Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA “sniffer” and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations—without affecting synaptic GABAergic transmission or resting GABA levels—slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks.

The cellular and circuit mechanisms that trigger and maintain epileptiform discharges remain the subject of intense debate. We have earlier reported a bell-shape dependence of spiking activity of interneuronal populations on tonic GABAA receptor conductance (Gtonic), suggesting an innate mechanism to enable slow self-sustained network oscillations. In the brain, Gtonic is controlled by the slow changes of the extracellular GABA concentration ([GABA]e), which in turn depends on spiking activity of interneurons. Here, we employ outside-out patch-clamp recordings of GABAA receptor and fluorescence imaging of a GABA sensor to show that periodic epileptiform discharges are preceded by [GABA]e rises. Computer simulations of spiking interneuronal networks reveal that incorporating extrasynaptic waves of [GABA]e readily enables periodic occurrences of synchronised interneuronal spiking, which are in phase with rises in Gtonic and can trigger short bursts of principal cell spiking. Simultaneous recording from multiple neurons and selective optogenetic stimulation of parvalbumin-positive (PV+) interneurons confirmed the modelling predictions, consistent with a causal relationship between synchronisation of interneuronal activity, inhibitory synaptic input, Gtonic, and interictal events. Our findings suggest a key role of [GABA]e dynamics in enabling and pacing regenerative rhythmic activity of brain networks.

Besides the usual classical method of hypnotising animals, which results in a hypnotic state manifesting itself in catalepsy, the authors were able, in the course of their research into the normal activity of the higher parts of the brain, to study in more detail the diverse and very delicate manifestations of the hypnotic state. This chapter offers a deep physiological analysis of hypnosis in animals and interprets some hypnotic phenomena on the basis of experimental data in the light of localisation and motion of hypnotic inhibition, phenomena of reciprocal induction and interactions of the cortex and subcortex. It includes the physiological analysis of hypnotic inhibition and negativism in the experimental dogs for explaining a number of phenomena observed in the psychiatric clinic, the psychopathological symptoms in patients with the catatonic form of schizophrenia in particular.

Seizures are complex pathological network events characterized by excessive and hypersynchronized activity of neurons, including a highly diverse population of GABAergic interneurons. Although the primary function of inhibitory interneurons under normal conditions is to restrain excitation in the brain, this system appears to fail intermittently, allowing runaway excitation. Recent developments in optogenetics, combined with genetic tools and advanced electrophysiological and imaging techniques, allow us for the first time to assess the causal roles of identified cell-types in network dynamics. While these methods have greatly increased our understanding of cortical microcircuits in epilepsy, the roles played by individual GABAergic cell-types in controlling ictogenesis remain incompletely resolved. Indeed, the ability of interneurons to suppress epileptic discharges varies across different subtypes, and an accumulating body of evidence paradoxically implicates some interneuron subtypes in the initiation and maintenance of epileptiform activity. Here, we bring together findings from this growing field and discuss what can be inferred regarding the causal role of different GABAergic cell-types in seizures.

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.

A major challenge in experimental epilepsy research is to reconcile the effects of anti-epileptic drugs (AEDs) on individual neurons with their network-level actions. Highlighting this difficulty, it is unclear why carbamazepine (CBZ), a frontline AED with a known molecular mechanism, has been reported to increase epileptiform activity in several clinical and experimental studies. We confirmed in an in vitro mouse model (in both sexes) that the frequency of interictal bursts increased after CBZ perfusion. To address the underlying mechanisms, we developed a method, activity clamp, to distinguish the response of individual neurons from network-level actions of CBZ. We first recorded barrages of synaptic conductances from neurons during epileptiform activity and then replayed them in pharmacologically isolated neurons under control conditions and in the presence of CBZ. CBZ consistently decreased the reliability of the second action potential in each burst of activity. Conventional current-clamp recordings using excitatory ramp or square-step current injections failed to reveal this effect. Network modeling showed that a CBZ-induced decrease of neuron recruitment during epileptic bursts can lead to an increase in burst frequency at the network level by reducing the refractoriness of excitatory transmission. By combining activity clamp with computer simulations, the present study provides a potential explanation for the paradoxical effects of CBZ on epileptiform activity. SIGNIFICANCE STATEMENT The effects of anti-epileptic drugs on individual neurons are difficult to separate from their network-level actions. Although carbamazepine (CBZ) has a known anti-epileptic mechanism, paradoxically, it has also been reported to increase epileptiform activity in clinical and experimental studies. To investigate this paradox during realistic neuronal epileptiform activity, we developed a method, activity clamp, to distinguish the effects of CBZ on individual neurons from network-level actions. We demonstrate that CBZ consistently decreases the reliability of the second action potential in each burst of epileptiform activity. Network modeling shows that this effect on individual neuronal responses could explain the paradoxical effect of CBZ at the network level.

Background: Novel treatments for drugresistant epilepsy are required.New method: Optogenetics is a combination of optical and genetic methods used to control the activity of specific populations of excitable cells using light with high temporal and spatial resolution. Derived from microbial organisms, 'opsin' genes encode lightactivated ion channels and pumps. Opsins can be genetically targeted to well-defined neuronal populations in mammalian brains using viral vectors. When exposed to light of an appropriate wavelength, the excitability of neurons can be increased or decreased optically on a millisecond timescale.Comparison with existing method(s): Alternative treatments for drugresistant epilepsy such as vagal, cortical or subcortical stimulation, focal cooling, callosotomy, or ketogenic diet have met with limited success, whereas optogenetic approaches have shown considerable preclinical promise.Conclusions: Several groups have reported that optogenetic approaches successfully attenuated epileptiform activity in different rodent models of epilepsy, providing proof of the principle that this approach may translate to an effective treatment for epilepsy patients. However, further studies are required to determine the optimal opsin, in which types (or subtypes) of neurons it should be expressed, and what are the most efficient temporal profiles of photostimulation. Although invasive due to the need to inject a viral vector into the brain and implant a device to deliver light to opsin-transduced neurons, this approach has the potential to be effective in suppressing spontaneous seizures while avoiding the sideeffects of anti epileptic drugs (AEDs) or the need to permanently excise regions of the brain. Optogenetic approaches may treat drugrefractory epilepsies. (C) 2015 Elsevier B.V. All rights reserved.

Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.

GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABA A receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg 2+ model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity.

Current antiepileptic treatments are aimed at stopping or preventing seizures rather than curing epilepsy. Here, we discuss how recent advances in genetics and neurophysiological research may not only help us better understand the pathophysiology of different epilepsies, but may also drive the development of novel approaches, including treatments that can modify the underlying disease. Although these new therapeutic strategies are still in the very early stages, rapid progress in this direction means that the ability to stop or even to reverse changes caused by epileptogenesis may not be that far away.

Focal epilepsy can result from both genetic and environmental factors. Acquired focal epilepsy occurs following a specific brain insult such as stroke, head injury or prolonged seizures (status epilepticus). The time from the insult to the development of seizures is termed the epileptogenic period during which there are changes in connectivity, neurotransmission and neuronal excitability. Epileptogenesis has conventionally been viewed as being associated with increased excitation and a loss of inhibition. This view has been reappraised in recent years due to a better understanding of the multifaceted roles of GABAergic signalling. There is growing evidence that loss of synaptic GABAA receptor-mediated inhibition observed in animal models of temporal lobe epilepsy is accompanied by compensatory upregulation of tonic currents mediated by extrasynaptic GABAA receptors. Here, we provide evidence for such a change in GABAA receptor-mediated inhibition during epileptogenesis and speculate on the possible functional impact that such a shift in inhibition will have. In particular, we argue that shifts from phasic to tonic inhibition in the hippocampus will lead to a maintenance of "inhibition" of the network but will alter network gain, decreasing network stability. Furthermore, changes in the subunit composition of extrasynaptic GABAA receptors during epileptogenesis have implications for targeted pharmacotherapy of epilepsy.

The spiking output of interneurons is key for rhythm generation in the brain. However, what controls interneuronal firing remains incompletely understood. Here we combine dynamic clamp experiments with neural network simulations to understand how tonic GABAA conductance regulates the firing pattern of CA3 interneurons. In baseline conditions, tonic GABAA depolarizes these cells, thus exerting an excitatory action while also reducing the excitatory postsynaptic potential (EPSP) amplitude through shunting. As a result, the emergence of weak tonic GABAA conductance transforms the interneuron firing pattern driven by individual EPSPs into a more regular spiking mode determined by the cell intrinsic properties. The increased regularity of spiking parallels stronger synchronization of the local network. With further increases in tonic GABAA conductance the shunting inhibition starts to dominate over excitatory actions and thus moderates interneuronal firing. The remaining spikes tend to follow the timing of suprathreshold EPSPs and thus become less regular again. The latter parallels a weakening in network synchronization. Thus, our observations suggest that tonic GABAA conductance can bidirectionally control brain rhythms through changes in the excitability of interneurons and in the temporal structure of their firing patterns.

Epilepsy is characterised by the propensity of the brain to generate spontaneous recurrent bursts of excessive neuronal activity, seizures. GABA-mediated inhibition is critical for restraining neuronal excitation in the brain, and therefore potentiation of GABAergic neurotransmission is commonly used to prevent seizures. However, data obtained in animal models of epilepsy and from human epileptic tissue suggest that GABA-mediated signalling contributes to interictal and ictal activity. Prolonged activation of GABAA receptors during epileptiform bursts may even initiate a shift in GABAergic neurotransmission from inhibitory to excitatory and so have a proconvulsant action. Direct targeting of the membrane mechanisms that reduce spiking in glutamatergic neurons may better control neuronal excitability in epileptic tissue. Manipulation of brain pH may be a promising approach and recent advances in gene therapy and optogenetics seem likely to provide further routes to effective therapeutic intervention.

Activation of GABA(A) receptors (GABA(A)Rs) produces two forms of inhibition: phasic inhibition generated by the rapid, transient activation of synaptic GABA(A)Rs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of perisynaptic or extrasynaptic GABA(A)Rs, which can detect extracellular GABA. Such tonic GABA(A)R-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABA(A) receptor openings. This tonic GABA(A)R conductance is resistant to the competitive GABA(A)R antagonist SR95531 (gabazine), which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker, picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to CSF concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABA(A)Rs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations.

Activation of GABA A receptors (GABA A Rs) produces two forms of inhibition: phasic inhibition generated by the rapid, transient activation of synaptic GABA A Rs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of perisynaptic or extrasynapticGABA A Rs,whichcandetectextracellularGABA.SuchtonicGABA A R-mediatedcurrentsareparticularlyevidentindentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABA A receptor openings. This tonic GABA A R conductance is resistant to the competitive GABA A R antagonist SR95531 (gabazine), which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker, picrotoxin. When slices are perfused with 200 n M GABA, a concentration that is comparable to CSF concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate