Plasticity of perisynaptic astroglia during ischemia-induced spreading depolarization.

High astroglial capacity for glutamate and potassium clearance aids in recovering spreading depolarization (SD)-evoked disturbance of ion homeostasis during stroke. Since perisynaptic astroglia cannot be imaged with diffraction-limited light microscopy, nothing is known about the impact of SD on the ultrastructure of a tripartite synapse. We used serial section electron microscopy to assess astroglial synaptic coverage in the sensorimotor cortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. At the subcellular level, astroglial mitochondria were remarkably resilient to SD compared to dendritic mitochondria that were fragmented by SD. Overall, 482 synapses in `Sham' during `SD' and `Recovery' groups were randomly selected and analyzed in 3D. Perisynaptic astroglia was present at the axon-spine interface (ASI) during SD and after recovery. Astrocytic processes were more likely found at large synapses on mushroom spines after recovery, while the length of the ASI perimeter surrounded by astroglia has also significantly increased at large synapses. These findings suggest that as larger synapses have a bigger capacity for neurotransmitter release during SD, they attract astroglial processes to their perimeter during recovery, limiting extrasynaptic glutamate escape and further enhancing the astrocytic ability to protect synapses in stroke.

Chronic basal forebrain activation improves spatial memory, boosts neurotrophin receptor expression, and lowers BACE1 and Aß42 levels in the cerebral cortex in mice.

The etiology of Alzheimer's dementia has been hypothesized in terms of basal forebrain cholinergic decline, and in terms of reflecting beta-amyloid neuropathology. To study these different biological elements, we activated the basal forebrain in 5xFAD Alzheimer's model mice and littermates. Mice received 5 months of 1 h per day intermittent stimulation of the basal forebrain, which includes cholinergic projections to the cortical mantle. Then, mice were behaviorally tested followed by tissue analysis. The 5xFAD mice performed worse in water-maze testing than littermates. Stimulated groups learned the water maze better than unstimulated groups. Stimulated groups had 2-3-fold increases in frontal cortex immunoblot measures of the neurotrophin receptors for nerve growth factor and brain-derived neurotrophic factor, and a more than 50% decrease in the expression of amyloid cleavage enzyme BACE1. Stimulation also led to lower Aß42 in 5xFAD mice. These data support a causal relationship between basal forebrain activation and both neurotrophin activation and reduced Aß42 generation and accumulation. The observation that basal forebrain activation suppresses Aß42 accumulation, combined with the known high-affinity antagonism of nicotinic receptors by Aß42, documents bidirectional antagonism between acetylcholine and Aß42.

Novel mechanism of hypoxic neuronal injury mediated by non-excitatory amino acids and astroglial swelling.

In ischemic stroke and post-traumatic brain injury (TBI), blood-brain barrier disruption leads to leaking plasma amino acids (AA) into cerebral parenchyma. Bleeding in hemorrhagic stroke and TBI also release plasma AA. Although excitotoxic AA were extensively studied, little is known about non-excitatory AA during hypoxic injury. Hypoxia-induced synaptic depression in hippocampal slices becomes irreversible with non-excitatory AA, alongside their intracellular accumulation and increased tissue electrical resistance. Four non-excitatory AA (l-alanine, glycine, l-glutamine, l-serine: AGQS) at plasmatic concentrations were applied to slices from mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Two-photon imaging, light transmittance (LT) changes, and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. radiatum were used to monitor synaptic function concurrently with cellular swelling and injury. During normoxia, AGQS-induced increase in LT was due to astroglial but not neuronal swelling. LT raise during hypoxia and AGQS manifested astroglial and neuronal swelling accompanied by a permanent loss of synaptic transmission and irreversible dendritic beading, signifying acute damage. Neuronal injury was not triggered by spreading depolarization which did not occur in our experiments. Hypoxia without AGQS did not cause cell swelling, leaving dendrites intact. Inhibition of NMDA receptors prevented neuronal damage and irreversible loss of synaptic function. Deleterious effects of AGQS during hypoxia were prevented by alanine-serine-cysteine transporters (ASCT2) and volume-regulated anion channels (VRAC) blockers. Our findings suggest that astroglial swelling induced by accumulation of non-excitatory AA and release of excitotoxins through antiporters and VRAC may exacerbate the hypoxia-induced neuronal injury.

Modulation of depression-related behaviors by adiponectin AdipoR1 receptors in 5-HT neurons.

The adipocyte-derived hormone adiponectin has a broad spectrum of functions beyond metabolic control. We previously reported that adiponectin acts in the brain to regulate depression-related behaviors. However, its underlying neural substrates have not been identified. Here we show that adiponectin receptor 1 (AdipoR1) is expressed in the dorsal raphe nucleus (DRN) and colocalized with tryptophan hydroxylase 2 (TPH2), a marker of serotonin (5-HT) neurons. Selective deletion of AdipoR1 in 5-HT neurons induced anhedonia in male mice, as indicated by reduced female urine sniffing time and saccharin preference, and behavioral despair in female mice and enhanced stress-induced decrease in sucrose preference in both sexes. The expression levels of TPH2 were downregulated with a concurrent reduction of 5-HT-immunoreactivity in the DRN and its two major projection regions, the hippocampus and medial prefrontal cortex (mPFC), in male but not female mice lacking AdipoR1 in 5-HT neurons. In addition, serotonin transporter (SERT) expression was upregulated in both DRN projection fields of male mice but only in the mPFC of female mice. These changes presumably lead to decreased 5-HT synthesis and/or increased 5-HT reuptake, thereby reducing 5-HT transmission. The augmented behavioral responses to the selective serotonin reuptake inhibitor fluoxetine but not desipramine, a selective norepinephrine reuptake inhibitor, observed in conditional knockout male mice supports deficient 5-HT transmission underlying depression-related phenotypes. Our results indicate that adiponectin acts on 5-HT neurons through AdipoR1 receptors to regulate depression-related behaviors in a sex-dependent manner.

The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention.

BACKGROUND: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. METHODS: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na+/K+ pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. RESULTS: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na+/K+ ATPase elicits SD. Elevated K+ or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. CONCLUSIONS: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.

Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization.

BACKGROUND: Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s METHODS: Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. RESULTS: Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. CONCLUSIONS: Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate.

Rapid Neuronal Ultrastructure Disruption and Recovery during Spreading Depolarization-Induced Cytotoxic Edema.

Two major pathogenic events that cause acute brain damage during neurologic emergencies of stroke, head trauma, and cardiac arrest are spreading depolarizing waves and the associated brain edema that course across the cortex injuring brain cells. Virtually nothing is known about how spreading depolarization (SD)-induced cytotoxic edema evolves at the ultrastructural level immediately after insult and during recovery. In vivo 2-photon imaging followed by quantitative serial section electron microscopy was used to assess synaptic circuit integrity in the neocortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. SD triggered a rapid fragmentation of dendritic mitochondria. A large increase in the density of synapses on swollen dendritic shafts implies that some dendritic spines were overwhelmed by swelling or merely retracted. The overall synaptic density was unchanged. The postsynaptic dendritic membranes remained attached to axonal boutons, providing a structural basis for the recovery of synaptic circuits. Upon immediate reperfusion, cytotoxic edema mainly subsides as affirmed by a recovery of dendritic ultrastructure. Dendritic recuperation from swelling and reversibility of mitochondrial fragmentation suggests that neurointensive care to improve tissue perfusion should be paralleled by treatments targeting mitochondrial recovery and minimizing the occurrence of SDs.

Which Spreading Depolarizations Are Deleterious To Brain Tissue?

Spreading depolarizations (SDs) are profound disruptions of cellular homeostasis that slowly propagate through gray matter and present an extraordinary metabolic challenge to brain tissue. Recent work has shown that SDs occur commonly in human patients in the neurointensive care setting and have established a compelling case for their importance in the pathophysiology of acute brain injury. The International Conference on Spreading Depolarizations (iCSD) held in Boca Raton, Florida, in September of 2018 included a discussion session focused on the question of "Which SDs are deleterious to brain tissue?" iCSD is attended by investigators studying various animal species including invertebrates, in vivo and in vitro preparations, diseases of acute brain injury and migraine, computational modeling, and clinical brain injury, among other topics. The discussion included general agreement on many key issues, but also revealed divergent views on some topics that are relevant to the design of clinical interventions targeting SDs. A draft summary of viewpoints offered was then written by a multidisciplinary writing group of iCSD members, based on a transcript of the session. Feedback of all discussants was then formally collated, reviewed and incorporated into the final document. It is hoped that this report will stimulate collection of data that are needed to develop a more nuanced understanding of SD in different pathophysiological states, as the field continues to move toward effective clinical interventions.

Reversible Disruption of Neuronal Mitochondria by Ischemic and Traumatic Injury Revealed by Quantitative Two-Photon Imaging in the Neocortex of Anesthetized Mice.

Mitochondria play a variety of functional roles in cortical neurons, from metabolic support and neuroprotection to the release of cytokines that trigger apoptosis. In dendrites, mitochondrial structure is closely linked to their function, and fragmentation (fission) of the normally elongated mitochondria indicates loss of their function under pathological conditions, such as stroke and brain trauma. Using in vivo two-photon microscopy in mouse brain, we quantified mitochondrial fragmentation in a full spectrum of cortical injuries, ranging from severe to mild. Severe global ischemic injury was induced by bilateral common carotid artery occlusion, whereas severe focal stroke injury was induced by Rose Bengal photosensitization. The moderate and mild traumatic injury was inflicted by focal laser lesion and by mild photo-damage, respectively. Dendritic and mitochondrial structural changes were tracked longitudinally using transgenic mice expressing fluorescent proteins localized either in cytosol or in mitochondrial matrix. In response to severe injury, mitochondrial fragmentation developed in parallel with dendritic damage signified by dendritic beading. Reconstruction from serial section electron microscopy confirmed mitochondrial fragmentation. Unlike dendritic beading, fragmentation spread beyond the injury core in focal stroke and focal laser lesion models. In moderate and mild injury, mitochondrial fragmentation was reversible with full recovery of structural integrity after 1-2 weeks. The transient fragmentation observed in the mild photo-damage model was associated with changes in dendritic spine density without any signs of dendritic damage. Our findings indicate that alterations in neuronal mitochondria structure are very sensitive to the tissue damage and can be reversible in ischemic and traumatic injuries. SIGNIFICANCE STATEMENT: During ischemic stroke or brain trauma, mitochondria can either protect neurons by supplying ATP and adsorbing excessive Ca2+, or kill neurons by releasing proapoptotic factors. Mitochondrial function is tightly linked to their morphology: healthy mitochondria are thin and long; dysfunctional mitochondria are thick (swollen) and short (fragmented). To date, fragmentation of mitochondria was studied either in dissociated cultured neurons or in brain slices, but not in the intact living brain. Using real-time in vivo two-photon microscopy, we quantified mitochondrial fragmentation during acute pathological conditions that mimic severe, moderate, and mild brain injury. We demonstrated that alterations in neuronal mitochondria structural integrity can be reversible in traumatic and ischemic injuries, highlighting mitochondria as a potential target for therapeutic interventions.

Chloride Cotransporters as a Molecular Mechanism underlying Spreading Depolarization-Induced Dendritic Beading.

Spreading depolarizations (SDs) are waves of sustained neuronal and glial depolarization that propagate massive disruptions of ion gradients through the brain. SD is associated with migraine aura and recently recognized as a novel mechanism of injury in stroke and brain trauma patients. SD leads to neuronal swelling as assessed in real time with two-photon laser scanning microscopy (2PLSM). Pyramidal neurons do not express aquaporins and thus display low inherent water permeability, yet SD rapidly induces focal swelling (beading) along the dendritic shaft by unidentified molecular mechanisms. To address this issue, we induced SD in murine hippocampal slices by focal KCl microinjection and visualized the ensuing beading of dendrites expressing EGFP by 2PLSM. We confirmed that dendritic beading failed to arise during large (100 mOsm) hyposmotic challenges, underscoring that neuronal swelling does not occur as a simple osmotic event. SD-induced dendritic beading was not prevented by pharmacological interference with the cytoskeleton, supporting the notion that dendritic beading may result entirely from excessive water influx. Dendritic beading was strictly dependent on the presence of Cl(-), and, accordingly, combined blockade of Cl(-)-coupled transporters led to a significant reduction in dendritic beading without interfering with SD. Furthermore, our in vivo data showed a strong inhibition of dendritic beading during pharmacological blockage of these cotransporters. We propose that SD-induced dendritic beading takes place as a consequence of the altered driving forces and thus activity for these cotransporters, which by transport of water during their translocation mechanism may generate dendritic beading independently of osmotic forces. SIGNIFICANCE STATEMENT: Spreading depolarization occurs during pathological conditions such as stroke, brain injury, and migraine and is characterized as a wave of massive ion translocation between intracellular and extracellular space in association with recurrent transient focal swelling (beading) of dendrites. Numerous ion channels have been demonstrated to be involved in generation and propagation of spreading depolarization, but the molecular machinery responsible for the dendritic beading has remained elusive. Using real-time in vitro and in vivo two-photon laser scanning microscopy, we have identified the transport mechanisms involved in the detrimental focal swelling of dendrites. These findings have clear clinical significance because they may point to a new class of pharmacological targets for prevention of neuronal swelling that consequently will serve as neuroprotective agents.

Astrocyte contributions to flow/pressure-evoked parenchymal arteriole vasoconstriction.

Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca(2+) dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca(2+) imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca(2+). Astrocytic Ca(2+) responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca(2+). In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca(2+)) and enhanced when high Ca(2+) or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K(+) and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone.

Adrenergic activation attenuates astrocyte swelling induced by hypotonicity and neurotrauma.

Edema in the central nervous system can rapidly result in life-threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, which affects astrocytes and is a primary trigger of acute post-traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, including the stress stimulus epinephrine protects neural parenchyma from damage, we characterized its effects on hypotonicity-induced cellular edema in cortical astrocytes by in vivo and in vitro imaging. After epinephrine administration, hypotonicity-induced swelling of astrocytes was markedly reduced and cytosolic 3'-5'-cyclic adenosine monophosphate (cAMP) was increased, as shown by a fluorescence resonance energy transfer nanosensor. Although, the kinetics of epinephrine-induced cAMP signaling was slowed in primary cortical astrocytes exposed to hypotonicity, the swelling reduction by epinephrine was associated with an attenuated hypotonicity-induced cytosolic Ca(2+) excitability, which may be the key to prevent astrocyte swelling. Furthermore, in a rat model of spinal cord injury, epinephrine applied locally markedly reduced neural edema around the contusion epicenter. These findings reveal new targets for the treatment of cellular edema in the central nervous system.

Evolution of neuronal and astroglial disruption in the peri-contusional cortex of mice revealed by in vivo two-photon imaging.

In traumatic brain injury mechanical forces applied to the cranium and brain cause irreversible primary neuronal and astroglial damage associated with terminal dendritic beading and spine loss representing acute damage to synaptic circuitry. Oedema develops quickly after trauma, raising intracranial pressure that results in a decrease of blood flow and consequently in cerebral ischaemia, which can cause secondary injury in the peri-contusional cortex. Spreading depolarizations have also been shown to occur after traumatic brain injury in humans and in animal models and are thought to accelerate and exacerbate secondary tissue injury in at-risk cortical territory. Yet, the mechanisms of acute secondary injury to fine synaptic circuitry within the peri-contusional cortex after mild traumatic brain injury remain unknown. A mild focal cortical contusion model in adult mouse sensory-motor cortex was implemented by the controlled cortical impact injury device. In vivo two-photon microscopy in the peri-contusional cortex was used to monitor via optical window yellow fluorescent protein expressing neurons, enhanced green fluorescent protein expressing astrocytes and capillary blood flow. Dendritic beading in the peri-contusional cortex developed slowly and the loss of capillary blood flow preceded terminal dendritic injury. Astrocytes were swollen indicating oedema and remained swollen during the next 24 h throughout the imaging session. There were no recurrent spontaneous spreading depolarizations in this mild traumatic brain injury model; however, when spreading depolarizations were repeatedly induced outside the peri-contusional cortex by pressure-injecting KCl, dendrites undergo rapid beading and recovery coinciding with passage of spreading depolarizations, as was confirmed with electrophysiological recordings in the vicinity of imaged dendrites. Yet, accumulating metabolic stress resulting from as few as four rounds of spreading depolarization significantly added to the fraction of beaded dendrites that were incapable to recover during repolarization, thus facilitating terminal injury. In contrast, similarly induced four rounds of spreading depolarization in another set of control healthy mice caused no accumulating dendritic injury as dendrites fully recovered from beading during repolarization. Taken together, our data suggest that in the mild traumatic brain injury the acute dendritic injury in the peri-contusional cortex is gated by the decline in the local blood flow, most probably as a result of developing oedema. Furthermore, spreading depolarization is a specific mechanism that could accelerate injury to synaptic circuitry in the metabolically compromised peri-contusional cortex, worsening secondary damage following traumatic brain injury.

Spreading depolarization causes reversible neuronal mitochondria fragmentation and swelling in healthy, normally perfused neocortex.

Mitochondrial function is tightly linked to their morphology, and fragmentation of dendritic mitochondria during noxious conditions suggests loss of function. In the normoxic cortex, spreading depolarization (SD) is a phenomenon underlying migraine aura. It is unknown whether mitochondria structure is affected by normoxic SD. In vivo two-photon imaging followed by quantitative serial section electron microscopy (ssEM) was used to monitor dendritic mitochondria in the normoxic cortex of urethane-anesthetized mature male and female mice during and after SD initiated by focal KCl microinjection. Structural dynamics of dendrites and their mitochondria were visualized by transfecting excitatory, glutamatergic neurons of the somatosensory cortex with bicistronic AAV, which induced tdTomoto labeling in neuronal cytoplasm and mitochondria labeling with roGFP. Normoxic SD triggered a rapid fragmentation of dendritic mitochondria alongside dendritic beading, both reversible; however, mitochondria took significantly longer to recover. Several rounds of SD resulted in transient mitochondrial fragmentation and dendritic beading without accumulating injury, as both recovered. SsEM corroborated normoxic SD-elicited dendritic and mitochondrial swelling and transformation of the filamentous mitochondrial network into shorter, swollen tubular and globular structures. Our results revealed normoxic SD-induced disruption of the dendritic mitochondrial structure that might impact mitochondrial bioenergetics during migraine with aura.

Specific regulation of NRG1 isoform expression by neuronal activity.

Neuregulin 1 (NRG1) is a trophic factor that has been implicated in neural development, neurotransmission, and synaptic plasticity. NRG1 has multiple isoforms that are generated by usage of different promoters and alternative splicing of a single gene. However, little is known about NRG1 isoform composition profile, whether it changes during development, or the underlying mechanisms. We found that each of the six types of NRG1 has a distinct expression pattern in the brain at different ages, resulting in a change in NRG1 isoform composition. In both human and rat, the most dominant are types III and II, followed by either type I or type V, while types IV and VI are the least abundant. The expression of NRG1 isoforms is higher in rat brains at ages of E13 and P5 (in particular type V), suggesting roles in early neural development and in the neonatal critical period. At the cellular level, the majority of NRG1 isoforms (types I, II, and III) are expressed in excitatory neurons, although they are also present in GABAergic neurons and astrocytes. Finally, the expression of each NRG1 isoform is distinctly regulated by neuronal activity, which causes significant increase in type I and IV NRG1 levels. Neuronal activity regulation of type IV expression requires a CRE cis-element in the 5' untranslated region (UTR) that binds to CREB. These results indicate that expression of NRG1 isoforms is regulated by distinct mechanisms, which may contribute to versatile functions of NRG1 and pathologic mechanisms of brain disorders such as schizophrenia.

Persistent astroglial swelling accompanies rapid reversible dendritic injury during stroke-induced spreading depolarizations.

Spreading depolarizations are a key event in the pathophysiology of stroke, resulting in rapid dendritic beading, which represents acute damage to synaptic circuitry. The impact of spreading depolarizations on the real-time injury of astrocytes during ischemia is less clear. We used simultaneous in vivo 2-photon imaging and electrophysiological recordings in adult mouse somatosensory cortex to examine spreading depolarization-induced astroglial structural changes concurrently with signs of neuronal injury in the early periods of focal and global ischemia. Astrocytes in the metabolically compromised ischemic penumbra-like area showed a long lasting swelling response to spontaneous spreading depolarizations despite rapid dendritic recovery in a photothrombotic occlusion model of focal stroke. Astroglial swelling was often facilitated by recurrent depolarizations and the magnitude of swelling strongly correlated with the total duration of depolarization. In contrast, spreading depolarization-induced astroglial swelling was transient in normoxic healthy tissue. In a model of transient global ischemia, the occurrence of a single spreading depolarization elicited by a bilateral common carotid artery occlusion coincided with astroglial swelling alongside dendritic beading. With immediate reperfusion, dendritic beading subsides. Astroglial swelling was either transient during short ischemic periods distinguished by a short-lasting spreading depolarization, or persistent during severe ischemia characterized by a long-lasting depolarization with the ultraslow negative voltage component. We propose that persistent astroglial swelling is initiated and exacerbated during spreading depolarization in brain tissue with moderate to severe energy deficits, disrupting astroglial maintenance of normal homeostatic function thus contributing to the negative outcome of ischemic stroke as astrocytes fail to provide neuronal support.

Recurrent spontaneous spreading depolarizations facilitate acute dendritic injury in the ischemic penumbra.

Spontaneous spreading depolarizations (SDs) occur in the penumbra surrounding ischemic core. These SDs, often referred to as peri-infarct depolarizations, cause vasoconstriction and recruitment of the penumbra into the ischemic core in the critical first hours after focal ischemic stroke; however, the real-time spatiotemporal dynamics of SD-induced injury to synaptic circuitry in the penumbra remain unknown. A modified cortical photothrombosis model was used to produce a square-shaped lesion surrounding a penumbra-like "area at risk" in middle cerebral artery territory of mouse somatosensory cortex. Lesioning resulted in recurrent spontaneous SDs. In vivo two-photon microscopy of green fluorescent protein-expressing neurons in this penumbra-like area at risk revealed that SDs were temporally correlated with rapid (<6 s) dendritic beading. Dendrites quickly (<3 min) recovered between SDs to near-control morphology until the occurrence of SD-induced terminal dendritic injury, signifying acute synaptic damage. SDs are characterized by a breakdown of ion homeostasis that can be recovered by ion pumps if the energy supply is adequate. Indeed, the likelihood of rapid dendritic recovery between SDs was correlated with the presence of nearby flowing blood vessels, but the presence of such vessels was not always sufficient for rapid dendritic recovery, suggesting that energy needs for recovery exceeded energy supply of compromised blood flow. We propose that metabolic stress resulting from recurring SDs facilitates acute injury at the level of dendrites and dendritic spines in metabolically compromised tissue, expediting penumbral recruitment into the ischemic core.

Capillary blood flow around microglial somata determines dynamics of microglial processes in ischemic conditions.

Microglia are the resident immune cells in the brain. Under normal conditions, resting ramified microglia constantly extend and retract fine processes while performing immunological surveillance. In ischemia, microglia become activated as demonstrated by morphological changes during deramification leading to transformation from ramified to amoeboid form. In vivo two-photon microscopy of enhanced green fluorescent protein (EGFP)-expressing microglia in mouse neocortex was used to examine microglial dynamics during the early periods of focal and global ischemia. A penumbra-like "area-at-risk" surrounded by a square-shaped area of severely hypoperfused tissue was created by laser-induced photothrombosis. The dynamics of microglial processes in the area-at-risk was strongly correlated with capillary blood flow (BF) measured within 10 µm of microglial somata. Changes in BF around distal microglial processes (>30 µm from somata) had no effect on microglial dynamics. A severe reduction of capillary BF near somata by 84% ± 6% resulted in initiation of microglial deramification, suggesting activation. A moderate decrease in BF near somata by 22% ± 5% or increase by 87% ± 10%, reflecting a redistribution of capillary BF, had no effect on microglial morphology. Complete BF loss during cardiac arrest (CA) or transient bilateral common carotid artery occlusion (BCCAO) entirely stalled all microglial processes without structural changes. Reperfusion after BCCAO induced recovery of microglial dynamics to preocclusion values. These findings suggest that during ischemia, the severe drop in BF around microglial somata coincides with morphological activation. However, this activation requires some residual BF, because complete perfusion loss (as during BCCAO and CA) did not support microglial deramification.

Potent inhibition of anoxic depolarization by the sodium channel blocker dibucaine.

Recurring waves of peri-infarct depolarizations (PIDs) propagate across gray matter in the hours and days following stroke, expanding the primary site of injury. Ischemic depolarization (termed anoxic depolarization or AD in live brain slices) is PID-like but immediately arises in the more metabolically compromised ischemic core. This causes dramatic neuronal and astrocyte swelling and dendritic beading with spine loss within minutes, resulting in acute cell death. AD is evoked in rodent neocortical slices by suppressing the Na(+)/K(+)-ATPase pump with either oxygen/glucose deprivation (OGD) or exposure to ouabain. The process driving AD and PIDs remains poorly understood. Here we show that dibucaine is a potent drug inhibiting AD because of its high binding affinity to the Na(+) channel. Field recording reveals that, when superfused with ouabain (5 min), neocortical slices pretreated with 1 µM dibucaine for 45 min display either no AD or delayed AD onset compared with untreated controls. If ouabain exposure is extended to 10 min, 1 µM dibucaine is still able to delay AD onset by ∼ 60%. Likewise, it delays OGD-evoked AD onset by ∼ 54% but does not depress action potentials (APs) or evoked orthodromic field potentials. Increasing dibucaine to 10 µM inhibits AP firing, gradually putting the slice into a stasis that inhibits AD onset but also renders the slice functionally quiescent. Two-photon microscopy reveals that 10 µM dibucaine pretreatment prevents or helps reverse ouabain-induced structural neuronal damage. Although the therapeutic range of dibucaine is quite narrow, dibucaine-like drugs could prove therapeutically useful in inhibiting PIDs and their resultant neuronal damage.