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Key points 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 because of 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 brain's extracellular space to neuronal signal processing via the effects on the amplitude and direction of GABAA transmembrane Cl currents. Extracellular chloride concentrations in the brain were measured using a new chloride‐sensitive organic fluorophore and two‐photon fluorescence lifetime imaging. In vivo, the extracellular chloride concentration was spatially heterogenous and only half of the cerebrospinal fluid chloride concentration Stable displacement of extracellular chloride by immobile extracellular anions was responsible for the low extracellular chloride concentration The changes in extracellular chloride were of sufficient magnitude to alter the conductance and reversal potential of GABAA chloride currents The 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 signalling suggests a previously unappreciated mechanism for modulating GABAA signalling. [ABSTRACT FROM AUTHOR]

Cerebral Vascular and Blood Brain-Barrier Abnormalities in a Mouse Model of Epilepsy and Tuberous Sclerosis Complex Guo D, Zhang B, Han L, Rensing NR, Wong M. Epilepsia. 2024;65(2):483-496. PMCID: PMC10922951. doi:10.1111/epi.17848 Objective: Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. Methods: Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. In vivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. Results: VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. Significance: Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC. [ABSTRACT FROM AUTHOR]

GABA, the primary inhibitory neurotransmitter, stimulates GABAA receptors (GABAARs) to increase the chloride conductance of the cytosolic membrane. The driving forces for membrane chloride currents are determined by the local differences between intracellular and extracellular chloride concentrations (Cli and Clo, respectively). While several strategies exist for the measurement of Cli, the field lacks tools for non-invasive measurement of Clo. We present the design and development of a fluorescent lifetime imaging (FLIM)-compatible small molecule, N(4-aminobutyl)phenanthridiunium (ABP) with the brightness, spectral features, sensitivity to chloride, and selectivity versus other anions to serve as a useful probe of Clo. ABP can be conjugated to dextran to ensure extracellular compartmentalization, and a second chloride-insensitive counter-label can be added for ratiometric imaging. We validate the utility of this novel sensor series in two sensor concentration-independent modes: FLIM or ratiometric intensity-based imaging. [ABSTRACT FROM AUTHOR]

Targeting Aberrant Dendritic Integration to Treat Cognitive Comorbidities of Epilepsy Masala N, Pofahl M, Haubrich AN, Islam KUS, Nikbakht N, Pasdarnavab M, Bohmbach K, Araki K, Kamali F, Henneberger C, Golcuk K, Ewell LA, Blaess S, Kelly T, Beck H. Brain. 2023;146(6):2399-2417. doi:10.1093/brain/awac455 Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed a marked increase in the fraction of hippocampal first-order CA1 pyramidal cell dendrites capable of generating dendritic spikes in the kainate model of chronic epilepsy. Moreover, in epileptic mice dendritic spikes were generated with lower input synchrony, and with a lower threshold. The Nav1.3/1.1 selective Na+ channel blocker ICA-121431 reversed dendritic hyperexcitability in epileptic mice, while the Nav1.2/1.6 preferring anticonvulsant S-Lic did not. We used in vivo two-photon imaging to determine if aberrant dendritic excitability is associated with altered place-related firing of CA1 neurons. We show that ICA-121431 improves degraded hippocampal spatial representations in epileptic mice. Finally, behavioural experiments show that reversing aberrant dendritic excitability with ICA-121431 reverses hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition. [ABSTRACT FROM AUTHOR]

Multiple Sources of Fast Traveling Waves During Human Seizures: Resolving a Controversy Schlafly ED, Marshall FA, Merricks EM, Eden UT, Cash SS, Schevon CA, Kramer MA. J Neurosci. 2022;42(36):6966-6982. doi:10.1523/JNEUROSCI.0338-22.2022 During human seizures organized waves of voltage activity rapidly sweep across the cortex. Two contradictory theories describe the source of these fast traveling waves: either a slowly advancing narrow region of multiunit activity (an ictal wavefront) or a fixed cortical location. Limited observations and different analyses prevent resolution of these incompatible theories. Here we address this disagreement by combining the methods and microelectrode array recordings (N = 11 patients, 2 females, N = 31 seizures) from previous human studies to analyze the traveling wave source. We find—inconsistent with both existing theories—a transient relationship between the ictal wavefront and traveling waves, and multiple stable directions of traveling waves in many seizures. Using a computational model that combines elements of both existing theories, we show that interactions between an ictal wavefront and fixed source reproduce the traveling wave dynamics observed in vivo. We conclude that combining both existing theories can generate the diversity of ictal traveling waves. Significance Statement: The source of voltage discharges that propagate across cortex during human seizures remains unknown. Two candidate theories exist, each proposing a different discharge source. Support for each theory consists of observations from a small number of human subject recordings, analyzed with separately developed methods. How the different, limited data and different analysis methods impact the evidence for each theory is unclear. To resolve these differences, we combine the unique, human microelectrode array recordings collected separately for each theory and analyze these combined data with a unified approach. We show that neither existing theory adequately describes the data. We then propose a new theory that unifies existing proposals and successfully reproduces the voltage discharge dynamics observed in vivo. [ABSTRACT FROM AUTHOR]

Brain-Wide Reconstruction of Inhibitory Circuits After Traumatic Brain Injury Frankowski JC, Tierno A, Pavani S, Cao Q, Lyon DC, Hunt RF. Nat Commun. 2022;13(1):1-6. doi:10.1038/s41467-022-31072-2 Despite the fundamental importance of understanding the brain's wiring diagram, our knowledge of how neuronal connectivity is rewired by traumatic brain injury remains remarkably incomplete. Here we use cellular resolution whole-brain imaging to generate brain-wide maps of the input to inhibitory neurons in a mouse model of traumatic brain injury. We find that somatostatin interneurons are converted into hyperconnected hubs in multiple brain regions, with rich local network connections but diminished long-range inputs, even at areas not directly damaged. The loss of long-range input does not correlate with cell loss in distant brain regions. Interneurons transplanted into the injury site receive orthotopic local and long-range input, suggesting the machinery for establishing distant connections remains intact even after a severe injury. Our results uncover a potential strategy to sustain and optimize inhibition after traumatic brain injury that involves spatial reorganization of the direct inputs to inhibitory neurons across the brain. [ABSTRACT FROM AUTHOR]

Presurgical Temporal Lobe Epilepsy Connectome Fingerprint for Seizure Outcome Prediction Morgan VL, Sainburg LE, Johnson GW, Janson A, Levine KK, Rogers BP, Chang C, Englot DJ. Brain Comm. 2022;4(3):fcac128. doi:10.1093/braincomms/fcac128 Temporal lobe epilepsy presents a unique situation where confident clinical localization of the seizure focus does not always result in a seizure-free or favourable outcome after mesial temporal surgery. In this work, magnetic resonance imaging derived functional and structural whole-brain connectivity was used to compute a network fingerprint that captures the connectivity profile characteristics that are common across a group of nine of these patients with seizure-free outcome. The connectivity profile was then computed for 38 left-out patients with the hypothesis that similarity to the fingerprint indicates seizure-free surgical outcome. Patient profile distance to the fingerprint was compared with 1-year seizure outcome and standard clinical parameters. Distance to the fingerprint was higher for patients with Engel III-IV 1-year outcome compared with those with Engel Ia, Ib-d, and II outcome (Kruskal-Wallis, P < 0.01; Wilcoxon rank-sum pcorr <0.05 Bonferroni-corrected). Receiver operator characteristic analysis revealed 100% sensitivity and 90% specificity in identifying patients with Engel III-IV outcome based on distance to the fingerprint in the left-out patients. Furthermore, distance to the fingerprint was not related to any individual clinical parameter including age at scan, duration of disease, total seizure frequency, presence of mesial temporal sclerosis, lateralizing ictal, interictal scalp electroencephalography, invasive stereo-encephalography, or positron emission tomography. And two published algorithms utilizing multiple clinical measures for predicting seizure outcome were not related to distance to the fingerprint, nor predictive of seizure outcome in this cohort. The functional and structural connectome fingerprint provides quantitative, clinically interpretable and significant information not captured by standard clinical assessments alone or in combinations. This automated and simple method may improve patient-specific prediction of seizure outcome in patients with a clinically identified focus in the mesial temporal lobe. [ABSTRACT FROM AUTHOR]

Original Article Citation Li, A. M., Hill, R. A. & Grutzendler, J. Intravital Imaging of Neocortical Heterotopia Reveals Aberrant Axonal Pathfinding and Myelination around Ectopic Neurons. Cereb Cortex 31, 4340-4356 (2021). doi:10.1093/cercor/bhab090. Neocortical heterotopia consist of ectopic neuronal clusters that are frequently found in individuals with cognitive disability and epilepsy. However, their pathogenesis remains poorly understood due in part to a lack of tractable animal models. We have developed an inducible model of focal cortical heterotopia that enables their precise spatiotemporal control and high-resolution optical imaging in live mice. Here, we report that heterotopia are associated with striking patterns of circumferentially projecting axons and increased myelination around neuronal clusters. Despite their aberrant axonal patterns, in vivo calcium imaging revealed that heterotopic neurons remain functionally connected to other brain regions, highlighting their potential to influence global neural networks. These aberrant patterns only form when heterotopia are induced during a critical embryonic temporal window, but not in early postnatal development. Our model provides a new way to investigate heterotopia formation in vivo and reveals features suggesting the existence of developmentally modulated, neuron-derived axon guidance and myelination factors. [ABSTRACT FROM AUTHOR]

Imaging modalities such as single-photon emission computed tomography (SPECT) and functional magnetic resonance imaging (fMRI) afford a comprehensive image of the brain. Experiments aimed at imaging seizures in head-fixed animals (as in the two studies highlighted here) are complicated by the fact that spontaneous recurrent seizures are rare in most animal models of epilepsy. Our data indicate that CBV-based imaging techniques should be more accurate than blood oxygen level dependent (BOLD)-based imaging techniques for seizure mapping in awake behaving animals. [Extracted from the article]

As with models of acquired epilepsies, Scn1a+/- mice have spontaneous seizures at a frequency that is incompatible with head-fixed imaging: approximately 1 seizure per 2 days. Recent studies have used two-photon calcium imaging (2 P imaging) in awake, behaving mice in head-fixed preparations to image seizures in vivo at high speed and cellular-level resolution to identify key seizure-related cell classes. Imaging Interneuron Impairment in Ictogenesis: A Spatiotemporal Sweet Spot of Seizure Sampling in Scn1a+/-. [Extracted from the article]

Epileptic networks are characterized by two outputs: brief interictal spikes and rarer, more prolonged seizures. Although either output state is readily modeled in silico and induced experimentally, the transition mechanisms are unknown, in part because no models exhibit both output states spontaneously. In silico small-world neural networks were built using single-compartment neurons whose physiological parameters were derived from dual whole-cell recordings of pyramidal cells in organotypic hippocampal slice cultures that were generating spontaneous seizure-like activity. In silico, neurons were connected by abundant local synapses and rare long-distance synapses. Activity-dependent synaptic depression and gradual recovery delimited synchronous activity. Full synaptic recovery engendered interictal population spikes that spread via long-distance synapses. When synaptic recovery was incomplete, postsynaptic neurons required coincident activation of multiple presynaptic terminals to reach firing threshold. Only local connections were sufficiently dense to spread activity under these conditions. This coalesced network activity into traveling waves whose velocity varied with synaptic recovery. Seizures were comprised of sustained traveling waves that were similar to those recorded during experimental and human neocortical seizures. Sustained traveling waves occurred only when wave velocity, network dimensions, and the rate of synaptic recovery enabled wave reentry into previously depressed areas at precisely ictogenic levels of synaptic recovery. Wide-field, cellular-resolution GCamP7b calcium imaging demonstrated similar initial patterns of activation in the hippocampus, although the anatomical distribution of traveling waves of synaptic activation was altered by the pattern of synaptic connectivity in the organotypic hippocampal cultures. [ABSTRACT FROM AUTHOR]

Toll-like Receptor 4 Signaling in Neurons Enhances Calcium-Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Currents and Drives Post-Traumatic Epileptogenesis Korgaonkar AA, Li Y, Sekhar D, et al. Ann Neurol. 2020;87(4):497-515. doi: 10.1002/ana.25698 Objective: Traumatic brain injury is a major risk factor for acquired epilepsies, and understanding the mechanisms underlying the early pathophysiology could yield viable therapeutic targets. Growing evidence indicates a role for inflammatory signaling in modifying neuronal excitability and promoting epileptogenesis. Here, we examined the effect of innate immune receptor Toll-like receptor 4 (TLR4) on excitability of the hippocampal dentate gyrus and epileptogenesis after brain injury. Methods: Slice and in vivo electrophysiology and western blots were conducted in rats subject to fluid percussion brain injury or sham injury. Results: The studies identify that TLR4 signaling in neurons augments dentate granule cell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (CP-AMPAR) currents after brain injury. Blocking TLR4 signaling in vivo shortly after brain injury reduced dentate network excitability and seizure susceptibility. When blocking of TLR4 signaling after injury was delayed; however, this treatment failed to reduce postinjury seizure susceptibility. Furthermore, TLR4 signal blocking was less efficacious in limiting seizure susceptibility when AMPAR currents, downstream targets of TLR4 signaling, were transiently enhanced. Paradoxically, blocking TLR4 signaling augmented both network excitability and seizure susceptibility in uninjured controls. Despite the differential effect on seizure susceptibility, TLR4 antagonism suppressed cellular inflammatory responses after injury without impacting sham controls. Interpretation: These findings demonstrate that independently of glia, the immune receptor TLR4 directly regulates post-traumatic neuronal excitability. Moreover, the TLR4-dependent early increase in dentate excitability is causally associated with epileptogenesis. Identification and selective targeting of the mechanisms underlying the aberrant TLR4-mediated increase in CP-AMPAR signaling after injury may prevent epileptogenesis after brain trauma. [ABSTRACT FROM AUTHOR]

GABA bouton subpopulations in the human dentate gyrus are differentially altered in mesial temporal lobe epilepsy Alhourani A, Fish KN, Wozny TA, Sudhakar V, Hamilton RL, Richardson RM. J Neuro. 2020;123(4):392-406. doi:10.1152/jn.00523.2018 Medically intractable temporal lobe epilepsy is a devastating disease, for which surgical removal of the seizure-onset zone is the only known cure. Multiple studies have found evidence of abnormal dentate gyrus network circuitry in human mesial temporal lobe epilepsy (MTLE). Principal neurons within the dentate gyrus gate entorhinal input into the hippocampus provide a critical step in information processing. Crucial to that role are GABA-expressing neurons, particularly parvalbumin (PV)-expressing basket cells (PVBCs) and chandelier cells (PVChCs), which provide strong, temporally coordinated inhibitory signals. Alterations in PVBC and PVChC boutons have been described in epilepsy, but the value of these studies has been limited due to methodological hurdles associated with studying human tissue. We developed a multilabel immunofluorescence confocal microscopy and a custom segmentation algorithm to quantitatively assess PVBC and PVChC bouton densities and to infer relative synaptic protein content in the human dentate gyrus. Using en bloc specimens from MTLE subjects with and without hippocampal sclerosis, paired with nonepileptic controls, we demonstrate the utility of this approach for detecting cell-type specific synaptic alterations. Specifically, we found increased density of PVBC boutons, while PVChC boutons decreased significantly in the dentate granule cell layer of subjects with hippocampal sclerosis compared with matched controls. In contrast, bouton densities for either PV-positive cell type did not differ between epileptic subjects without sclerosis and matched controls. These results may explain conflicting findings from previous studies that have reported both preserved and decreased PV bouton densities and establish a new standard for quantitative assessment of interneuron boutons in epilepsy. [ABSTRACT FROM AUTHOR]

Role of Paroxysmal Depolarization in Focal Seizure Activity Tryba AK, Merricks EM, Lee S, et al. J Neuroph. 2019;122(5):1861-1873. doi.org/10.1152/jn.00392.2019 We analyze the role of inhibition in sustaining focal epileptic seizure activity. We review ongoing seizure activity at the mesoscopic scale that can be observed with microelectrode arrays as well as at the macroscale of standard clinical electroencephalogram. We provide clinical, experimental, and modeling data to support the hypothesis that paroxysmal depolarization (PD) is a critical component of the ictal machinery. We present dual-patch recordings in cortical cultures showing reduced synaptic transmission associated with presynaptic occurrence of PD, and we find that the PD threshold is cell size related. We further find evidence that optically evoked PD activity in parvalbumin neurons can promote propagation of neuronal excitation in neocortical networks in vitro. Spike sorting results from microelectrode array measurements around ictal wave propagation in human focal seizures demonstrate a strong increase in putative inhibitory firing with an approaching excitatory wave, followed by a sudden reduction of firing at passage. At the macroscopic level, we summarize evidence that this excitatory ictal wave activity is strongly correlated with oscillatory activity across a centimeter-sized cortical network. We summarize Wilson–Cowan-type modeling showing how inhibitory function is crucial for this behavior. Our findings motivated us to develop a network motif of neurons in silico, governed by a reduced version of the Hodgkin–Huxley formalism, to show how feedforward, feedback, PD, and local failure of inhibition contribute to observed dynamics across network scales. The presented multidisciplinary evidence suggests that the PD not only is a cellular marker or epiphenomenon but actively contributes to seizure activity. NEW & NOTEWORTHY: We present mechanisms of ongoing focal seizures across meso- and macroscales of microelectrode array and standard clinical recordings, respectively. We find modeling, experimental, and clinical evidence for a dual role of inhibition across these scales: local failure of inhibition allows propagation of a mesoscopic ictal wave, whereas inhibition elsewhere remains intact and sustains macroscopic oscillatory activity. We present evidence for PD as a mechanism behind this dual role of inhibition in shaping ictal activity. [ABSTRACT FROM AUTHOR]

In secondary epilepsy, a seizure-prone neural network evolves during the latent period between brain injury and the onset of spontaneous seizures. The nature of the evolution is largely unknown, and even its completeness at the onset of seizures has recently been challenged by measures of gradually decreasing intervals between subsequent seizures. Sequential calcium imaging of neuronal activity, in the pyramidal cell layer of mouse hippocampal in vitro preparations, during early post-traumatic epileptogenesis demonstrated rapid increases in the fraction of neurons that participate in interictal activity. This was followed by more gradual increases in the rate at which individual neurons join each developing seizure, the pairwise correlation of neuronal activities as a function of the distance separating the pair, and network-wide measures of functional connectivity. These data support the continued evolution of synaptic connectivity in epileptic networks beyond the latent period: early seizures occur when recurrent excitatory pathways are largely polysynaptic, while ongoing synaptic remodeling after the onset of epilepsy enhances intranetwork connectivity as well as the onset and spread of seizure activity. [ABSTRACT FROM AUTHOR]

Great advancements have been made in understanding the basic mechanisms of ictogenesis using single-cell electrophysiology (e.g., patch clamp, sharp electrode), large-scale electrophysiology (e.g., electroencephalography [EEG], field potential recording), and large-scale imaging (magnetic resonance imaging [ MRI], positron emission tomography [ PET], calcium imaging of acetoxymethyl ester [AM] dye-loaded tissue). Until recently, it has been challenging to study experimentally how population rhythms emerge from cellular activity. Newly developed optical imaging technologies hold promise for bridging this gap by making it possible to simultaneously record the many cellular elements that comprise a neural circuit. Furthermore, easily accessible genetic technologies for targeting expression of fluorescent protein-based indicators make it possible to study, in animal models of epilepsy, epileptogenic changes to neural circuits over long periods. In this review, we summarize some of the latest imaging tools (fluorescent probes, gene delivery methods, and microscopy techniques) that can lead to the advancement of cell- and circuit-level understanding of epilepsy, which in turn may inform and improve development of next generation antiepileptic and antiepileptogenic drugs. [ABSTRACT FROM AUTHOR]

Gelatinase Biosensor Reports Cellular Remodeling During Epileptogenesis Bouquier N, Girard B, Arias JA, Fagni L, Bertaso F, Perroy J. Front Synaptic Neurosci. 2020;12:15. doi:10.3389/fnsyn.2020.00015 Epileptogenesis is the gradual process responsible for converting a healthy brain into an epileptic brain. This process can be triggered by a wide range of factors, including brain injury or tumors, infections, and status epilepticus. Epileptogenesis results in aberrant synaptic plasticity, neuroinflammation, and seizure-induced cell death. As matrix metalloproteinases (MMPs) play a crucial role in cellular plasticity by remodeling the extracellular matrix, gelatinases (MMP-2 and MMP-9) were recently highlighted as key players in epileptogenesis. In this work, we engineered a biosensor to report in situ gelatinase activity in a model of epileptogenesis. This biosensor encompasses a gelatinase-sensitive activatable cell penetrating peptide (ACPP) coupled to a TAMRA fluorophore, allowing fluorescence uptake in cells displaying endogenous gelatinase activities. In a preclinical mouse model of temporal lobe epilepsy, the intrahippocampal kainate injection, ACPPs revealed a localized distribution of gelatinase activities, refining temporal cellular changes during epileptogenesis. The activity was found particularly but not only in the ipsilateral hippocampus, starting from the CA1 area and spreading to dentate gyrus from the early stages throughout chronic epilepsy, notably in neurons and microglial cells. Thus, our work shows that ACPPs are suitable molecular imaging probes for detecting the spatiotemporal pattern of gelatinase activity during epileptogenesis, suggesting their possible use as vectors to target cellular reactive changes with treatment for epileptogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Seizures are thought to originate from a failure of inhibition to quell hyperactive neural circuits, but the nature of this failure remains unknown. Here we combine high-speed two-photon imaging with electrophysiological recordings to directly evaluate the interaction between populations of interneurons and principal cells during the onset of seizure-like activity in mouse hippocampal slices. Both calcium imaging and dual patch clamp recordings reveal that in vitro seizure-like events (SLEs) are preceded by pre-ictal bursts of activity in which interneurons predominate. Corresponding changes in intracellular chloride concentration were observed in pyramidal cells using the chloride indicator Clomeleon. These changes were measurable at SLE onset and became very large during the SLE. Pharmacological manipulation of GABAergic transmission, either by blocking GABAA receptors or by hyperpolarizing the GABAA reversal potential, converted SLEs to short interictal-like bursts. Together, our results support a model in which pre-ictal GABAA receptor-mediated chloride influx shifts EGABA to produce a positive feedback loop that contributes to the initiation of seizure activity. [Copyright &y& Elsevier]

We examined how proprioceptive contributions to perception of hand path straightness are influenced by visual, motor and attentional sources of performance variability during horizontal planar reaching. Subjects held the handle of a robot that constrained goal-directed movements of the hand to the paths of controlled curvature. Subjects attempted to detect the presence of hand path curvature during both active (subject driven) and passive (robot driven) movements that either required active muscle force production or not. Subjects were less able to discriminate curved from straight paths when actively reaching for a target versus when the robot moved their hand through the same curved paths. This effect was especially evident during robot-driven movements requiring concurrent activation of lengthening but not shortening muscles. Subjects were less likely to report curvature and were more variable in reporting when movements appeared straight in a novel “visual channel” condition previously shown to block adaptive updating of motor commands in response to deviations from a straight-line hand path. Similarly, compromised performance was obtained when subjects simultaneously performed a distracting secondary task (key pressing with the contralateral hand). The effects compounded when these last two treatments were combined. It is concluded that environmental, intrinsic and attentional factors all impact the ability to detect deviations from a rectilinear hand path during goal-directed movement by decreasing proprioceptive contributions to limb state estimation. In contrast, response variability increased only in experimental conditions thought to impose additional attentional demands on the observer. Implications of these results for perception and other sensorimotor behaviors are discussed. [ABSTRACT FROM AUTHOR]

Abstract: We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available. [Copyright &y& Elsevier]

Previous studies have demonstrated the presence of functional glycine receptors (GlyRs) in hippocampus. In this work, we examine the baseline activity and activity-dependent modulation of GlyRs in region CA1. We find that strychnine-sensitive GlyRs are open in the resting CA1 pyramidal cell, creating a state of tonic inhibition that "shunts" the magnitude of EPSPs evoked by electrical stimulation of the Schaffer collateral inputs. This GlyR-mediated shunting conductance is independent of the presynaptic stimulation rate; however, pairs of presynaptic and postsynaptic action potentials, repeated at frequencies above 5 Hz, reduce the GlyR-mediated conductance and increase peak EPSP magnitudes to levels at least 20% larger than those seen with presynaptic stimulation alone. We refer to this phenomenon as rate-dependent efficacy (RDE). Exogenous GlyR agonists (glycine, taurine) block RDE by preventing the closure of postsynaptic GlyRs. The GlyR antagonist strychnine blocks postsynaptic GlyRs under all conditions, occluding RDE. During RDE, GlyRs are less responsive to local glycine application, suggesting that a reduction in the number or sensitivity of membrane-inserted GlyRs underlies RDE. By extending the RDE induction protocol to include 500 paired presynaptic and postsynaptic spikes, we can induce long-term synaptic depression (LTD). Manipulations that lead to reduced functionality of GlyRs, either pharmacologically or through RDE, also lead to increased LTD. This result suggests that RDE contributes to long-term synaptic plasticity in the hippocampus. [ABSTRACT FROM AUTHOR]

Abstract: In the dynamic clamp technique, a typically nonlinear feedback system delivers electrical current to an excitable cell that represents the actions of “virtual” ion channels (e.g., channels that are gated by local membrane potential or by electrical activity in neighboring biological or virtual neurons). Since the conception of this technique, there have been a number of different implementations of dynamic clamp systems, each with differing levels of flexibility and performance. Embedded hardware-based systems typically offer feedback that is very fast and precisely timed, but these systems are often expensive and sometimes inflexible. PC-based systems, on the other hand, allow the user to write software that defines an arbitrarily complex feedback system, but real-time performance in PC-based systems can be deteriorated by imperfect real-time performance. Here, we systematically evaluate the performance requirements for artificial dynamic clamp knock-in of transient sodium and delayed rectifier potassium conductances. Specifically, we examine the effects of controller time step duration, differential equation integration method, jitter (variability in time step), and latency (the time lag from reading inputs to updating outputs). Each of these control system flaws is artificially introduced in both simulated and real dynamic clamp experiments. We demonstrate that each of these errors affect dynamic clamp accuracy in a way that depends on the time constants and stiffness of the differential equations being solved. In simulations, time steps above 0.2ms lead to catastrophic alteration of spike shape, but the frequency–current relationship is much more robust. Latency (the part of the time step that occurs between measuring membrane potential and injecting re-calculated membrane current) is a crucial factor as well. Experimental data are substantially more sensitive to inaccuracies than simulated data. [Copyright &y& Elsevier]

Intraventricular haemorrhage is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability and/or hydrocephalus. Animal models suggest that brain tissue shrinkage, with subsequent vascular stretch and tear, is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for intraventricular haemorrhage are biomarkers of hypoxic–ischaemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction in these conditions. This is because immature neurons express the chloride, salt and water transporter NKCC1, which subserves regulatory volume increases in non-neural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic–ischaemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters are diminished. Consequentially, mature neurons swell (cytotoxic oedema), whereas immature neurons might shrink. After hypoxic–ischaemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, in addition to prevention of neuronal shrinkage and the ensuing intraventricular haemorrhage, in premature infants. [ABSTRACT FROM AUTHOR]

Objective: The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive 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. Here, we quantify the fraction of ictal signal from each source. Methods: 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. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. 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 of spontaneous seizures was then used to quantify the cellular contributions to population‐level ictal signal. Results: The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. Significance: This work provides a critical link between single neuron activity and population measures of seizure activity. [ABSTRACT FROM AUTHOR]

Seizure initiation is the least understood and most disabling element of epilepsy. Studies of ictogenesis require high speed recordings at cellular resolution in the area of seizure onset. However, in vivo seizure onset areas cannot be determined at the level of resolution necessary to enable such studies. To circumvent these challenges, we used novel GCaMP7-based calcium imaging in the organotypic hippocampal slice culture model of post-traumatic epilepsy in mice. Organotypic hippocampal slice cultures generate spontaneous, recurrent seizures in a preparation in which it is feasible to image the activity of the entire network (with no unseen inputs existing). Chronic calcium imaging of the entire hippocampal network, with paired electrophysiology, revealed three patterns of seizure onset: (i) low amplitude fast activity; (ii) sentinel spike; and (iii) spike burst and low amplitude fast activity onset. These patterns recapitulate common features of human seizure onset, including low voltage fast activity and spike discharges. Weeks-long imaging of seizure activity showed a characteristic evolution in onset type and a refinement of the seizure onset zone. Longitudinal tracking of individual neurons revealed that seizure onset is stochastic at the single neuron level, suggesting that seizure initiation activates neurons in non-stereotyped sequences seizure to seizure. This study demonstrates for the first time that transitions to seizure are not initiated by a small number of neuronal 'bad actors' (such as overly connected hub cells), but rather by network changes which enable the onset of pathology among large populations of neurons. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Synchronous activation of neural networks is an important physiological mechanism, and dysregulation of synchrony forms the basis of epilepsy. We analyzed the propagation of synchronous activity through chronically epileptic neural networks. Electrocorticographic recordings from epileptic patients demonstrate remarkable variance in the pathways of propagation between sequential interictal spikes (IISs). Calcium imaging in chronically epileptic slice cultures demonstrates that pathway variance depends on the presence of GABAergic inhibition and that spike propagation becomes stereotyped following GABA receptor blockade. Computer modeling suggests that GABAergic quenching of local network activations leaves behind regions of refractory neurons, whose late recruitment forms the anatomical basis of variability during subsequent network activation. Targeted path scanning of slice cultures confirmed local activations, while ex vivo recordings of human epileptic tissue confirmed the dependence of interspike variance on GABA-mediated inhibition. These data support the hypothesis that the paths by which synchronous activity spreads through an epileptic network change with each activation, based on the recent history of localized activity that has been successfully inhibited. [ABSTRACT FROM AUTHOR]

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported. We found a corresponding variance in the GABAA receptor reversal potential (EGABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl-]i) in individual dendrites. We determined [Cl-]i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl--sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl--sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of EGABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs. [ABSTRACT FROM AUTHOR]