Chloride dynamics alter the input-output properties of neurons.

Fast synaptic inhibition is a critical determinant of neuronal output, with subcellular targeting of synaptic inhibition able to exert different transformations of the neuronal input-output function. At the receptor level, synaptic inhibition is primarily mediated by chloride-permeable Type A GABA receptors. Consequently, dynamics in the neuronal chloride concentration can alter the functional properties of inhibitory synapses. How differences in the spatial targeting of inhibitory synapses interact with intracellular chloride dynamics to modulate the input-output function of neurons is not well understood. To address this, we developed computational models of multi-compartment neurons that incorporate experimentally parametrised mechanisms to account for neuronal chloride influx, diffusion, and extrusion. We found that synaptic input (either excitatory, inhibitory, or both) can lead to subcellular variations in chloride concentration, despite a uniform distribution of chloride extrusion mechanisms. Accounting for chloride changes resulted in substantial alterations in the neuronal input-output function. This was particularly the case for peripherally targeted dendritic inhibition where dynamic chloride compromised the ability of inhibition to offset neuronal input-output curves. Our simulations revealed that progressive changes in chloride concentration mean that the neuronal input-output function is not static but varies significantly as a function of the duration of synaptic drive. Finally, we found that the observed effects of dynamic chloride on neuronal output were mediated by changes in the dendritic reversal potential for GABA. Our findings provide a framework for understanding the computational effects of chloride dynamics on dendritically targeted synaptic inhibition.

Excitatory GABAergic signalling is associated with benzodiazepine resistance in status epilepticus.

Status epilepticus is defined as a state of unrelenting seizure activity. Generalized convulsive status epilepticus is associated with a rapidly rising mortality rate, and thus constitutes a medical emergency. Benzodiazepines, which act as positive modulators of chloride (Cl-) permeable GABAA receptors, are indicated as first-line treatment, but this is ineffective in many cases. We found that 48% of children presenting with status epilepticus were unresponsive to benzodiazepine treatment, and critically, that the duration of status epilepticus at the time of treatment is an important predictor of non-responsiveness. We therefore investigated the cellular mechanisms that underlie acquired benzodiazepine resistance, using rodent organotypic and acute brain slices. Removing Mg2+ ions leads to an evolving pattern of epileptiform activity, and eventually to a persistent state of repetitive discharges that strongly resembles clinical EEG recordings of status epilepticus. We found that diazepam loses its antiseizure efficacy and conversely exacerbates epileptiform activity during this stage of status epilepticus-like activity. Interestingly, a low concentration of the barbiturate phenobarbital had a similar exacerbating effect on status epilepticus-like activity, while a high concentration of phenobarbital was effective at reducing or preventing epileptiform discharges. We then show that the persistent status epilepticus-like activity is associated with a reduction in GABAA receptor conductance and Cl- extrusion capability. We explored the effect on intraneuronal Cl- using both gramicidin, perforated-patch clamp recordings and Cl- imaging. This showed that during status epilepticus-like activity, reduced Cl- extrusion capacity was further exacerbated by activity-dependent Cl- loading, resulting in a persistently high intraneuronal Cl-. Consistent with these results, we found that optogenetic stimulation of GABAergic interneurons in the status epilepticus-like state, actually enhanced epileptiform activity in a GABAAR dependent manner. Together our findings describe a novel potential mechanism underlying benzodiazepine-resistant status epilepticus, with relevance to how this life-threatening condition should be managed in the clinic.

Model systems for investigating disease processes in neurocysticercosis.

Neurocysticercosis (NCC) occurs following brain infection by larvae of the cestode Taenia solium. It is the leading cause of preventable epilepsy worldwide and therefore constitutes a critical health challenge with significant global relevance. Despite this, much is still unknown about many key pathogenic aspects of the disease, including how cerebral infection with T. solium results in the development of seizures. Over the past century, valuable mechanistic insights have been generated using both clinical studies and animal models. In this review, we critically assess model systems for investigating disease processes in NCC. We explore the respective strengths and weaknesses of each model and summarize how they have contributed to current knowledge of the disease. We call for the continued development of animal models of NCC, with a focus on novel strategies for understanding this debilitating but often neglected disorder.

Neuronal Chloride Regulation via KCC2 Is Modulated through a GABAB Receptor Protein Complex.

GABAB receptors are G-protein-coupled receptors that mediate inhibitory synaptic actions through a series of downstream target proteins. It is increasingly appreciated that the GABAB receptor forms part of larger signaling complexes, which enable the receptor to mediate multiple different effects within neurons. Here we report that GABAB receptors can physically associate with the potassium-chloride cotransporter protein, KCC2, which sets the driving force for the chloride-permeable ionotropic GABAA receptor in mature neurons. Using biochemical, molecular, and functional studies in rodent hippocampus, we show that activation of GABAB receptors results in a decrease in KCC2 function, which is associated with a reduction in the protein at the cell surface. These findings reveal a novel "crosstalk" between the GABA receptor systems, which can be recruited under conditions of high GABA release and which could be important for the regulation of inhibitory synaptic transmission.SIGNIFICANCE STATEMENT Synaptic inhibition in the brain is mediated by ionotropic GABAA receptors (GABAARs) and metabotropic GABAB receptors (GABABRs). To fully appreciate the function and regulation of these neurotransmitter receptors, we must understand their interactions with other proteins. We describe a novel association between the GABABR and the potassium-chloride cotransporter protein, KCC2. This association is significant because KCC2 sets the intracellular chloride concentration found in mature neurons and thereby establishes the driving force for the chloride-permeable GABAAR. We demonstrate that GABABR activation can regulate KCC2 at the cell surface in a manner that alters intracellular chloride and the reversal potential for the GABAAR. Our data therefore support an additional mechanism by which GABABRs are able to modulate fast synaptic inhibition.

Tight Coupling of Astrocyte pH Dynamics to Epileptiform Activity Revealed by Genetically Encoded pH Sensors.

UNLABELLED: Astrocytes can both sense and shape the evolution of neuronal network activity and are known to possess unique ion regulatory mechanisms. Here we explore the relationship between astrocytic intracellular pH dynamics and the synchronous network activity that occurs during seizure-like activity. By combining confocal and two-photon imaging of genetically encoded pH reporters with simultaneous electrophysiological recordings, we perform pH measurements in defined cell populations and relate these to ongoing network activity. This approach reveals marked differences in the intracellular pH dynamics between hippocampal astrocytes and neighboring pyramidal neurons in rodent in vitro models of epilepsy. With three different genetically encoded pH reporters, astrocytes are observed to alkalinize during epileptiform activity, whereas neurons are observed to acidify. In addition to the direction of pH change, the kinetics of epileptiform-associated intracellular pH transients are found to differ between the two cell types, with astrocytes displaying significantly more rapid changes in pH. The astrocytic alkalinization is shown to be highly correlated with astrocytic membrane potential changes during seizure-like events and mediated by an electrogenic Na(+)/HCO3 (-) cotransporter. Finally, comparisons across different cell-pair combinations reveal that astrocytic pH dynamics are more closely related to network activity than are neuronal pH dynamics. This work demonstrates that astrocytes exhibit distinct pH dynamics during periods of epileptiform activity, which has relevance to multiple processes including neurometabolic coupling and the control of network excitability. SIGNIFICANCE STATEMENT: Dynamic changes in intracellular ion concentrations are central to the initiation and progression of epileptic seizures. However, it is not known how changes in intracellular H(+) concentration (ie, pH) differ between different cell types during seizures. Using recently developed pH-sensitive proteins, we demonstrate that astrocytes undergo rapid alkalinization during periods of seizure-like activity, which is in stark contrast to the acidification that occurs in neighboring neurons. Rapid astrocytic pH changes are highly temporally correlated with seizure activity, are mediated by an electrogenic Na(+)/HCO3- cotransporter, and are more tightly coupled to network activity than are neuronal pH changes. As pH has profound effects on signaling in the nervous system, this work has implications for our understanding of seizure dynamics.

Methodological standards for in vitro models of epilepsy and epileptic seizures. A TASK1-WG4 report of the AES/ILAE Translational Task Force of the ILAE.

In vitro preparations are a powerful tool to explore the mechanisms and processes underlying epileptogenesis and ictogenesis. In this review, we critically review the numerous in vitro methodologies utilized in epilepsy research. We provide support for the inclusion of detailed descriptions of techniques, including often ignored parameters with unpredictable yet significant effects on study reproducibility and outcomes. In addition, we explore how recent developments in brain slice preparation relate to their use as models of epileptic activity.

Excitatory effects of parvalbumin-expressing interneurons maintain hippocampal epileptiform activity via synchronous afterdischarges.

Epileptic seizures are characterized by periods of hypersynchronous, hyperexcitability within brain networks. Most seizures involve two stages: an initial tonic phase, followed by a longer clonic phase that is characterized by rhythmic bouts of synchronized network activity called afterdischarges (ADs). Here we investigate the cellular and network mechanisms underlying hippocampal ADs in an effort to understand how they maintain seizure activity. Using in vitro hippocampal slice models from rats and mice, we performed electrophysiological recordings from CA3 pyramidal neurons to monitor network activity and changes in GABAergic signaling during epileptiform activity. First, we show that the highest synchrony occurs during clonic ADs, consistent with the idea that specific circuit dynamics underlie this phase of the epileptiform activity. We then show that ADs require intact GABAergic synaptic transmission, which becomes excitatory as a result of a transient collapse in the chloride (Cl(-)) reversal potential. The depolarizing effects of GABA are strongest at the soma of pyramidal neurons, which implicates somatic-targeting interneurons in AD activity. To test this, we used optogenetic techniques to selectively control the activity of somatic-targeting parvalbumin-expressing (PV(+)) interneurons. Channelrhodopsin-2-mediated activation of PV(+) interneurons during the clonic phase generated excitatory GABAergic responses in pyramidal neurons, which were sufficient to elicit and entrain synchronous AD activity across the network. Finally, archaerhodopsin-mediated selective silencing of PV(+) interneurons reduced the occurrence of ADs during the clonic phase. Therefore, we propose that activity-dependent Cl(-) accumulation subverts the actions of PV(+) interneurons to perpetuate rather than terminate pathological network hyperexcitability during the clonic phase of seizures.

Adenosine release during seizures attenuates GABAA receptor-mediated depolarization.

Seizure-induced release of the neuromodulator adenosine is a potent endogenous anticonvulsant mechanism, which limits the extension of seizures and mediates seizure arrest. For this reason several adenosine-based therapies for epilepsy are currently under development. However, it is not known how adenosine modulates GABAergic transmission in the context of seizure activity. This may be particularly relevant as strong activation of GABAergic inputs during epileptiform activity can switch GABA(A) receptor (GABA(A)R) signaling from inhibitory to excitatory, which is a process that plays a significant role in intractable epilepsies. We used gramicidin-perforated patch-clamp recordings to investigate the role of seizure-induced adenosine release in the modulation of postsynaptic GABA(A)R signaling in pyramidal neurons of rat hippocampus. Consistent with previous reports, GABA(A)R responses during seizure activity transiently switched from hyperpolarizing to depolarizing and excitatory. We found that adenosine released during the seizure significantly attenuated the depolarizing GABA(A)R responses and also reduced the extent of the after-discharge phase of the seizure. These effects were mimicked by exogenous adenosine administration and could not be explained by a change in chloride homeostasis mechanisms that set the reversal potential for GABA(A)Rs, or by a change in the conductance of GABA(A)Rs. Rather, A(1)R-dependent activation of potassium channels increased the cell's membrane conductance and thus had a shunting effect on GABA(A)R currents. As depolarizing GABA(A)R signaling has been implicated in seizure initiation and progression, the adenosine-induced attenuation of depolarizing GABA(A)R signaling may represent an important mechanism by which adenosine can limit seizure activity.

A genetically targeted ion sensor reveals distinct seizure-related chloride and pH dynamics in GABAergic interneuron populations.

Intracellular chloride and pH play fundamental roles in determining a neuron's synaptic inhibition and excitability. Yet it has been difficult to measure changes in these ions during periods of heightened network activity, such as occur in epilepsy. Here we develop a version of the fluorescent reporter, ClopHensorN, to enable simultaneous quantification of chloride and pH in genetically defined neurons during epileptiform activity. We compare pyramidal neurons to the major GABAergic interneuron subtypes in the mouse hippocampus, which express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal polypeptide (VIP). Interneuron populations exhibit higher baseline chloride, with PV interneurons exhibiting the highest levels. During an epileptiform discharge, however, all subtypes converge upon a common elevated chloride level. Concurrent with these dynamics, epileptiform activity leads to different degrees of intracellular acidification, which reflect baseline pH. Thus, a new optical tool for dissociating chloride and pH reveals neuron-specific ion dynamics during heightened network activity.

Active cortical networks promote shunting fast synaptic inhibition in vivo.

Fast synaptic inhibition determines neuronal response properties in the mammalian brain and is mediated by chloride-permeable ionotropic GABA-A receptors (GABAARs). Despite their fundamental role, it is still not known how GABAARs signal in the intact brain. Here, we use in vivo gramicidin recordings to investigate synaptic GABAAR signaling in mouse cortical pyramidal neurons under conditions that preserve native transmembrane chloride gradients. In anesthetized cortex, synaptic GABAARs exert classic hyperpolarizing effects. In contrast, GABAAR-mediated synaptic signaling in awake cortex is found to be predominantly shunting. This is due to more depolarized GABAAR equilibrium potentials (EGABAAR), which are shown to result from the high levels of synaptic activity that characterize awake cortical networks. Synaptic EGABAAR observed in awake cortex facilitates the desynchronizing effects of inhibitory inputs upon local networks, which increases the flexibility of spiking responses to external inputs. Our findings therefore suggest that GABAAR signaling adapts to optimize cortical functions.

Mouse Organotypic Brain Slice Cultures: A Novel Model for Studying Neuroimmune Responses to Cryptococcal Brain Infections.

Cryptococcal meningitis affects millions of people worldwide and is especially prevalent in regions with a high burden of HIV/AIDS. The study of the pathophysiology of this often fatal disease has been significantly hindered by the lack of reliable experimental models, especially at the level of the brain, which is the main organ of injury. Here we outline our novel protocol for the use of hippocampal organotypic brain slice cultures (HOCs) to study the host-fungal interactions during cryptococcal infections of the brain. HOCs are a powerful platform for investigating neuroimmune interactions as they allow for the preservation of all innate neuroglial cells including microglia, astrocytes, and neurons, all of which maintain their three-dimensional architecture and functional connectivity. We made HOCs from neonatal mice and infected these with a fluorescent strain of Cryptococcus neoformans for 24 h. Using immunofluorescent staining, we confirmed the presence and morphology of microglia, astrocytes, and neurons in HOCs prior to infection. Using fluorescent and light microscopy, we also confirmed that Cryptococcus neoformans encapsulates and buds in vitro, as it would in a host. Finally, we demonstrate that infection of HOCs with Cryptococcus neoformans results in close association of the fungal cells with host microglial cells. Our results demonstrate the utility of HOCs as a model to study the pathophysiology and host neuroimmune responses in neurocryptococcosis, which may assist in improving our collective understanding of the pathogenesis of this disease.

Cell type-specific gene expression dynamics during human brain maturation.

The human brain undergoes protracted post-natal maturation, guided by dynamic changes in gene expression. To date, studies exploring these processes have used bulk tissue analyses, which mask cell type-specific gene expression dynamics. Here, using single nucleus (sn)RNA-Sseq on temporal lobe tissue, including samples of African ancestry, we build a joint paediatric and adult atlas of 54 cell subtypes, which we verify with spatial transcriptomics. We explore the differences in cell states between paediatric and adult cell types, revealing the genes and pathways that change during brain maturation. Our results highlight excitatory neuron subtypes, including the LTK and FREM subtypes, that show elevated expression of genes associated with cognition and synaptic plasticity in paediatric tissue. The new resources we present here improve our understanding of the brain during a critical period of its development and contribute to global efforts to build an inclusive cell map of the brain.

Why won't it stop? The dynamics of benzodiazepine resistance in status epilepticus.

Status epilepticus is a life-threatening neurological emergency that affects both adults and children. Approximately 36% of episodes of status epilepticus do not respond to the current preferred first-line treatment, benzodiazepines. The proportion of episodes that are refractory to benzodiazepines is higher in low-income and middle-income countries (LMICs) than in high-income countries (HICs). Evidence suggests that longer episodes of status epilepticus alter brain physiology, thereby contributing to the emergence of benzodiazepine resistance. Such changes include alterations in GABAA receptor function and in the transmembrane gradient for chloride, both of which erode the ability of benzodiazepines to enhance inhibitory synaptic signalling. Often, current management guidelines for status epilepticus do not account for these duration-related changes in pathophysiology, which might differentially impact individuals in LMICs, where the average time taken to reach medical attention is longer than in HICs. In this Perspective article, we aim to combine clinical insights and the latest evidence from basic science to inspire a new, context-specific approach to efficiently managing status epilepticus.

Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells.

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings give technical insights on how iPSC-derived neurons and photoreceptors can be derived from SCA7 patients and demonstrate that these cells express molecular and electrophysiological differences that may be indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.

The transition to status epilepticus: how the brain meets the demands of perpetual seizure activity.

The pathophysiology leading to the development of status epilepticus (SE) remains a topic of significant scientific interest and clinical relevance. The use of multiple experimental and computational models has shown that SE relies on a complex interaction between mechanisms that operate at both a cellular and network level. In this narrative review, we will summarise the current knowledge on the factors that play a key role in allowing SE to develop and persist. These include pathological adaptations to changing ion dynamics, neuroenergetics, receptor expression and neurotransmission, which enable the brain to meet the extensive demands required to maintain ongoing synchronous hyperexcitability. We will examine how these processes converge to enable synapses to support seizure perpetuation. Lastly, we will use the concept of a perpetuating network to highlight how connections between brain regions can provide positive feedback loops that can serve to propagate seizure activity. We hope this review will collate the findings of previous research and help fuel further studies into the mechanisms that underlie how the brain can make the transition to SE.

When a Good Cop Turns Bad: The Pro-Ictal Action of Parvalbumin Expressing Interneurons During Seizures.

KCC2 Overexpression Prevents the Paradoxical Seizure-Promoting Action of Somatic Inhibition Magloire, V., Cornford, J., Lieb, A., Kullmann, D. M., and Pavlov, I. Nat. Commun. 10, 1225. doi:10.1038/s41467-019-08933-4. Although cortical interneurons are apparently well-placed to suppress seizures, several recent reports have highlighted a paradoxical role of perisomatic-targeting parvalbumin-positive (PV+) interneurons in ictogenesis. Here, we use an acute in vivo model of focal cortical seizures in awake behaving mice, together with closed-loop optogenetic manipulation of PV+ interneurons, to investigate their function during seizures. We show that photo-depolarization of PV+ interneurons rapidly switches from an anti-ictal to a pro-ictal effect within a few seconds of seizure initiation. The pro-ictal effect of delayed photostimulation of PV+ interneurons was not shared with dendrite-targeting somatostatin-positive (SOM+) interneurons. We also show that this switch can be prevented by overexpression of the neuronal potassium-chloride co-transporter KCC2 in principal cortical neurons. These results suggest that strategies aimed at improving the ability of principal neurons to maintain a trans-membrane chloride gradient in the face of excessive network activity can prevent interneurons from contributing to seizure perpetuation.

Divergent paths to seizure-like events.

Much debate exists about how the brain transitions into an epileptic seizure. One source of confusion is that there are likely to be critical differences between experimental seizure models. To address this, we have compared the evolving activity patterns in two widely used in vitro models of epileptic discharges. Brain slices from young adult mice were prepared in the same way and bathed either in 0 Mg2+ or 100 µmol/L 4AP artificial cerebrospinal fluid. We have found that while local field potential recordings of epileptiform discharges in the two models appear broadly similar, patch-clamp analysis reveals an important difference in the relative degree of glutamatergic involvement. 4AP affects parvalbumin-expressing interneurons more than other cortical populations, destabilizing their resting state and inducing spontaneous bursting behavior. Consequently, the most prominent pattern of transient discharge ("interictal event") in this model is almost purely GABAergic, although the transition to seizure-like events (SLEs) involves pyramidal recruitment. In contrast, interictal discharges in 0 Mg2+ are only maintained by a very large glutamatergic component that also involves transient discharges of the interneurons. Seizure-like events in 0 Mg2+ have significantly higher power in the high gamma frequency band (60-120Hz) than these events do in 4AP, and are greatly delayed in onset by diazepam, unlike 4AP events. We, therefore, conclude that the 0 Mg2+ and 4AP models display fundamentally different levels of glutamatergic drive, demonstrating how ostensibly similar pathological discharges can arise from different sources. We contend that similar interpretative issues will also be relevant to clinical practice.

Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis.

Fast synaptic inhibition in the nervous system depends on the transmembrane flux of Cl- ions based on the neuronal Cl- driving force. Established theories regarding the determinants of Cl- driving force have recently been questioned. Here, we present biophysical models of Cl- homeostasis using the pump-leak model. Using numerical and novel analytic solutions, we demonstrate that the Na+/K+-ATPase, ion conductances, impermeant anions, electrodiffusion, water fluxes and cation-chloride cotransporters (CCCs) play roles in setting the Cl- driving force. Our models, together with experimental validation, show that while impermeant anions can contribute to setting [Cl-]i in neurons, they have a negligible effect on the driving force for Cl- locally and cell-wide. In contrast, we demonstrate that CCCs are well-suited for modulating Cl- driving force and hence inhibitory signaling in neurons. Our findings reconcile recent experimental findings and provide a framework for understanding the interplay of different chloride regulatory processes in neurons.

How do we use in vitro models to understand epileptiform and ictal activity? A report of the TASK1-WG4 group of the ILAE/AES Joint Translational Task Force.

In vitro brain tissue preparations allow the convenient and affordable study of brain networks and have allowed us to garner molecular, cellular, and electrophysiologic insights into brain function with a detail not achievable in vivo. Preparations from both rodent and human postsurgical tissue have been utilized to generate in vitro electrical activity similar to electrographic activity seen in patients with epilepsy. A great deal of knowledge about how brain networks generate various forms of epileptiform activity has been gained, but due to the multiple in vitro models and manipulations used, there is a need for a standardization across studies. Here, we describe epileptiform patterns generated using in vitro brain preparations, focusing on issues and best practices pertaining to recording, reporting, and interpretation of the electrophysiologic patterns observed. We also discuss criteria for defining in vitro seizure-like patterns (i.e., ictal) and interictal discharges. Unifying terminologies and definitions are proposed. We suggest a set of best practices for reporting in vitro studies to favor both efficient across-lab comparisons and translation to in vivo models and human studies.