Microglia-neuron-vascular interactions in ischemia.

Cerebral ischemia is a devastating condition that results in impaired blood flow in the brain leading to acute brain injury. As the most common form of stroke, occlusion of cerebral arteries leads to a characteristic sequence of pathophysiological changes in the brain tissue. The mechanisms involved, and comorbidities that determine outcome after an ischemic event appear to be highly heterogeneous. On their own, the processes leading to neuronal injury in the absence of sufficient blood supply to meet the metabolic demand of the cells are complex and manifest at different temporal and spatial scales. While the contribution of non-neuronal cells to stroke pathophysiology is increasingly recognized, recent data show that microglia, the main immune cells of the central nervous system parenchyma, play previously unrecognized roles in basic physiological processes beyond their inflammatory functions, which markedly change during ischemic conditions. In this review, we aim to discuss some of the known microglia-neuron-vascular interactions assumed to contribute to the acute and delayed pathologies after cerebral ischemia. Because the mechanisms of neuronal injury have been extensively discussed in several excellent previous reviews, here we focus on some recently explored pathways that may directly or indirectly shape neuronal injury through microglia-related actions. These discoveries suggest that modulating gliovascular processes in different forms of stroke and other neurological disorders might have presently unexplored therapeutic potential in combination with neuroprotective and flow restoration strategies.

Microglial control of neuronal development via somatic purinergic junctions.

Microglia, the resident immune cells of the brain, play important roles during development. Although bi-directional communication between microglia and neuronal progenitors or immature neurons has been demonstrated, the main sites of interaction and the underlying mechanisms remain elusive. By using advanced methods, here we provide evidence that microglial processes form specialized contacts with the cell bodies of developing neurons throughout embryonic, early postnatal, and adult neurogenesis. These early developmental contacts are highly reminiscent of somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. The formation and maintenance of these junctions is regulated by functional microglial P2Y12 receptors, and deletion of P2Y12Rs disturbs proliferation of neuronal precursors and leads to aberrant cortical cytoarchitecture during development and in adulthood. We propose that early developmental formation of somatic purinergic junctions represents an important interface for microglia to monitor the status of immature neurons and control neurodevelopment.

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions.

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1ß (IL-1ß), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

Shaping Neuronal Fate: Functional Heterogeneity of Direct Microglia-Neuron Interactions.

The functional contribution of microglia to normal brain development, healthy brain function, and neurological disorders is increasingly recognized. However, until recently, the nature of intercellular interactions mediating these effects remained largely unclear. Recent findings show microglia establishing direct contact with different compartments of neurons. Although communication between microglia and neurons involves intermediate cells and soluble factors, direct membrane contacts enable a more precisely regulated, dynamic, and highly effective form of interaction for fine-tuning neuronal responses and fate. Here, we summarize the known ultrastructural, molecular, and functional features of direct microglia-neuron interactions and their roles in brain disease.

Microglia alter the threshold of spreading depolarization and related potassium uptake in the mouse brain.

Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signaling and to reduce the incidence of spreading depolarization (SD) during cerebral ischemia. However, the mechanisms through which microglia interfere with SD remained unexplored. Here, we identify microglia as essential modulators of the induction and evolution of SD in the physiologically intact brain in vivo. Confocal- and super-resolution microscopy revealed that a series of SDs induced rapid morphological changes in microglia, facilitated microglial process recruitment to neurons and increased the density of P2Y12 receptors (P2Y12R) on recruited microglial processes. In line with this, depolarization and hyperpolarization during SD were microglia- and P2Y12R-dependent. An absence of microglia was associated with altered potassium uptake after SD and increased the number of c-fos-positive neurons, independently of P2Y12R. Thus, the presence of microglia is likely to be essential to maintain the electrical elicitation threshold and to support the full evolution of SD, conceivably by interfering with the extracellular potassium homeostasis of the brain through sustaining [K+]e re-uptake mechanisms.

Microglia monitor and protect neuronal function through specialized somatic purinergic junctions.

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.

Brainstem nucleus incertus controls contextual memory formation.

Hippocampal pyramidal cells encode memory engrams, which guide adaptive behavior. Selection of engram-forming cells is regulated by somatostatin-positive dendrite-targeting interneurons, which inhibit pyramidal cells that are not required for memory formation. Here, we found that γ-aminobutyric acid (GABA)-releasing neurons of the mouse nucleus incertus (NI) selectively inhibit somatostatin-positive interneurons in the hippocampus, both monosynaptically and indirectly through the inhibition of their subcortical excitatory inputs. We demonstrated that NI GABAergic neurons receive monosynaptic inputs from brain areas processing important environmental information, and their hippocampal projections are strongly activated by salient environmental inputs in vivo. Optogenetic manipulations of NI GABAergic neurons can shift hippocampal network state and bidirectionally modify the strength of contextual fear memory formation. Our results indicate that brainstem NI GABAergic cells are essential for controlling contextual memories.

New Insights into Microglia-Neuron Interactions: A Neuron's Perspective.

Microglia are the primary immune cells of the central nervous system. However, recent data indicate that microglia also contribute to diverse physiological and pathophysiological processes that extend beyond immune-related functions and there is a growing interest to understand the mechanisms through which microglia interact with other cells in the brain. In particular, the molecular processes that contribute to microglia-neuron communication in the healthy brain and their role in common brain diseases have been intensively studied during the last decade. In line with this, fate-mapping studies, genetic models and novel pharmacological approaches have revealed the origin of microglial progenitors, demonstrated the role of self-maintaining microglial populations during brain development or in adulthood, and identified the unexpectedly long lifespan of microglia that may profoundly change our view about senescence and age-related human diseases. Despite the exponentially increasing knowledge about microglia, the role of these cells in health and disease is still extremely controversial and the precise molecular targets for intervention are not well defined. This is in part due to the lack of microglia-specific manipulation approaches until very recently and to the high level of complexity of the interactions between microglia and other cells in the brain that occur at different temporal and spatial scales. In this review, we briefly summarize the known physiological roles of microglia-neuron interactions in brain homeostasis and attempt to outline some major directions and challenges of future microglia research.

Co-transmission of acetylcholine and GABA regulates hippocampal states.

The basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter. Yet, we find that mouse hippocampal cholinergic terminals invariably establish GABAergic synapses, and their cholinergic vesicles dock at those synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes composite postsynaptic potentials, which are mutually cross-regulated by presynaptic autoreceptors. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. Therefore, the differentially regulated GABAergic and cholinergic co-transmission suggests a hitherto unrecognised level of control over cortical states. This novel model of hippocampal cholinergic neurotransmission may lead to alternative pharmacotherapies after cholinergic deinnervation seen in neurodegenerative disorders.

Mitochondrial Ultrastructure Is Coupled to Synaptic Performance at Axonal Release Sites.

Mitochondrial function in neurons is tightly linked with metabolic and signaling mechanisms that ultimately determine neuronal performance. The subcellular distribution of these organelles is dynamically regulated as they are directed to axonal release sites on demand, but whether mitochondrial internal ultrastructure and molecular properties would reflect the actual performance requirements in a synapse-specific manner, remains to be established. Here, we examined performance-determining ultrastructural features of presynaptic mitochondria in GABAergic and glutamatergic axons of mice and human. Using electron-tomography and super-resolution microscopy we found, that these features were coupled to synaptic strength: mitochondria in boutons with high synaptic activity exhibited an ultrastructure optimized for high rate metabolism and contained higher levels of the respiratory chain protein cytochrome-c (CytC) than mitochondria in boutons with lower activity. The strong, cell type-independent correlation between mitochondrial ultrastructure, molecular fingerprints and synaptic performance suggests that changes in synaptic activity could trigger ultrastructural plasticity of presynaptic mitochondria, likely to adjust their performance to the actual metabolic demand.