Harnessing olfactory bulb oscillations to perform fully brain-based sleep-scoring and real-time monitoring of anaesthesia depth.

Real-time tracking of vigilance states related to both sleep or anaesthesia has been a goal for over a century. However, sleep scoring cannot currently be performed with brain signals alone, despite the deep neuromodulatory transformations that accompany sleep state changes. Therefore, at heart, the operational distinction between sleep and wake is that of immobility and movement, despite numerous situations in which this one-to-one mapping fails. Here we demonstrate, using local field potential (LFP) recordings in freely moving mice, that gamma (50-70 Hz) power in the olfactory bulb (OB) allows for clear classification of sleep and wake, thus providing a brain-based criterion to distinguish these two vigilance states without relying on motor activity. Coupled with hippocampal theta activity, it allows the elaboration of a sleep scoring algorithm that relies on brain activity alone. This method reaches over 90% homology with classical methods based on muscular activity (electromyography [EMG]) and video tracking. Moreover, contrary to EMG, OB gamma power allows correct discrimination between sleep and immobility in ambiguous situations such as fear-related freezing. We use the instantaneous power of hippocampal theta oscillation and OB gamma oscillation to construct a 2D phase space that is highly robust throughout time, across individual mice and mouse strains, and under classical drug treatment. Dynamic analysis of trajectories within this space yields a novel characterisation of sleep/wake transitions: whereas waking up is a fast and direct transition that can be modelled by a ballistic trajectory, falling asleep is best described as a stochastic and gradual state change. Finally, we demonstrate that OB oscillations also allow us to track other vigilance states. Non-REM (NREM) and rapid eye movement (REM) sleep can be distinguished with high accuracy based on beta (10-15 Hz) power. More importantly, we show that depth of anaesthesia can be tracked in real time using OB gamma power. Indeed, the gamma power predicts and anticipates the motor response to stimulation both in the steady state under constant anaesthetic and dynamically during the recovery period. Altogether, this methodology opens the avenue for multi-timescale characterisation of brain states and provides an unprecedented window onto levels of vigilance.

Validating the theoretical bases of sleep reactivation during sharp-wave ripples and their association with emotional valence.

Sleep is important for memory consolidation, and an abundant literature suggests that reactivation in the hippocampus during sleep is instrumental to this process. Yet, the current interpretation of activity during sharp-waves ripples (SWRs), as replay of wake experiences, relies on hypotheses that, while widely accepted, have only recently begun to be tested directly. Moreover, this theory has been mainly studied in the context of pure spatial learning, and it is still not clear how emotional valence can fit into this conceptual framework when considering reward- or punishment-based learning. In this review, we will present recent experimental arguments validating the interpretation of sleep replay as reactivation of awake experiences and examine the evidence showing that the emotional valence is also replayed during sleep in a coordinated fashion with hippocampal SWRs. Finally, we will detail recent experiments showing that brain-computer interfaces can be used to modify the emotional valence associated with sleep replay.

Astroglial Connexin 43 Hemichannels Modulate Olfactory Bulb Slow Oscillations.

An emergent concept in neurosciences consists in considering brain functions as the product of dynamic interactions between neurons and glial cells, particularly astrocytes. Although the role played by astrocytes in synaptic transmission and plasticity is now largely documented, their contribution to neuronal network activity is only beginning to be appreciated. In mouse olfactory bulb slices, we observed that the membrane potential of mitral cells oscillates between UP and DOWN states at a low frequency (<1 Hz). Such slow oscillations are correlated with glomerular local field potentials, indicating spontaneous local network activity. Using a combination of genetic and pharmacological tools, we showed that the activity of astroglial connexin 43 hemichannels, opened in an activity-dependent manner, increases UP state amplitude and impacts mitral cell firing rate. This effect requires functional adenosine A1 receptors, in line with the observation that ATP is released via connexin 43 hemichannels. These results highlight a new mechanism of neuroglial interaction in the olfactory bulb, where astrocyte connexin hemichannels are both targets and modulators of neuronal circuit function. SIGNIFICANCE STATEMENT: An emergent concept in neuroscience consists in considering brain function as the product of dynamic interactions between neurons and glial cells, particularly astrocytes. A typical feature of astrocytes is their high expression level of connexins, the molecular constituents of gap junction channels and hemichannels. Although hemichannels represent a powerful medium for intercellular communication between astrocytes and neurons, their function in physiological conditions remains largely unexplored. Our results show that in the olfactory bulb, connexin 43 hemichannel function is promoted by neuronal activity and, in turn, modulates neuronal network slow oscillations. This novel mechanism of neuroglial interaction could influence olfactory information processing by directly impacting the output of the olfactory bulb.

Interaction Between Hippocampus and Cerebellum Crus I in Sequence-Based but not Place-Based Navigation.

To examine the cerebellar contribution to human spatial navigation we used functional magnetic resonance imaging and virtual reality. Our findings show that the sensory-motor requirements of navigation induce activity in cerebellar lobules and cortical areas known to be involved in the motor loop and vestibular processing. By contrast, cognitive aspects of navigation mainly induce activity in a different cerebellar lobule (VIIA Crus I). Our results demonstrate a functional link between cerebellum and hippocampus in humans and identify specific functional circuits linking lobule VIIA Crus I of the cerebellum to medial parietal, medial prefrontal, and hippocampal cortices in nonmotor aspects of navigation. They further suggest that Crus I belongs to 2 nonmotor loops, involved in different strategies: place-based navigation is supported by coherent activity between left cerebellar lobule VIIA Crus I and medial parietal cortex along with right hippocampus activity, while sequence-based navigation is supported by coherent activity between right lobule VIIA Crus I, medial prefrontal cortex, and left hippocampus. These results highlight the prominent role of the human cerebellum in both motor and cognitive aspects of navigation, and specify the cortico-cerebellar circuits by which it acts depending on the requirements of the task.

Plasticity of astroglial networks in olfactory glomeruli.

Several recent findings have shown that neurons as well as astrocytes are organized into networks. Indeed, astrocytes are interconnected through connexin-formed gap junction channels allowing exchanges of ions and signaling molecules. The aim of this study is to characterize astrocyte network properties in mouse olfactory glomeruli where neuronal connectivity is highly ordered. Dye-coupling experiments performed in olfactory bulb acute slices (P16-P22) highlight a preferential communication between astrocytes within glomeruli and not between astrocytes in adjacent glomeruli. Such organization relies on the oriented morphology of glomerular astrocytes to the glomerulus center and the enriched expression of two astroglial connexins (Cx43 and Cx30) within the glomeruli. Glomerular astrocytes detect neuronal activity showing membrane potential fluctuations correlated with glomerular local field potentials. Accordingly, gap junctional coupling of glomerular networks is reduced when neuronal activity is silenced by TTX treatment or after early sensory deprivation. Such modulation is lost in Cx30 but not in Cx43 KO mice, indicating that Cx30-formed channels are the molecular targets of this activity-dependent modulation. Extracellular potassium is a key player in this neuroglial interaction, because (i) the inhibition of dye coupling observed in the presence of TTX or after sensory deprivation is restored by increasing [K(+)](e) and (ii) treatment with a K(ir) channel blocker inhibits dye spread between glomerular astrocytes. Together, these results demonstrate that extracellular potassium generated by neuronal activity modulates Cx30-mediated gap junctional communication between glomerular astrocytes, indicating that strong neuroglial interactions take place at this first relay of olfactory information processing.

[Animal models of trauma and post-traumatic stress disorder]. / Les modèles animaux du traumatisme et du trouble de stress post-traumatique.

Initially believed to be specific to humans emerging from life-threatening events, Post-traumatic stress disorder (PTSD) has been found to occur in wild animals and can also be experimentally induced in laboratory rodents. This article aims to highlight and discuss the evolution and relevance of animal models in PTSD research. Studies by LeDoux, Davis, and McGaugh have made significant contributions to our understanding of PTSD. By focusing on fear responses in rodents and aversive Pavlovian conditioning, they suggested that PTSD could result from excessively efficient aversive learning processes, with a significant role played by amygdala. However, numerous studies have shown that this explanation fails to account for the complexity of processes involved in PTSD. Current hypotheses focus on deficits in extinction retention, perception of safety signals, or emotional regulation. This review will specifically address the animal models that closely resemble human PTSD and explore reasons for their limited utilization, as a majority of animal studies continues to employ classical Pavlovian conditioning protocols. Furthermore, this review will present cutting-edge experimental studies that tackle previously challenging questions in animal research. Specifically, we will examine the relationship between respiration and the maintenance of fear states, offering a potential explanation for the efficacy of meditation and breath control techniques in emotion regulation. We will also shed light on recent findings on decoding neural activity related to internal representations in animals, thus enabling now the exploration of rumination, a characteristic symptom of PTSD previously inaccessible to animal studies.

Title: Les modèles animaux du traumatisme et du trouble de stress post-traumatique. Abstract: Le trouble de stress post-traumatique (TSPT) est généralement associé à menace vitale et est parfois considéré comme une condition spécifiquement humaine. Cependant de nombreuses études ont montré qu'il pouvait être observé chez des animaux en milieu sauvage et pouvait être induit en laboratoire chez des rongeurs. Cet article vise à présenter et discuter l'évolution et la pertinence des modèles animaux dans l'étude du TSPT. Les études de LeDoux, Davis et McGaugh sur la peur et le conditionnement aversif pavlovien chez le rongeur ont apporté une immense contribution à la compréhension du TSPT. Initialement, il a été proposé que le TSPT résulterait d'un apprentissage aversif trop efficace, impliquant en particulier l'amygdale. Néanmoins, de nombreuses études ont révélé que cette hypothèse n'était pas suffisante pour expliquer la complexité des processus mis en jeu dans le TSPT. Les théories actuelles suggèrent plutôt des déficits dans la capacité à maintenir l'extinction, la perception des signaux de sécurité ou la régulation émotionnelle. Nous examinerons plus précisément les modèles animaux qui se rapprochent le plus du TSPT humain et nous discuterons des raisons pour lesquelles leur utilisation reste limitée. En effet, la plupart des études chez l'animal continuent de s'appuyer majoritairement sur des protocoles classiques de conditionnement pavlovien. Enfin, cette revue mettra en lumière de nouvelles études expérimentales permettant d'aborder des questions auparavant difficiles à étudier chez l'animal. Nous examinerons notamment les liens entre respiration et maintien des états de peur, offrant une explication potentielle à l'efficacité des techniques de méditation et de contrôle de la respiration dans la régulation des émotions. De plus, nous présenterons des résultats récents sur le décodage de l'activité neuronale liée aux représentations internes chez l'animal, offrant ainsi la possibilité d'étudier les ruminations, symptômes caractéristiques du TSPT qui étaient auparavant inaccessibles à l'expérimentation animale.

Spatial learning and action planning in a prefrontal cortical network model.

The interplay between hippocampus and prefrontal cortex (PFC) is fundamental to spatial cognition. Complementing hippocampal place coding, prefrontal representations provide more abstract and hierarchically organized memories suitable for decision making. We model a prefrontal network mediating distributed information processing for spatial learning and action planning. Specific connectivity and synaptic adaptation principles shape the recurrent dynamics of the network arranged in cortical minicolumns. We show how the PFC columnar organization is suitable for learning sparse topological-metrical representations from redundant hippocampal inputs. The recurrent nature of the network supports multilevel spatial processing, allowing structural features of the environment to be encoded. An activation diffusion mechanism spreads the neural activity through the column population leading to trajectory planning. The model provides a functional framework for interpreting the activity of PFC neurons recorded during navigation tasks. We illustrate the link from single unit activity to behavioral responses. The results suggest plausible neural mechanisms subserving the cognitive "insight" capability originally attributed to rodents by Tolman & Honzik. Our time course analysis of neural responses shows how the interaction between hippocampus and PFC can yield the encoding of manifold information pertinent to spatial planning, including prospective coding and distance-to-goal correlates.

Breathing-driven prefrontal oscillations regulate maintenance of conditioned-fear evoked freezing independently of initiation.

Brain-body interactions are thought to be essential in emotions but their physiological basis remains poorly understood. In mice, regular 4 Hz breathing appears during freezing after cue-fear conditioning. Here we show that the olfactory bulb (OB) transmits this rhythm to the dorsomedial prefrontal cortex (dmPFC) where it organizes neural activity. Reduction of the respiratory-related 4 Hz oscillation, via bulbectomy or optogenetic perturbation of the OB, reduces freezing. Behavioural modelling shows that this is due to a specific reduction in freezing maintenance without impacting its initiation, thus dissociating these two phenomena. dmPFC LFP and firing patterns support the region's specific function in freezing maintenance. In particular, population analysis reveals that network activity tracks 4 Hz power dynamics during freezing and reaches a stable state at 4 Hz peak that lasts until freezing termination. These results provide a potential mechanism and a functional role for bodily feedback in emotions and therefore shed light on the historical James-Cannon debate.

Principal component analysis of ensemble recordings reveals cell assemblies at high temporal resolution.

Simultaneous recordings of many single neurons reveals unique insights into network processing spanning the timescale from single spikes to global oscillations. Neurons dynamically self-organize in subgroups of coactivated elements referred to as cell assemblies. Furthermore, these cell assemblies are reactivated, or replayed, preferentially during subsequent rest or sleep episodes, a proposed mechanism for memory trace consolidation. Here we employ Principal Component Analysis to isolate such patterns of neural activity. In addition, a measure is developed to quantify the similarity of instantaneous activity with a template pattern, and we derive theoretical distributions for the null hypothesis of no correlation between spike trains, allowing one to evaluate the statistical significance of instantaneous coactivations. Hence, when applied in an epoch different from the one where the patterns were identified, (e.g. subsequent sleep) this measure allows to identify times and intensities of reactivation. The distribution of this measure provides information on the dynamics of reactivation events: in sleep these occur as transients rather than as a continuous process.

Toward safer thrombolytic agents in stroke: molecular requirements for NMDA receptor-mediated neurotoxicity.

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (K(D)=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.

The Theta Rhythm Mixes and Matches Gamma Oscillations Cycle by Cycle.

In the hippocampus, gamma power modulation by the theta rhythm is interpreted as the signature of temporally coordinated inputs that reflect ongoing processing. In this issue of Neuron, Lopes-Dos-Santos et al. (2018) develop a new methodology demonstrating that theta cycles can be viewed as individual computational units characterized by typical gamma profiles.

Slow-wave sleep: From the cell to the clinic.

In recent decades, increasing evidence has positioned slow-wave sleep (SWS) as a major actor in neurophysiological phenomena such as glucose metabolism, hormone release, immunity and memory. This proposed role for SWS, coupled with observations of impaired SWS in several pathologies as well as in aging, has led some researchers to implement methods that could specifically enhance SWS. This review aims to gather the current knowledge extending from the cell to the clinic, in order to construct an overview of what is currently known about so-called SWS. We slowly expand the view from the molecular processes underlying SWS to the cell unit and assembly to cortical manifestations. We then describe its role in physiology and cognition to finally assess its association with clinical aspects. Finally, we address practical considerations for several techniques that could be used to manipulate SWS, in order to improve our understanding of SWS and possibly help the development of treatments for SWS clinical disorders.

Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis.

BACKGROUND: Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions. METHODS AND RESULTS: In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4 degrees C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor-related protein family. CONCLUSIONS: We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke.

Equivocal roles of tissue-type plasminogen activator in stroke-induced injury.

Stroke represents a major health problem in the ever-ageing population of industrialized nations. Each year, over three million people in the USA alone suffer from this affliction. Stroke, which results from the obstruction of an intra- or extra-cerebral artery, induces irreversible neuronal damage. The clot-busting drug tissue-type plasminogen activator (tPA) is the only FDA-approved therapy for acute stroke. Although tPA has been successfully used to treat myocardial infarction due to clot formation, its use in the treatment of occlusive cerebrovascular diseases remains controversial. Indeed, tPA is clearly beneficial as a thrombolytic agent. However, increasing evidence suggests that tPA could have direct and deleterious effects on neurons and glial cells.

4-Hz oscillations synchronize prefrontal-amygdala circuits during fear behavior.

Fear expression relies on the coordinated activity of prefrontal and amygdala circuits, yet the mechanisms allowing long-range network synchronization during fear remain unknown. Using a combination of extracellular recordings, pharmacological and optogenetic manipulations, we found that freezing, a behavioral expression of fear, temporally coincided with the development of sustained, internally generated 4-Hz oscillations in prefrontal-amygdala circuits. 4-Hz oscillations predict freezing onset and offset and synchronize prefrontal-amygdala circuits. Optogenetic induction of prefrontal 4-Hz oscillations coordinates prefrontal-amygdala activity and elicits fear behavior. These results unravel a sustained oscillatory mechanism mediating prefrontal-amygdala coupling during fear behavior.

Oxygen glucose deprivation switches the transport of tPA across the blood-brain barrier from an LRP-dependent to an increased LRP-independent process.

BACKGROUND AND PURPOSE: Despite uncontroversial benefit from its thrombolytic activity, the documented neurotoxic effect of tissue plasminogen activator (tPA) raises an important issue: the current emergency stroke treatment might not be optimum if exogenous tPA can enter the brain and thus add to the deleterious effects of endogenous tPA within the cerebral parenchyma. Here, we aimed at determining whether vascular tPA crosses the blood-brain barrier (BBB) during cerebral ischemia, and if so, by which mechanism. METHODS: First, BBB permeability was assessed in vivo by measuring Evans Blue extravasation following intravenous injection at 0 or 3 hours after middle cerebral artery electrocoagulation in mice. Second, the passage of vascular tPA was investigated in an in vitro model of BBB, subjected or not to oxygen and glucose deprivation (OGD). RESULTS: We first demonstrated that after focal permanent ischemia in mice, the BBB remains impermeable to Evans Blue in the early phase (relative to the therapeutic window of tPA), whereas at later time points massive Evans Blue extravasation occurs. Then, the passage of tPA during these 2 phases, was investigated in vitro and we show that in control conditions, tPA crosses the intact BBB by a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent transcytosis, whereas OGD leads to an exacerbation of tPA passage, which switches to a LRP-independent process. CONCLUSIONS: We evidence 2 different mechanisms through which vascular tPA can reach the brain parenchyma, depending on the state of the BBB. As discussed, these data show the importance of taking the side effects of blood-derived tPA into account and offer a basis to improve the current thrombolytic strategy.

From necessity to sufficiency in memory research: when sleep helps to understand wake experiences.

Memory is the ability to adapt our behavior by using the stored information, previously encoded. The first investigations of the neuronal bases of the memory trace concerned its properties (location, cellular and molecular mechanisms, among others). However, to understand how this is achieved at the scale of neurons, we must provide evidence about the necessity of a neuronal subpopulation to support the memory trace, but also its sufficiency. Here, we will present past and recent studies that provide information about the neuronal nature of memories. We will show that research on sleep, when cells assembly supposedly carrying information from the past are replayed, could also provide valuable information about the memory processes at stake during wake.

Explicit memory creation during sleep demonstrates a causal role of place cells in navigation.

Hippocampal place cells assemblies are believed to support the cognitive map, and their reactivations during sleep are thought to be involved in spatial memory consolidation. By triggering intracranial rewarding stimulations by place cell spikes during sleep, we induced an explicit memory trace, leading to a goal-directed behavior toward the place field. This demonstrates that place cells' activity during sleep still conveys relevant spatial information and that this activity is functionally significant for navigation.

Selective blockade of endothelin-B receptors exacerbates ischemic brain damage in the rat.

BACKGROUND AND PURPOSE: Endothelins act through 2 receptors, namely, ET(A) and ET(B). In the cerebral circulation, ET(A) mediates marked and prolonged vasoconstriction, and its blockade increases cerebral blood flow (CBF) and reduces ischemic brain damage. However, the role of ET(B) receptors remains unclear. In this study we examined, in rats, the kinetics of expression of ET(B) and the effects of ET(B) blockade on changes in CBF and brain damage after focal cerebral ischemia and N-methyl-D-aspartate (NMDA)-induced excitotoxic injury. METHODS: Rats were subjected to transient (60 minutes) focal cerebral ischemia or cortical injection of NMDA. The selective ET(B) antagonist BQ-788 was injected intracerebroventricularly 30 minutes before and 30 minutes after the onset of ischemia. Cortical perfusion was monitored by laser-Doppler flowmetry. The volume of infarction or NMDA-induced cortical lesion was assessed at 24 hours after the insult. The reverse transcription-polymerase chain reaction technique was used to assess ET(B) expression. RESULTS: Cerebral ischemia failed to alter the expression of ET(B) mRNA in both acute and chronic stages. Administration of BQ-788 resulted in an increase in infarction volume (178%; P<0.05) accompanied by a decrease in residual CBF (-26.7% versus control; P<0.01). In these animals we found a positive correlation between the volume of infarction and the severity of the decrease in CBF. NMDA-induced cortical lesions were not affected by the administration of BQ-788. CONCLUSIONS: Our results suggest that the ET(B) antagonist BQ-788 induces deleterious effects that are mediated by the reduction of residual blood flow after ischemia and argue that the optimal therapeutic strategy in stroke would be to target the use of selective ET(A) antagonists and not mixed ET(A)/ET(B) antagonists.

2,7-Bis-(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride, tPA stop, prevents tPA-enhanced excitotoxicity both in vitro and in vivo.

Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidino-benzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-D-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.