The fasciola cinereum of the hippocampal tail as an interventional target in epilepsy.

Targeted tissue ablation involving the anterior hippocampus is the standard of care for patients with drug-resistant mesial temporal lobe epilepsy. However, a substantial proportion continues to suffer from seizures even after surgery. We identified the fasciola cinereum (FC) neurons of the posterior hippocampal tail as an important seizure node in both mice and humans with epilepsy. Genetically defined FC neurons were highly active during spontaneous seizures in epileptic mice, and closed-loop optogenetic inhibition of these neurons potently reduced seizure duration. Furthermore, we specifically targeted and found the prominent involvement of FC during seizures in a cohort of six patients with epilepsy. In particular, targeted lesioning of the FC in a patient reduced the seizure burden present after ablation of anterior mesial temporal structures. Thus, the FC may be a promising interventional target in epilepsy.

Neural and behavioural state switching during hippocampal dentate spikes.

Distinct brain and behavioural states are associated with organized neural population dynamics that are thought to serve specific cognitive functions1-3. Memory replay events, for example, occur during synchronous population events called sharp-wave ripples in the hippocampus while mice are in an 'offline' behavioural state, enabling cognitive mechanisms such as memory consolidation and planning4-11. But how does the brain re-engage with the external world during this behavioural state and permit access to current sensory information or promote new memory formation? Here we found that the hippocampal dentate spike, an understudied population event that frequently occurs between sharp-wave ripples12, may underlie such a mechanism. We show that dentate spikes are associated with distinctly elevated brain-wide firing rates, primarily observed in higher order networks, and couple to brief periods of arousal. Hippocampal place coding during dentate spikes aligns to the mouse's current spatial location, unlike the memory replay accompanying sharp-wave ripples. Furthermore, inhibiting neural activity during dentate spikes disrupts associative memory formation. Thus, dentate spikes represent a distinct brain state and support memory during non-locomotor behaviour, extending the repertoire of cognitive processes beyond the classical offline functions.

Retrograde endocannabinoid signaling at inhibitory synapses in vivo.

Endocannabinoid (eCB)-mediated suppression of inhibitory synapses has been hypothesized, but this has not yet been demonstrated to occur in vivo because of the difficulty in tracking eCB dynamics and synaptic plasticity during behavior. In mice navigating a linear track, we observed location-specific eCB signaling in hippocampal CA1 place cells, and this was detected both in the postsynaptic membrane and the presynaptic inhibitory axons. All-optical in vivo investigation of synaptic responses revealed that postsynaptic depolarization was followed by a suppression of inhibitory synaptic potentials. Furthermore, interneuron-specific cannabinoid receptor deletion altered place cell tuning. Therefore, rapid, postsynaptic, activity-dependent eCB signaling modulates inhibitory synapses on a timescale of seconds during behavior.

Acetylcholine receptor based chemogenetics engineered for neuronal inhibition and seizure control assessed in mice.

Epilepsy is a prevalent disorder involving neuronal network hyperexcitability, yet existing therapeutic strategies often fail to provide optimal patient outcomes. Chemogenetic approaches, where exogenous receptors are expressed in defined brain areas and specifically activated by selective agonists, are appealing methods to constrain overactive neuronal activity. We developed BARNI (Bradanicline- and Acetylcholine-activated Receptor for Neuronal Inhibition), an engineered channel comprised of the α7 nicotinic acetylcholine receptor ligand-binding domain coupled to an α1 glycine receptor anion pore domain. Here we demonstrate that BARNI activation by the clinical stage α7 nicotinic acetylcholine receptor-selective agonist bradanicline effectively suppressed targeted neuronal activity, and controlled both acute and chronic seizures in male mice. Our results provide evidence for the use of an inhibitory acetylcholine-based engineered channel activatable by both exogenous and endogenous agonists as a potential therapeutic approach to treating epilepsy.

Sudden unexpected death in epilepsy is prevented by blocking postictal hypoxia.

Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack of preventative treatments. Based on the observation that discrete seizures lead to local and postictal vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain we reasoned those similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of seizure-induced premature mortality, ibuprofen and nicardipine extended life while systemic drug levels remained high enough to be effective. We also examined the potential role of spreading depolarization in the acute model of seizure-induced premature mortality. These data provide a proof-of-principle for the neurovascular hypothesis of SUDEP rather than spreading depolarization and the use of currently available drugs to prevent it.

A tool for monitoring cell type-specific focused ultrasound neuromodulation and control of chronic epilepsy.

Focused ultrasound (FUS) is a powerful tool for noninvasive modulation of deep brain activity with promising therapeutic potential for refractory epilepsy; however, tools for examining FUS effects on specific cell types within the deep brain do not yet exist. Consequently, how cell types within heterogeneous networks can be modulated and whether parameters can be identified to bias these networks in the context of complex behaviors remains unknown. To address this, we developed a fiber Photometry Coupled focused Ultrasound System (PhoCUS) for simultaneously monitoring FUS effects on neural activity of subcortical genetically targeted cell types in freely behaving animals. We identified a parameter set that selectively increases activity of parvalbumin interneurons while suppressing excitatory neurons in the hippocampus. A net inhibitory effect localized to the hippocampus was further confirmed through whole brain metabolic imaging. Finally, these inhibitory selective parameters achieved significant spike suppression in the kainate model of chronic temporal lobe epilepsy, opening the door for future noninvasive therapies.

A consensus statement on detection of hippocampal sharp wave ripples and differentiation from other fast oscillations.

Decades of rodent research have established the role of hippocampal sharp wave ripples (SPW-Rs) in consolidating and guiding experience. More recently, intracranial recordings in humans have suggested their role in episodic and semantic memory. Yet, common standards for recording, detection, and reporting do not exist. Here, we outline the methodological challenges involved in detecting ripple events and offer practical recommendations to improve separation from other high-frequency oscillations. We argue that shared experimental, detection, and reporting standards will provide a solid foundation for future translational discovery.

Ripple-selective GABAergic projection cells in the hippocampus.

Ripples are brief high-frequency electrographic events with important roles in episodic memory. However, the in vivo circuit mechanisms coordinating ripple-related activity among local and distant neuronal ensembles are not well understood. Here, we define key characteristics of a long-distance projecting GABAergic cell group in the mouse hippocampus that selectively exhibits high-frequency firing during ripples while staying largely silent during theta-associated states when most other GABAergic cells are active. The high ripple-associated firing commenced before ripple onset and reached its maximum before ripple peak, with the signature theta-OFF, ripple-ON firing pattern being preserved across awake and sleep states. Controlled by septal GABAergic, cholinergic, and CA3 glutamatergic inputs, these ripple-selective cells innervate parvalbumin and cholecystokinin-expressing local interneurons while also targeting a variety of extra-hippocampal regions. These results demonstrate the existence of a hippocampal GABAergic circuit element that is uniquely positioned to coordinate ripple-related neuronal dynamics across neuronal assemblies.

A fluorescent sensor for spatiotemporally resolved imaging of endocannabinoid dynamics in vivo.

Endocannabinoids (eCBs) are retrograde neuromodulators with important functions in a wide range of physiological processes, but their in vivo dynamics remain largely uncharacterized. Here we developed a genetically encoded eCB sensor called GRABeCB2.0. GRABeCB2.0 consists of a circular-permutated EGFP and the human CB1 cannabinoid receptor, providing cell membrane trafficking, second-resolution kinetics with high specificity for eCBs, and shows a robust fluorescence response at physiological eCB concentrations. Using GRABeCB2.0, we monitored evoked and spontaneous changes in eCB dynamics in cultured neurons and acute brain slices. We observed spontaneous compartmentalized eCB transients in cultured neurons and eCB transients from single axonal boutons in acute brain slices, suggesting constrained, localized eCB signaling. When GRABeCB2.0 was expressed in the mouse brain, we observed foot shock-elicited and running-triggered eCB signaling in the basolateral amygdala and hippocampus, respectively. In a mouse model of epilepsy, we observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. GRABeCB2.0 is a robust probe for eCB release in vivo.

Supramammillary regulation of locomotion and hippocampal activity.

Locomotor speed is a basic input used to calculate one's position, but where this signal comes from is unclear. We identified neurons in the supramammillary nucleus (SuM) of the rodent hypothalamus that were highly correlated with future locomotor speed and reliably drove locomotion when activated. Robust locomotion control was specifically identified in Tac1 (substance P)­expressing (SuMTac1+) neurons, the activation of which selectively controlled the activity of speed-modulated hippocampal neurons. By contrast, Tac1-deficient (SuMTac1−) cells weakly regulated locomotion but potently controlled the spike timing of hippocampal neurons and were sufficient to entrain local network oscillations. These findings emphasize that the SuM not only regulates basic locomotor activity but also selectively shapes hippocampal neural activity in a manner that may support spatial navigation.

In vivo endocannabinoid dynamics at the timescale of physiological and pathological neural activity.

The brain's endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling in vivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling in vivo.

Alternating sources of perisomatic inhibition during behavior.

Interneurons expressing cholecystokinin (CCK) and parvalbumin (PV) constitute two key GABAergic controllers of hippocampal pyramidal cell output. Although the temporally precise and millisecond-scale inhibitory regulation of neuronal ensembles delivered by PV interneurons is well established, the in vivo recruitment patterns of CCK-expressing basket cell (BC) populations has remained unknown. We show in the CA1 of the mouse hippocampus that the activity of CCK BCs inversely scales with both PV and pyramidal cell activity at the behaviorally relevant timescales of seconds. Intervention experiments indicated that the inverse coupling of CCK and PV GABAergic systems arises through a mechanism involving powerful inhibitory control of CCK BCs by PV cells. The tightly coupled complementarity of two key microcircuit regulatory modules demonstrates a novel form of brain-state-specific segregation of inhibition during spontaneous behavior.

In vivo assessment of mechanisms underlying the neurovascular basis of postictal amnesia.

Long-lasting confusion and memory difficulties during the postictal state remain a major unmet problem in epilepsy that lacks pathophysiological explanation and treatment. We previously identified that long-lasting periods of severe postictal hypoperfusion/hypoxia, not seizures per se, are associated with memory impairment after temporal lobe seizures. While this observation suggests a key pathophysiological role for insufficient energy delivery, it is unclear how the networks that underlie episodic memory respond to vascular constraints that ultimately give rise to amnesia. Here, we focused on cellular/network level analyses in the CA1 of hippocampus in vivo to determine if neural activity, network oscillations, synaptic transmission, and/or synaptic plasticity are impaired following kindled seizures. Importantly, the induction of severe postictal hypoperfusion/hypoxia was prevented in animals treated by a COX-2 inhibitor, which experimentally separated seizures from their vascular consequences. We observed complete activation of CA1 pyramidal neurons during brief seizures, followed by a short period of reduced activity and flattening of the local field potential that resolved within minutes. During the postictal state, constituting tens of minutes to hours, we observed no changes in neural activity, network oscillations, and synaptic transmission. However, long-term potentiation of the temporoammonic pathway to CA1 was impaired in the postictal period, but only when severe local hypoxia occurred. Lastly, we tested the ability of rats to perform object-context discrimination, which has been proposed to require temporoammonic input to differentiate between sensory experience and the stored representation of the expected object-context pairing. Deficits in this task following seizures were reversed by COX-2 inhibition, which prevented severe postictal hypoxia. These results support a key role for hypoperfusion/hypoxia in postictal memory impairments and identify that many aspects of hippocampal network function are resilient during severe hypoxia except for long-term synaptic plasticity.

Quantitative T2 MRI is predictive of neurodegeneration following organophosphate exposure in a rat model.

Organophosphorus compounds, such as chemical warfare nerve agents and pesticides, are known to cause neurological damage. This study measured nerve agent-related neuropathology and determined whether quantitative T2 MRI could be used as a biomarker of neurodegeneration. Quantitative T2 MRI was performed using a 9.4 T MRI on rats prior to and following soman exposure. T2 images were taken at least 24 h prior, 1 h and 18-24 h after soman exposure. Rats were pre- and post-treated with HI-6 dimethanesulfonate and atropine methyl nitrate. A multicomponent T2 acquisition and analysis was performed. Brains were stained with Fluoro-Jade C to assess neurodegeneration. Rats exposed to soman developed behavioral expression of electrographic seizures. At 18-24 h after soman exposure, significant increases in T2, a possible marker of edema, were found in multiple regions. The largest changes were in the piriform cortex (before: 47.7 ± 1.4 ms; 18-24 h: 82.3 ± 13.4 ms). Fluoro-Jade C staining showed significant neurodegeneration 18-24 h post exposure. The piriform cortex had the strongest correlation between the change in relaxation rate and percent neurodegeneration (r = 0.96, p < 0.001). These findings indicate there is regionally specific neurodegeneration 24 h after exposure to soman. The high correlation between T2 relaxivity and histopathology supports the use of T2 as a marker of injury.

Dynamic oxygen changes during status epilepticus and subsequent endogenous kindling.

OBJECTIVE: Brain tissue oxygen (partial oxygen pressure [pO2 ]) levels are tightly regulated to stay within the normoxic zone, with deviations on either side resulting in impaired brain function. Whereas pathological events such as ischemic attacks and brief seizures have previously been shown to result in pO2 levels well below the normoxic zone, oxygen levels during prolonged status epilepticus (SE) and the subsequent endogenous kindling period are unknown. METHODS: We utilized two models of acquired temporal lobe epilepsy in rats: intrahippocampal kainic acid infusion and prolonged perforant pathway stimulation. Local tissue oxygen was measured in the dorsal hippocampus using an optode during and for several weeks following SE. RESULTS: We observed hyperoxia in the hippocampus during induced SE in both models. Following termination of SE, 88% of rats initiated focal self-generated spiking activity in the hippocampus within the first 7 days, which was associated with dynamic oxygen changes. Self-generated and recurring epileptiform activity subsequently organized into higher-frequency bursts that became progressively longer and were ultimately associated with behavioral seizures that became more severe with time and led to postictal hypoxia. SIGNIFICANCE: Induced SE and self-generated recurrent epileptiform activity can have profound and opposing effects on brain tissue oxygenation that may serve as a biomarker for ongoing pathological activity in the brain.

Resolving the Micro-Macro Disconnect to Address Core Features of Seizure Networks.

Current drug treatments for epilepsy attempt to broadly restrict excitability to mask a symptom, seizures, with little regard for the heterogeneous mechanisms that underlie disease manifestation across individuals. Here, we discuss the need for a more complete view of epilepsy, outlining how key features at the cellular and microcircuit level can significantly impact disease mechanisms that are not captured by the most common methodology to study epilepsy, electroencephalography (EEG). We highlight how major advances in neuroscience tool development now enable multi-scale investigation of fundamental questions to resolve the currently controversial understanding of seizure networks. These findings will provide essential insight into what has emerged as a disconnect between the different levels of investigation and identify new targets and treatment options.

Fast oxygen dynamics as a potential biomarker for epilepsy.

Changes in brain activity can entrain cerebrovascular dynamics, though this has not been extensively investigated in pathophysiology. We assessed whether pathological network activation (i.e. seizures) in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS) could alter dynamic fluctuations in local oxygenation. Spontaneous absence seizures in an epileptic rat model robustly resulted in brief dips in cortical oxygenation and increased spectral oxygen power at frequencies greater than 0.08 Hz. Filtering oxygen data for these fast dynamics was sufficient to distinguish epileptic vs. non-epileptic rats. Furthermore, this approach distinguished brain regions with seizures from seizure-free brain regions in the epileptic rat strain. We suggest that fast oxygen dynamics may be a useful biomarker for seizure network identification and could be translated to commonly used clinical tools that measure cerebral hemodynamics.

Neurodegeneration and Pathology in Epilepsy: Clinical and Basic Perspectives.

Epilepsy is commonly associated with a number of neurodegenerative and pathological alterations in those areas of the brain that are involved in repeated electrographic seizures. These most prominently include neuron loss and an increase in astrocyte number and size but may also include enhanced blood-brain barrier permeability, the formation of new capillaries, axonal sprouting, and central inflammation. In animal models in which seizures are either repeatedly elicited or are self-generated, a similar set of neurodegenerative and pathological alterations in brain anatomy are observed. The primary causal agent responsible for these alterations may be the cascade of events that follow a seizure and lead to an hypoperfusion/hypoxic episode. While epilepsy has long and correctly been considered an electrical disorder, the vascular system likely plays an important causal role in the neurodegeneration and pathology that occur as a consequence of repeated seizures.

Postictal hypoperfusion/hypoxia provides the foundation for a unified theory of seizure-induced brain abnormalities and behavioral dysfunction.

A recent article by Farrell et al. characterizes the phenomenon, mechanisms, and treatment of a local and severe hypoperfusion/hypoxia event that occurs in brain regions following a focal seizure. Given the well-established role of cerebral ischemia/hypoxia in brain damage and behavioral dysfunction in other clinical settings (e.g., stroke, cerebral vasospasm), we put forward a new theory: postictal hypoperfusion/hypoxia is responsible for the negative consequences associated with seizures. Fortunately, inhibition of two separate molecular targets, cyclooxygenase-2 (COX-2) and l-type calcium channels, can prevent the expression of postictal hypoperfusion/hypoxia. These inhibitors are important experimental tools used to separate the seizure from the resulting hypoperfusion/hypoxia and can allow researchers to address the contribution of this phenomenon to negative outcomes associated with seizures. Herein we address the implications of this postictal stroke-like event in acute behavioral dysfunction (e.g., Todd's paresis) and sudden unexpected death in epilepsy (SUDEP). Moreover, anatomic alterations such as increased blood-brain barrier permeability, glial activation, central inflammation, and neuronal loss could also be a consequence of repeated hypoperfusion/hypoxic events and, in turn, underlie chronic interictal cognitive and behavioral comorbidities (e.g., memory deficits, anxiety, depression, and psychosis) and exacerbate epileptogenesis. Thus these seemingly disparate and clinically important observations may share a common point of origin: postictal hypoperfusion/hypoxia.