to preclinical investigations of hypercapnia with normoxia, with controlled intracranial pressure, as a therapeutic option for children with status epilepticus after hemorrhagic TBI.

neuroscience↗

bioRxiv2023

Release of extracellular matrix components after human traumatic brain injury

Most research on the evolution of damage after traumatic brain injury (TBI) focuses on cellular effects, but the analysis of human tissue slices and animal research have shown that TBI causes concomitant damage in the extracellular matrix, which can play a significant role in both short-term consequences such as edema, and late effects such as post-traumatic epilepsy (PTE). To test the hypothesis that traumatic brain injury (TBI) in human patients causes disruption of sulfated glycosaminoglycan (sGAG) in the extracellular matrix, we measured levels of these substances in the ventricular cerebrospinal fluid (CSF) in patients with severe TBI in the acute post-injury period, along with concomitant levels in blood and urine. We assessed whether levels corresponded to parenchymal injury load, distance of traumatic brain lesions from the ventricle, presence of polytrauma, or host demographic factors. MethodsSamples of CSF, blood, and urine were obtained within 72 hours of injury in patients who received external ventricular drains as part of their treatment of severe TBI, and levels of chondroitin and heparan sGAGs were measured, along with their disaccharide constituents. Basic demographic information, presence and severity of polytrauma, brain injury load based on imaging findings, and distance of radiologically visible parenchymal injury from the ventricle were analyzed for correlation with total subtype sGAG levels in each patient. ResultsLevels were measured in 14 patients ranging in age from 17-90 years. CSF sGAG levels were variable among patients, and sGAG levels were higher in plasma than in CSF and variable in urine. Patients with polytrauma had non-significantly higher blood sGAG compared to patients with isolated head injury. Subcategories of CSF sGAG levels correlated with distance from the ventricle of parenchymal injury but not with brain injury load, which may reflect rapid metabolism in the parenchyma, contamination by blood, or bulk directional CSF flow from the ventricle to the subarachnoid space. ConclusionThis study is the first to measure sGAG levels in ventricular CSF and also provides the first measurements in patients with TBI. Damage to the extracellular matrix may play a major role in acute and chronic injury sequelae, and these data demonstrate elevation locally of intracranial sGAGS after severe TBI and also suggest rapid local metabolism of these breakdown products. The consequences of extracellular matrix breakdown may provide unique therapeutic and preventive avenues to mitigate post-injury sequelae.

neuroscience↗

bioRxiv2023

A dynamic balance between neuronal death and clearance after acute brain injury

After acute brain injury, neuronal apoptosis may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. The size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for two weeks. Altering phagocytosis rates, e.g. by changing the number of microglia, dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for vital stains. Canonically neuroprotective interventions such as seizure blockade and neurotoxic maneuvers such as perinatal ethanol exposure were mediated by effects on microglial activity and the membrane permeability of apoptotic neurons, and had either no or opposing effects on healthy surviving neurons. SignificanceAfter acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus longitudinal assays of healthy cells, such as assessment of the fluorescence emission of transgenically-expressed proteins, provide more accurate estimates of cell death than do single-time-point anatomical or biochemical assays. More accurate estimates of death rates will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.

neuroscience↗

bioRxiv2023

2-photon imaging of fluorescent proteins in living swine

A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.

neuroscience↗

bioRxiv2022

Displacement of extracellular chloride by sulfated glycosaminoglycans of the brain extracellular matrix

GABA is the primary inhibitory neurotransmitter. Membrane currents evoked by GABAA receptor activation have uniquely small driving forces: their reversal potential (EGABA) is very close to the resting membrane potential. As a consequence, GABAA currents can flow in either direction, depending on both the membrane potential and the local intra and extracellular concentrations of the primary permeant ion, chloride (Cl). Local cytoplasmic Cl concentrations vary widely due to displacement of mobile Cl ions by relatively immobile anions. Here we use new reporters of extracellular chloride (Cl-o) to demonstrate that Cl is displaced in the extracellular space by high and spatially heterogenous concentrations of immobile anions including sulfated glycosaminoglycans (sGAGs). Cl-o varies widely, and the mean Cl-o is only half the canonical concentration, i.e. the Cl concentration in the cerebrospinal fluid. These unexpectedly low and heterogenous Cl-o domains provide a mechanism to link the varied but highly stable distribution of sGAGs and other immobile anions in the brains extracellular space to neuronal signal processing via the effects on the amplitude and direction of GABAA transmembrane Cl currents. Key Points SummaryO_LIExtracellular chloride concentrations in the brain were measured using a new chloride-sensitive organic fluorophore and two photon fluorescence lifetime imaging. C_LIO_LIIn vivo, the extracellular chloride concentration was spatially heterogenous and only half of the CSF chloride concentration C_LIO_LIStable displacement of extracellular chloride by immobile extracellular anions was responsible for the low extracellular chloride concentration C_LIO_LIThe changes in extracellular chloride were of sufficient magnitude to alter the conductance and reversal potential of GABAA chloride currents C_LIO_LIThe stability of the extracellular matrix, the impact of the component immobile anions including sulfated glycosaminoglycans on extracellular chloride concentrations, and the consequent effect on GABAA signaling suggests a previously unappreciated mechanism for modulating GABAA signaling. C_LI

neuroscience↗

bioRxiv2022

Cellular contributions to ictal population signals

ObjectiveThe amplitude of ictal activity is a defining feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using e.g. EEG, ECoG, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. MethodsSpontaneous seizure activity was assessed in an awake, behaving mouse model of focal cortical injury and in organotypic hippocampal slice cultures (OHSC), an in vitro preparation from which recurrent seizures can be readily captured. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging was then performed during spontaneous seizures in vivo and in vitro to quantify the cellular contributions to the population-level calcium signal. ResultsPopulation signal may increase from three potential sources: 1) increased synchrony, i.e. more co-active neurons, 2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential, and 3) altered subthreshold state, which includes all lower levels of activity. The largest contributor to the signal recorded at seizure onset was increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. The relative contribution of synchrony increased as seizures progressed, but cell intrinsic alterations in both the subthreshold and active states remained the largest driver of the ictal signal. SignificanceWe introduce here a novel method for the quantification of the relative contributions of inter-versus intra-cellular changes to provide a critical link between single neuron activity and population measures of seizure activity. Key PointsO_LINeuronal calcium as measured by GCaMP reports a range of membrane depolarizations, from EPSPs to action potential firing and paroxysmal depolarizing shifts C_LIO_LIThe mean population calcium signal is highly correlated with the electrographic local field potential C_LIO_LIIncreased calcium signal during seizure onset is largely driven by increased subthreshold calcium within individual neurons C_LIO_LIRecruitment of newly active neurons is a minor contributor to the increasing population-level signal during the transition to frank seizure C_LI

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bioRxiv2022

Robust, long-term video EEG monitoring in a porcine model of post-traumatic epilepsy.

To date, post-traumatic epilepsy (PTE) research in large animal models has been limited. Recent advances in neocortical microscopy have made possible new insights into neocortical PTE. However, it is very difficult to engender convincing neocortical PTE in rodents. Thus, large animal models that develop neocortical PTE may provide useful insights that also can be more comparable to human patients. Because gyrencephalic species have prolonged latent periods, long-term video EEG recording is required. Here, we report a fully subcutaneous EEG implant with synchronized video in freely ambulatory swine for up to 14 months during epileptogenesis following bilateral cortical impact injuries or sham surgery The advantages of this system include the availability of a commercially available system that is simple to install, a low failure rate after surgery for EEG implantation, radiotelemetry that enables continuous monitoring of freely ambulating animals, excellent synchronization to video to EEG, and a robust signal to noise ratio. The disadvantages of this system in this species and age are the accretion of skull bone which entirely embedded a subset of skull screws and EEG electrodes, and the inability to rearrange the EEG electrode array. These disadvantages may be overcome by splicing a subdural electrode strip to the electrode leads so that skull growth is less likely to interfere with long-term signal capture and by placing two implants for a more extensive montage. This commercially available system in this bilateral cortical impact swine model may be useful to a wide range of investigators studying epileptogenesis in PTE. SignificancePost-traumatic epilepsy (PTE) is a cause of significant morbidity after traumatic brain injury (TBI) and is often drug-resistant. Robust, informative animal models would greatly facilitate PTE research. Ideally, this biofidelic model of PTE would utilize a species that approximates human brain anatomy, brain size, glial populations, and inflammatory pathways. An ideal model would also incorporate feasible methods for long-term video EEG recording required to quantify seizure activity. Here, we describe the first model of PTE in swine and describe a method for robust long-term video EEG monitoring for up to 14 months post-TBI. The relatively easy "out-of-the-box" radiotelemetry system and surgical techniques described here will be adaptable by a wide array of investigators studying the pathogenesis and treatment of PTE.

neuroscience↗

bioRxiv2020

Unique actions of GABA arising from cytoplasmic chloride microdomains

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (EGABA) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl- ([Cl-i]). We determined [Cl-]i by: 1) two-photon imaging of the Cl- sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl- sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vivo. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.

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