Extracellular glutamate and GABA transients at the transition from interictal spiking to seizures.

Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal ∼1.5 mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint.

Anti-seizure gene therapy for focal cortical dysplasia.

Focal cortical dysplasias are a common subtype of malformation of cortical development, which frequently presents with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. Focal cortical dysplasia type II is typically caused by somatic mutations resulting in mammalian target of rapamycin (mTOR) hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe focal cortical dysplasia. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established focal cortical dysplasia model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human Ras homolog enriched in brain (RHEB) plasmid, an activator of mTOR complex 1. We characterized the model by quantifying electrocorticographic and behavioural abnormalities, both in mice developing spontaneous generalized seizures and in mice only exhibiting interictal discharges. Injection of AAV9-CAMK2A-EKC in the dysplastic region resulted in a robust decrease (∼64%) in the frequency of seizures. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal discharges or behaviour in mice without generalized seizures. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability.

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network.

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.

On-demand cell-autonomous gene therapy for brain circuit disorders.

Several neurodevelopmental and neuropsychiatric disorders are characterized by intermittent episodes of pathological activity. Although genetic therapies offer the ability to modulate neuronal excitability, a limiting factor is that they do not discriminate between neurons involved in circuit pathologies and "healthy" surrounding or intermingled neurons. We describe a gene therapy strategy that down-regulates the excitability of overactive neurons in closed loop, which we tested in models of epilepsy. We used an immediate early gene promoter to drive the expression of Kv1.1 potassium channels specifically in hyperactive neurons, and only for as long as they exhibit abnormal activity. Neuronal excitability was reduced by seizure-related activity, leading to a persistent antiepileptic effect without interfering with normal behaviors. Activity-dependent gene therapy is a promising on-demand cell-autonomous treatment for brain circuit disorders.

Long-term potentiation in neurogliaform interneurons modulates excitation-inhibition balance in the temporoammonic pathway.

Apical dendrites of pyramidal neurons integrate information from higher-order cortex and thalamus, and gate signalling and plasticity at proximal synapses. In the hippocampus, neurogliaform cells and other interneurons located within stratum lacunosum-moleculare (SLM) mediate powerful inhibition of CA1 pyramidal neuron distal dendrites. Is the recruitment of such inhibition itself subject to use-dependent plasticity, and if so, what induction rules apply? Here we show that interneurons in mouse SLM exhibit Hebbian NMDA receptor-dependent long-term potentiation (LTP). Such plasticity can be induced by selective optogenetic stimulation of afferents in the temporoammonic pathway from the entorhinal cortex (EC), but not by equivalent stimulation of afferents from the thalamic nucleus reuniens. We further show that theta-burst patterns of afferent firing induces LTP in neurogliaform interneurons identified using neuron-derived neurotrophic factor (Ndnf)-Cre mice. Theta-burst activity of EC afferents led to an increase in disynaptic feed-forward inhibition, but not monosynaptic excitation, of CA1 pyramidal neurons. Activity-dependent synaptic plasticity in SLM interneurons thus alters the excitation-inhibition balance at EC inputs to the apical dendrites of pyramidal neurons, implying a dynamic role for these interneurons in gating CA1 dendritic computations. KEY POINTS: Electrogenic phenomena in distal dendrites of principal neurons in the hippocampus have a major role in gating synaptic plasticity at afferent synapses on proximal dendrites. Apical dendrites also receive powerful feed-forward inhibition, mediated in large part by neurogliaform neurons. Here we show that theta-burst activity in afferents from the entorhinal cortex (EC) induces 'Hebbian' long-term potentiation (LTP) at excitatory synapses recruiting these GABAergic cells. LTP in interneurons innervating apical dendrites increases disynaptic inhibition of principal neurons, thus shifting the excitation-inhibition balance in the temporoammonic (TA) pathway in favour of inhibition, with implications for computations and learning rules in proximal dendrites.

Cholinergic modulation of Up-Down states in the mouse medial entorhinal cortex in vitro.

Cholinergic tone is high during wake and rapid eye movement sleep and lower during slow wave sleep (SWS). Nevertheless, the low tone of acetylcholine during SWS modulates sharp wave ripple incidence in the hippocampus and slow wave activity in the neocortex. Linking the hippocampus and neocortex, the medial entorhinal cortex (mEC) regulates the coupling between these structures during SWS, alternating between silent Down states and active Up states, which outlast neocortical ones. Here, we investigated how low physiological concentrations of acetylcholine (ACh; 100-500 nM) modulate Up and Down states in a mEC slice preparation. We find that ACh has a dual effect on mEC activity: it prolongs apparent Up state duration as recorded in individual cells and decreases the total synaptic charge transfer, without affecting the duration of detectable synaptic activity. The overall outcome of ACh application is excitatory and we show that ACh increases Up state incidence via muscarinic receptor activation. The mean firing rate of principal neurons increased in around half of the cells while the other half showed a decrease in firing rate. Using two-photon calcium imaging of population activity, we found that population-wide network events are more frequent and rhythmic during ACh and confirmed that ACh modulates cell participation in these network events, consistent with a role for cholinergic modulation in regulating information flow between the hippocampus and neocortex during SWS.

Dendritic NMDA receptors in parvalbumin neurons enable strong and stable neuronal assemblies.

Parvalbumin-expressing (PV+) GABAergic interneurons mediate feedforward and feedback inhibition and have a key role in gamma oscillations and information processing. The importance of fast synaptic recruitment and action potential initiation and repolarization, and rapid synchronous GABA release by PV+ cells, is well established. In contrast, the functional significance of PV+ cell NMDA receptors (NMDARs), which generate relatively slow postsynaptic currents, is unclear. Underlining their potential importance, several studies implicate PV+ cell NMDAR disruption in impaired network function and circuit pathologies. Here, we show that dendritic NMDARs underlie supralinear integration of feedback excitation from local pyramidal neurons onto mouse CA1 PV+ cells. Furthermore, by incorporating NMDARs at feedback connections onto PV+ cells in spiking networks, we show that these receptors enable cooperative recruitment of PV+ interneurons, strengthening and stabilising principal cell assemblies. Failure of this phenomenon provides a parsimonious explanation for cognitive and sensory gating deficits in pathologies with impaired PV+ NMDAR signalling.

A genetically encoded fluorescent sensor for in vivo imaging of GABA.

Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

KCC2 overexpression prevents the paradoxical seizure-promoting action of somatic inhibition.

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.

GABAergic Interneurons in Seizures: Investigating Causality With Optogenetics.

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.

Distinct mechanisms of Up state maintenance in the medial entorhinal cortex and neocortex.

The medial entorhinal cortex (mEC) is a key structure which controls the communication between the hippocampus and the neocortex. During slow-wave sleep, it stands out from other cortical regions by exhibiting persistent activity that outlasts neocortical Up states, decoupling the entorhinal cortex-hippocampal interaction from the neocortex. Here, we compared the mechanisms involved in the maintenance of the Up state in the barrel cortex (BC) and mEC using whole cell recordings in acute mouse brain slices. Bath application of an NMDA receptor antagonist abolished Up states in the BC, and reduced the incidence but not the duration of Up states in the mEC. Conversely, blockade of kainate receptors decreased Up state duration in the mEC, but not in the BC. Voltage clamp recordings demonstrated the presence of a non-NMDA glutamate receptor-mediated slow excitatory postsynaptic current, sensitive to the selective kainate receptor antagonist UBP-302, in layer III neurons of the mEC, which was not observed in the BC. Moreover, we found that kainate receptor-mediated currents assist in recovery back to the Up state membrane potential following a current-induced hyperpolarisation of individual cells in the mEC. Finally, we were able to generate Up state activity in a network model of exponential integrate-and-fire neurons only supported by AMPA and kainate receptor-mediated currents. We propose that synaptic kainate receptors are responsible for the unique properties of mEC Up states.

Astrocytic GABA transporter activity modulates excitatory neurotransmission.

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.

Optogenetic approaches to treat epilepsy.

BACKGROUND: Novel treatments for drug-resistant 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 light-activated 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 drug-resistant 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 pre-clinical 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 side-effects of anti-epileptic drugs (AEDs) or the need to permanently excise regions of the brain. Optogenetic approaches may treat drug-refractory epilepsies.

Grafted neuronal precursor cells differentiate and integrate in injured hippocampus in experimental pneumococcal meningitis.

Bacterial meningitis (BM) frequently causes persisting neurofunctional sequelae. Autopsy studies in patients dying from BM show characteristic apoptotic brain injury to the stem cell niche in the subgranular zone of the hippocampal dentate gyrus (DG), and this form of brain damage is associated with learning and memory deficits in experimental BM. With an eye to potential regenerative therapies, the survival, migration, and differentiation of neuronal precursor cells (NPCs) were evaluated after engraftment into the injured hippocampus in vitro and in vivo in an infant rat model of pneumococcal meningitis. Green fluorescent protein (GFP)-expressing NPCs were grafted into the DG of organotypic hippocampal slice cultures injured by challenge with live Streptococcus pneumoniae. Seven days after engraftment, NPCs had migrated from the site of injection into the injured granular layer of the DG and electro-functionally integrated into the hippocampal network. In vivo, GFP-expressing NPCs migrated within 1 week from the injection site in the hilus region to the injured granular layer of the hippocampal DG and showed neuronal differentiation at 2 and 4 weeks after transplantation. Hippocampal injury induced by BM guides grafted NPCs to the area of brain damage and provides a microenvironment for neuronal differentiation and functional integration.

Intrinsic activity and positive feedback in motor circuits in organotypic spinal cord slice cultures.

In co-cultures of embryonic rat spinal cord slices and skeletal muscle, spinal motoneurons innervate muscle fibres and drive muscle contractions. However, multi-electrode array (MEA) recordings show that muscle contractions often appear in the absence of population activity in the spinal cord networks. Such uncorrelated muscle activity persists when the population bursts in the neuronal networks are prevented by un-coupling the network with the glutamatergic antagonists CNQX and D-APV. By contrast, the uncorrelated muscle activity is fully suppressed by the muscular nicotinic antagonist D-tubocurarine. Together, these findings confirm the previous finding that motoneurons drive muscle fibres in this preparation and suggest that they are intrinsically spiking in the absence of synaptic input. Intracellular recordings from spinal neurons support this suggestion. Analysing the correlated muscle activity, we found that in 15% of the population bursts, muscle activity appears at the beginning or before neuronal activity, suggesting that in these cases motoneurons initiate the population activity. Both the total number of population bursts and the percentage of such bursts that are initiated by muscle activity are reduced by a block of nicotinic receptors. Uncorrelated muscle and neuronal activity is reduced by the gap junction blocker carbenoxolone, suggesting that electrical coupling is involved in the generation of this activity. Together, these findings suggest that intrinsic firing of motoneurons may contribute to the activation of population bursts through cholinergic positive feedback loops in cultured spinal networks.

Delayed changes of sleep duration after rewarded olfactory discrimination learning in the rat.

The aim of this experiment was to determine if a task of associative olfactory learning, based on the ethological repertory of rats and learnt rapidly in 5 successive trials, could modify slow wave sleep (SWS) and/or paradoxical sleep (PS) duration after learning and/or after a retrieval-reactivation test 24 h later. Somnopolygraphic recordings were performed for 20 h per day on trained and control (submitted to a pseudo-learning test) rats. SWS and PS durations were analyzed per 20 h and per 4 h time-periods. Compared to control rats, after learning, trained rats showed a significant increase in SWS duration counterbalanced by a significant decrease in wake duration focused on the 5-8 h post-training time-window and a significant decrease in PS duration during the 17-20 h post-training time-window. After the retrieval-reactivation test trained rats only showed a decreased PS duration compared to control rats submitted to a pseudo-retrieval test. Thus, a rather simple learning task succeeded in eliciting an increase in SWS duration in a limited time-window. As the learning task used can be compared to human associate-paired learning, this result sustains the hypothesis of a link between declarative memory and SWS. In control rats, changes in PS duration might be linked to odorized-environment exposure.

Modulation of intrinsic spiking in spinal cord neurons.

The vertebrate spinal cord is equipped with a number of neuronal networks that underlie repetitive patterns of behavior as locomotion. Activity in such networks is mediated not only by intrinsic cellular properties but also by synaptic coupling. In this study, we focused on the modulation of the intrinsic activity by 5-hydroxytryptamine (5-HT, serotonin) and the cholinergic agonist muscarine in spinal cord cultures (embryonic age 14 rats). We investigated theses cultures (slices and dissociated cells) at the network level using multielectrode arrays (MEAs) and at the cellular level using whole cell patch clamp. All cultures showed bursting network activity and intrinsic activity when gamma-aminobutyric acid, glycine, and glutamate transmission was blocked. Using MEAs, we observed an increase of the intrinsic activity in the ventral part of the slices with 5-HT and muscarine. In single-cell recordings we found that 43 and 35% of the cells that were silent in the absence of fast synaptic activity were transformed into intrinsically spiking cells by 5-HT and muscarine, respectively. We tested the hypothesis that these neuromodulators act via modulation of the persistent sodium currents (I(NaP)) in these neurons. We found that 5-HT increased threefold the amplitude of I(NaP), specifically in the nonintrinsically spiking cells, and thus switched these cells into intrinsically spiking cells via activation of 5-HT(2) receptor and the phospholipase C pathway. In contrast, the effect of muscarine on nonintrinsically spiking neurons seems to be independent of I(NaP). We conclude from these findings that serotoninergic and cholinergic modulation can turn silent into spontaneously spiking neurons and thus initiate new sources of activity for rhythm generation in spinal networks.

Local oscillations of spiking activity in organotypic spinal cord slice cultures.

The origin of rhythm generation in mammalian spinal cord networks is still poorly understood. We have previously proposed that disinhibition-induced rhythms are based on intrinsic firing, recurrent excitation and several mechanisms to de-activate the network. In order to clarify these mechanisms we here investigated spontaneous spike discharge oscillations in rat spinal cord slice cultures using multi-electrode arrays and patch clamp. Episodes of such oscillations at 8.5 Hz spontaneously appeared in the ventral parts of the cultured slices. The rising phase of their initial cycles was entirely based on AMPA/kainate receptor-dependent recurrent excitation. Initial oscillations were changed into persistent activity by bicuculline and other blockers of GABA A, but not by blockers of glycine receptors, suggesting a role for GABAergic synaptic inhibition in network de-activation during oscillation cycles. Blockade of glycine receptors by strychnine caused a prolongation of oscillations and their spreading in the slice, suggesting that these receptors are mainly involved in the spatial and temporal restriction of oscillations. In most cultures, oscillations reappeared under disinhibition after an initial phase of persistent activity. Both spontaneous and disinhibition-induced oscillations were facilitated by riluzole, which enhances fast sodium current inactivation and thus leads to early cessation of firing during strong depolarization (depolarization block). In single cell recordings, episodes of strong depolarization were mostly seen during oscillations induced by disinhibition, but occasionally also during spontaneous oscillations. We conclude that both GABA A-mediated synaptic inhibition and depolarization block contribute to the de-activation of spinal cord networks during oscillation cycles.