Chris G. Dulla

Both phasic and tonic GABAergic activities in the cortex are shaped by the clearance of GABA from the extracellular space by GABA transporters (GATs). However, estimates of the time-course of GAT activity from GAT currents, GABA _A , and GABA _B electrophysiological recordings range from tens of milliseconds to multiple seconds. These estimates may reflect receptor properties rather than extracellular GABA dynamics. Understanding how long GABA is free in the extracellular space is key to understanding how inhibition controls activity but has been technically challenging. Here, we utilize the fluorescent GABA sensor, iGABASnFr, to enable us to optically assay extracellular GABA dynamics following bursts of synaptic activity. In the mouse cerebral cortex of either sex, we show that GABA rapidly disperses from release sites via diffusion, with very little role for active transport by GATs. Following diffusion from the sites of release, GABA then accumulates in the extracellular space, where GATs require hundreds of milliseconds to remove it. For areas of high-density GABA release or during bursts of activity, this clearance is further slowed. Continued GABA release and summation in the extracellular space for bursts of activity contrasts with recorded inhibitory postsynaptic current (IPSC) signals, which rapidly depress with additional stimuli, suggesting a disconnect between IPSCs and extracellular GABA. Lastly, the resulting extracellular accumulation of GABA can act heterosynaptically by activating and/or desensitizing GABA _A receptors. Together, this raises new questions about how GABAergic inhibition works to shape network function and suggests a spatiotemporal continuum of extracellular GABA.

Astrocytes modulate brain processes such as neurotransmitter signaling, ion homeostasis, vascular tone, metabolism and synaptic transmission, and serve a critical role in complex brain functions. However, it remains unclear on which spatial scales these functions are organized in astrocytes and their networks: for instance, whether astrocytic interactions with neurons are organized into units at the level of individual perisynaptic processes and synapses, astrocytic domains covering synapse populations, or astrocytic networks controlling local neuronal circuits. Here we analyze astrocytic morphological features and molecular heterogeneity to define the multiple spatial scales on which astrocytes operate. We further discuss evidence regarding their intracellular and intercellular signaling, their role in neurotransmitter homeostasis and their neurotransmitter-mediated dialog with neurons, supporting the existence of astrocytic functional units with distinct spatial scales. We propose that astrocytes constitute a multilayered system of functional units that operate across multiple spatial scales, thereby increasing the degrees of freedom in brain information processing.

The extracellular matrix (ECM) is composed of proteoglycans and glycoproteins that regulate the external environment surrounding neurons, glia, and the vascular system. The ECM is vital for maintaining the structure and function of the brain and also acts as a reservoir for various signaling molecules and neurotransmitters, modulating synaptic transmission and plasticity. Recent research highlights the important role of ECM proteins in both brain development and various neurological diseases, including epilepsy. Alterations in ECM composition and subsequent remodeling may disrupt physiological network excitability and synaptic connectivity, triggering neuroinflammation with active participation from glial cells, including astrocytes and microglia, and blood–brain barrier function. This influences neuronal function and contributes to the formation of epileptic foci and the development of drug‐resistant epilepsy. Understanding the intricate interplay between the ECM and epilepsy may enable the identification of novel biomarkers and development of therapeutic strategies targeting this dynamic microenvironment. In this review, we provide a summary of the discussions held at the XVII Workshop on Neurobiology of Epilepsy (WONOEP), organized in 2023 by the International League Against Epilepsy, on the role of the extracellular space in epilepsy. Specifically, we summarize recent advances in understanding the ECM, its alterations in epilepsy, advanced imaging and omics tools for ECM analysis, and the implications for the development of ECM‐based therapeutics and biomarkers for acquired and genetic forms of epilepsy. To complement the material presented at WONOEP, we performed targeted, nonsystematic literature searches in PubMed using topic‐specific terms (e.g., “perineuronal nets”, “extracellular matrix”, “traumatic brain injury”, “post‐traumatic epilepsy”, “chloride”) and citation tracking to ensure that key experimental and translational studies relevant to these themes were included.

Summary Paragraph: Variants in ion channel genes are common causes of pediatric epilepsy, often leading to intractable seizures, developmental delay and other comorbidities, which increases risk of death. Pathogenic variants in the SCN8A gene, which encodes a voltage-gated sodium channel critical for action potential generation in the brain, account for ∼1% of genetic epilepsies. The voltage sensor in SCN8A domain 1 is encoded by one of two developmentally-regulated mutually exclusive alternative exons, 5N and 5A. We observe that variants in these exons are more likely to cause infantile spasms, a severe seizure type, than variants elsewhere in SCN8A , and that some pathogenic variants affect exon 5 splicing, impacting patient phenotype. Molecular and evolutionary analyses implicate the exon sequences of these and other voltage-gated ion channel alternative exons in splicing regulation. We identified antisense oligonucleotides (ASOs) that shift splicing of SCN8A exon 5N to 5A or vice versa. These ASOs normalize neuronal activity in patient-derived iPSC neurons, and reduce seizures and motor impairment and extend lifespan in a new exon 5N mutant mouse model. Our results demonstrate that splice-switching ASOs can effectively reduce the expression of pathogenic isoforms and rescue both seizure and non-seizure phenotypes. Similar approaches should be applicable to pediatric genetic epilepsies caused by mutations in other ion channel alternative exons.

Glutamate, the primary excitatory neurotransmitter in the central nervous system (CNS), is regulated by the excitatory amino acid transporters glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST). Following traumatic brain injury, extracellular glutamate levels increase, contributing to excitotoxicity, circuit dysfunction, and morbidity. Increased neuronal glutamate release and compromised astrocyte-mediated uptake contribute to elevated glutamate, but the mechanistic and spatiotemporal underpinnings of these changes are not well established. Using the controlled cortical impact model of TBI and iGluSnFR glutamate imaging, we quantified extracellular glutamate dynamics after injury. Three days postinjury, glutamate release was increased, and glutamate uptake and GLT-1 expression were reduced. Seven and 14 days postinjury, glutamate dynamics were comparable between sham and controlled cortical impact animals. Changes in peak glutamate response were unique to specific cortical layers and proximity to injury. This was likely driven by increases in glutamate release, which was spatially heterogeneous, rather than reduced uptake, which was spatially uniform. The astrocyte K+ channel, Kir4.1, regulates activity-dependent slowing of glutamate uptake. Surprisingly, Kir4.1 was unchanged after controlled cortical impact and accordingly, activity-dependent slowing of glutamate uptake was unaltered. This dynamic glutamate dysregulation after traumatic brain injury underscores a brief period in which disrupted glutamate uptake may contribute to dysfunction and highlights a potential therapeutic window to restore glutamate homeostasis.

The hippocampus forms memories of our experiences by registering processed sensory information in coactive populations of excitatory principal cells or ensembles. Fast-spiking parvalbumin-expressing inhibitory neurons (PV INs) in the dentate gyrus (DG)-CA3/CA2 circuit contribute to memory encoding by exerting precise temporal control of excitatory principal cell activity through mossy fiber-dependent feed-forward inhibition. PV INs respond to input-specific information by coordinating changes in their intrinsic excitability, input-output synaptic-connectivity, synaptic-physiology and synaptic-plasticity, referred to here as experience-dependent PV IN plasticity, to influence hippocampal functions. PV IN impairments in early life, when neural circuitry is highly sensitive to experience-dependent refinement, are thought to result in imbalanced excitation and inhibition, impaired cognition, network hyperexcitability and seizures: hallmarks of neurodevelopmental disorders (NDDs) such as Autism Spectrum Disorder and epilepsy. Discovery of transcriptional regulators of experience-dependent PV IN plasticity in the adult hippocampus may permit reversal of these developmental impairments. Here, in a screen designed to capture the PV IN intrinsic program induced by increased mossy fiber inputs, a trigger for experience-dependent PV IN plasticity, we identify the homeobox gene Meis2 as a regulator of experience-dependent PV IN plasticity gene (XPG) in the adult DG-CA3/CA2 circuit. We found that a significant number of upregulated XPGs also exhibit haploinsufficiency in ASDs, epilepsies, and schizophrenia. We demonstrate that virally-mediated rescue of experience-dependent Meis2 upregulation in CA3/CA2 PV INs in a NDD risk mouse model in adulthood is sufficient to restore experience-dependent PV IN plasticity, spatial and social memory, ensemble specificity, suppression of network hyperexcitability and seizures. Together, these findings suggest that experience-dependent PV IN plasticity is a convergent mechanism for NDD risk genes that can be re-instated in adulthood to reverse developmental deficits in circuitry, network excitability and cognition.

Glutamate, the primary excitatory neurotransmitter in the CNS, is regulated by the excitatory amino acid transporters (EAATs) GLT-1 and GLAST. Following traumatic brain injury (TBI), extracellular glutamate levels increase, contributing to excitotoxicity, circuit dysfunction, and morbidity. Increased neuronal glutamate release and compromised astrocyte-mediated uptake contribute to elevated glutamate, but the mechanistic and spatiotemporal underpinnings of these changes are not well established. Using the controlled cortical impact (CCI) model of TBI and iGluSnFR glutamate imaging, we quantified extracellular glutamate dynamics after injury. Three days post-injury, glutamate release was increased, and glutamate uptake and GLT-1 expression were reduced. 7- and 14-days post-injury, glutamate dynamics were comparable between sham and CCI animals. Changes in peak glutamate response were unique to specific cortical layers and proximity to injury. This was likely driven by increases in glutamate release, which was spatially heterogenous, rather than reduced uptake, which was spatially uniform. The astrocyte K ⁺ channel, Kir4.1, regulates activity-dependent slowing of glutamate uptake. Surprisingly, Kir4.1 was unchanged after CCI and accordingly, activity-dependent slowing of glutamate uptake was unaltered. This dynamic glutamate dysregulation after TBI underscores a brief period in which disrupted glutamate uptake may contribute to dysfunction and highlights a potential therapeutic window to restore glutamate homeostasis.

Parvalbumin-positive interneurons (PV-INs) regulate neuronal and circuit activity, and their dysfunction is observed across neurological conditions, including traumatic brain injury (TBI), epilepsy, Alzheimer’s disease, and schizophrenia. PV-INs are particularly vulnerable to cell loss, potentially due to their increased metabolic demands arising from their uniquely high level of electrical activity, which render them susceptible to metabolic pressure. Here, we use single-nucleus RNA-sequencing (snRNAseq) data from a rodent model of TBI, as well as human TBI data, and demonstrate PV-INs have unique metabolic specializations that are lost after injury and can be rescued by in vivo treatment with the glycolytic inhibitor, 2-deoxyglucose. We generated a novel PV-IN transcriptional identity module comprised primarily of genes encoding specialized ion channels, metabolic enzymes, and synaptic machinery, that identifies heterogenous subsets of injury-associated PV-INs with loss of PV-IN transcriptional identity. We show that changes in metabolic specialization are coupled to changes in transcriptional identity in PV-INs and implicate the PV-IN-enriched transcriptional co-activator, Ppargc1a , as a key driver of PV-IN transcriptional metabolic dysfunction. We also identify a family of long non-coding RNAs enriched in this subset of transcriptionally dysfunctional PV-INs that negatively correlates with PV-IN metabolic specialization. Lastly, we utilize these tools to interrogate a published human TBI snRNAseq data set and find nearly identical changes, underscoring the importance of PV-IN metabolic dysfunction in the pathology of TBI.

Traumatic brain injury (TBI) is the leading cause of death in young people and can cause cognitive and motor dysfunction and disruptions in functional connectivity between brain regions. In human TBI patients and rodent models of TBI, functional connectivity is decreased after injury. Recovery of connectivity after TBI is associated with improved cognition and memory, suggesting an important link between connectivity and functional outcome. We examined widespread alterations in functional connectivity following TBI using simultaneous widefield mesoscale GCaMP7c calcium imaging and electrocorticography (ECoG) in mice injured using the controlled cortical impact (CCI) model of TBI. Combining CCI with widefield cortical imaging provides us with unprecedented access to characterize network connectivity changes throughout the entire injured cortex over time. Our data demonstrate that CCI profoundly disrupts functional connectivity immediately after injury, followed by partial recovery over 3 weeks. Examining discrete periods of locomotion and stillness reveals that CCI alters functional connectivity and reduces theta power only during periods of behavioral stillness. Together, these findings demonstrate that TBI causes dynamic, behavioral state-dependent changes in functional connectivity and ECoG activity across the cortex.