Corrigendum: GABA tonic currents and glial cells are altered during epileptogenesis in a mouse model of Dravet syndrome.

[This corrects the article DOI: 10.3389/fncel.2022.919493.].

GABA tonic currents and glial cells are altered during epileptogenesis in a mouse model of Dravet syndrome.

Dravet Syndrome (DS) is a rare autosomic encephalopathy with epilepsy linked to Nav1.1 channel mutations and defective GABAergic signaling. Effective therapies for this syndrome are lacking, urging a better comprehension of the mechanisms involved. In a recognized mouse model of DS, we studied GABA tonic current, a form of inhibition largely neglected in DS, in brain slices from developing mice before spontaneous seizures are reported. In neurons from the temporal cortex (TeCx) and CA1 region, GABA tonic current was reduced in DS mice compared to controls, while in the entorhinal cortex (ECx) it was not affected. In this region however allopregnanonole potentiation of GABA tonic current was reduced in DS mice, suggesting altered extrasynaptic GABAA subunits. Using THIP as a selective agonist, we found reduced δ subunit mediated tonic currents in ECx of DS mice. Unexpectedly in the dentate gyrus (DG), a region with high δ subunit expression, THIP-evoked currents in DS mice were larger than in controls. An immunofluorescence study confirmed that δ subunit expression was reduced in ECx and increased in DG of DS mice. Finally, considering the importance of neuroinflammation in epilepsy and neurodevelopmental disorders, we evaluated classical markers of glia activation. Our results show that DS mice have increased Iba1 reactivity and GFAP expression in both ECx and DG, compared to controls. Altogether we report that before spontaneous seizures, DS mice develop significant alterations of GABA tonic currents and glial cell activation. Understanding all the mechanisms involved in these alterations during disease maturation and progression may unveil new therapeutic targets.

Interneuron-specific signaling evokes distinctive somatostatin-mediated responses in adult cortical astrocytes.

The signaling diversity of GABAergic interneurons to post-synaptic neurons is crucial to generate the functional heterogeneity that characterizes brain circuits. Whether this diversity applies to other brain cells, such as the glial cells astrocytes, remains unexplored. Using optogenetics and two-photon functional imaging in the adult mouse neocortex, we here reveal that parvalbumin- and somatostatin-expressing interneurons, two key interneuron classes in the brain, differentially signal to astrocytes inducing weak and robust GABAB receptor-mediated Ca2+ elevations, respectively. Furthermore, the astrocyte response depresses upon parvalbumin interneuron repetitive stimulations and potentiates upon somatostatin interneuron repetitive stimulations, revealing a distinguished astrocyte plasticity. Remarkably, the potentiated response crucially depends on the neuropeptide somatostatin, released by somatostatin interneurons, which activates somatostatin receptors at astrocytic processes. Our study unveils, in the living brain, a hitherto unidentified signaling specificity between interneuron subtypes and astrocytes opening a new perspective into the role of astrocytes as non-neuronal components of inhibitory circuits.

Interneuronal Network Activity at the Onset of Seizure-Like Events in Entorhinal Cortex Slices.

The onset of focal seizures in humans and in different animal models of focal epilepsy correlates with reduction of neuronal firing and enhanced interneuronal network activity. Whether this phenomenon contributes to seizure generation is still unclear. We used the in vitro entorhinal cortex slices bathed in 4-aminopirydine (4-AP) as an experimental paradigm model to evaluate the correlation between interneuronal GABAergic network activity and seizure-like events. Epileptiform discharges were recorded in layer V-VI pyramidal neurons and fast-spiking interneurons in slices from male and female mice and in the isolated female guinea pig brain preparation during perfusion with 4-AP. We observed that 90% of seizure-like events recorded in principal cells were preceded by outward currents coupled with extracellular potassium shifts, abolished by pharmacological blockade of GABAA receptors. Potassium elevations associated to GABAA receptor-mediated population events were confirmed in the entorhinal cortex of the in vitro isolated whole guinea pig brain. Fast-rising and sustained extracellular potassium increases associated to interneuronal network activity consistently preceded the initiation of seizure-like events. We conclude that in the 4-AP seizure model, interneuronal network activity occurs before 4-AP-induced seizures and therefore supports a role of interneuron activity in focal seizure generation.SIGNIFICANCE STATEMENT The paper focuses on the mechanisms of ictogenesis, a topic that requires a step beyond the simplistic view that seizures, and epilepsy, are due to an increase of excitatory network activity. Focal temporal lobe seizures in humans and in several experimental epilepsies likely correlate with a prevalent activation of interneurons. The potassium channel blocker 4-aminopyridine reliably induces seizure-like events in temporal lobe structures. Herein, we show that a majority of seizures in the entorhinal cortex starts with interneuronal network activity accompanied by a fast and sustained increase in extracellular potassium. Our new findings reinforce and add a new piece of evidence to the proposal that limbic seizures can be supported by GABAergic hyperactivity.

Unaltered Network Activity and Interneuronal Firing During Spontaneous Cortical Dynamics In Vivo in a Mouse Model of Severe Myoclonic Epilepsy of Infancy.

Severe myoclonic epilepsy of infancy (SMEI) is associated with loss of function of the SCN1A gene encoding the NaV1.1 sodium channel isoform. Previous studies in Scn1a(-/+) mice during the pre-epileptic period reported selective reduction in interneuron excitability and proposed this as the main pathological mechanism underlying SMEI. Yet, the functional consequences of this interneuronal dysfunction at the circuit level in vivo are unknown. Here, we investigated whether Scn1a(-/+) mice showed alterations in cortical network function. We found that various forms of spontaneous network activity were similar in Scn1a(-/+) during the pre-epileptic period compared with wild-type (WT) in vivo. Importantly, in brain slices from Scn1a(-/+) mice, the excitability of parvalbumin (PV) and somatostatin (SST) interneurons was reduced, epileptiform activity propagated more rapidly, and complex synaptic changes were observed. However, in vivo, optogenetic reduction of firing in PV or SST cells in WT mice modified ongoing network activities, and juxtasomal recordings from identified PV and SST interneurons showed unaffected interneuronal firing during spontaneous cortical dynamics in Scn1a(-/+) compared with WT. These results demonstrate that interneuronal hypoexcitability is not observed in Scn1a(-/+) mice during spontaneous activities in vivo and suggest that additional mechanisms may contribute to homeostatic rearrangements and the pathogenesis of SMEI.

A brain slice experimental model to study the generation and the propagation of focally-induced epileptiform activity.

The early cellular events that in a brain network lead to seizure generation and govern seizure propagation are probably based on different cellular mechanisms. Experimental models in which these events can be separately studied would contribute to improve our understanding of epilepsy. We recently described an in vitro model in entorhinal cortex slices from young rats in which focal seizure-like discharges (SLDs) can be induced in spatially defined regions and at predictable times by local NMDA applications performed in the presence of 4-amimopyridine (4-AP) and low extracellular Mg(2+). Through the use of single-dual cell patch-clamp and field potential recordings, and Ca(2+) imaging from large ensembles of neurons, interneurons and astrocytes, we here extend this model to entorhinal and temporal cortex slices of rat and mouse brain, providing evidence that multiple SLDs exhibiting the typical tonic-clonic discharge pattern can be also evoked in these cortical regions by successive NMDA applications. Importantly, the temporal cortex is more accessible to viral vector injections than the entorhinal cortex: this makes it feasible in the former region the selective expression in inhibitory interneurons or principal neurons of genetically encoded Ca(2+) indicators (GECI) or light-gated opsins. In this model, an optogenetic approach allows to activate specific neuronal types at spatially defined locations, i.e., the focus or the propagating region, and at precise time, i.e., before or during SLD. The NMDA/4-AP model can, therefore, represent a valuable tool to gain insights into the role of specific cell populations in seizure generation, propagation and cessation.

The inhibitory neurotransmitter GABA evokes long-lasting Ca(2+) oscillations in cortical astrocytes.

Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca(2+) response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser-scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15-20) and adult (P30-60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca(2+) oscillations. This response was mediated by GABAB receptors and involved both Gi/o protein and inositol 1,4,5-trisphosphate (IP3 ) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABAB receptor-mediated Ca(2+) elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long-lasting Ca(2+) oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch-clamp recordings it was found that Ca(2+) oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP3 R2(-/-) mice, in which astrocytic GABAB receptor-mediated Ca(2+) elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons.

Parvalbumin-Positive Inhibitory Interneurons Oppose Propagation But Favor Generation of Focal Epileptiform Activity.

Parvalbumin (Pv)-positive inhibitory interneurons effectively control network excitability, and their optogenetic activation has been reported to block epileptic seizures. An intense activity in GABAergic interneurons, including Pv interneurons, before seizures has been described in different experimental models of epilepsy, raising the hypothesis that an increased GABAergic inhibitory signal may, under certain conditions, initiate seizures. It is therefore unclear whether the activity of Pv interneurons enhances or opposes epileptiform activities. Here we use a mouse cortical slice model of focal epilepsy in which the epileptogenic focus can be identified and the role of Pv interneurons in the generation and propagation of seizure-like ictal events is accurately analyzed by a combination of optogenetic, electrophysiological, and imaging techniques. We found that a selective activation of Pv interneurons at the focus failed to block ictal generation and induced postinhibitory rebound spiking in pyramidal neurons, enhancing neuronal synchrony and promoting ictal generation. In contrast, a selective activation of Pv interneurons distant from the focus blocked ictal propagation and shortened ictal duration at the focus. We revealed that the reduced ictal duration was a direct consequence of the ictal propagation block, probably by preventing newly generated afterdischarges to travel backwards to the original focus of ictal initiation. Similar results were obtained upon individual Pv interneuron activation by intracellular depolarizing current pulses. The functional dichotomy of Pv interneurons here described opens new perspectives to our understanding of how local inhibitory circuits govern generation and spread of focal epileptiform activities.