in the past , the central nervous system ( cns ) was considered a site of immune privilege . microglia , first described by achcarro and subsequently thoroughly characterized by his successor , rio - hortega , are now known to play a pivotal role in innate immunity within the cns . in addition , emerging evidence demonstrates the presence of a conduit that enables communication between the brain parenchyma and cervical lymph nodes via the glymphatic system ( an interstitial fluid ( isf)/cerebrospinal fluid ( csf ) exchange system located in the perivascular space ) ( figure 1 ) . along with brain microglia , this exchange system facilitates immune surveillance of brain isf . these findings suggest that the cns immune system is more constitutively active than previously envisioned . a currently supported view concerning the origin of microglia postulates that they are derived from erythromyeloid progenitors in yolk sac that enter the brain during different developmental stages in the brain . yolk sac erythromyeloid progenitors express the chemokine receptor cx3cr1 . to closely analyze the fate of erythromyeloid progenitors in the cns during embryogenesis , kierdorf et  al . used cx3cr1gfp / wt mice , which contain a gfp knock - in on one allele of the cx3cr1 gene . they discovered that mouse microglia were derived from primitive c - kit erythromyeloid precursors present in the yolk sac of embryos at embryonic age of 7.5 to 8.0 days ( e7.5e8.0 ) ( figure 2(a ) ) . these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . microglial maturation is accompanied by a reduction in cd31 expression and concomitant upregulation of the macrophage marker , f4/80 , and macrophage colony stimulating factor receptor ( mcsf - r ) . proliferating a2 cells develop into microglia and invade the developing brain at e9.5 , a process that requires activity of matrix metalloproteinase . after entering the cns , inhibition of microglia - mediated synaptic pruning results in aberrant brain circuitry with excessive dendritic spines and immature synapses . within the postnatal brain , microglia are maintained through cellular division or expansion of progenitors that colonized the brain during cns development . microglia form an autonomous population of cells within the brain that demonstrate activities independent of circulating monocyte activity . resident microglia are distributed in the brain parenchyma , and use motile processes to survey the microenvironment ( left ) . the brain parenchyma is equipped with the glymphatic system , which clears interstitial solutes through the exchange of isf and csf along the perivascular space between the wall of the veins and astrocytic endfeet . subsequently , csf is absorbed into the meningeal lymphatic vessels , which are located in the vicinity of the venous sinus . figure 2.(a ) schematic representation of microglial development : microglia are derived from c - kit - positive erythromyeloid precursors ( emps ) in the yolk sac at e7.5e8.0 . these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . proliferating a2 cells develop into embryonic microglia . before completion of the blood  brain barrier ( bbb ) , they invade the developing brain in a manner requiring matrix metalloproteinase ( mmp ) activity at e9.5 . unless the bbb is disrupted , microglia and monocytes are independent of each other in the postnatal stage . ( b ) multiple roles of microglia in the postnatal brain : microglia in the postnatal brain are multifunctional . primarily , they are involved in immune surveillance , and when necessary , they initiate an inflammatory response . although this contributes to the elimination of pathogens , an excessive inflammatory response may result in bystander tissue damage . microglia also phagocytose pathogens and apoptotic neurons and exert scavenging actions on tissue debris under pathological conditions . microglia recognizes apoptotic cells by phosphatidylserine and other apoptotic signals . with regard to synaptic refinement in addition , microglia - derived tnf can exert neuroprotective as well as neurodestructive action within different contexts . microglia also play a role in angiogenesis through by synthesizing vascular endothelial growth factor ( vegf ) . resident microglia are distributed in the brain parenchyma , and use motile processes to survey the microenvironment ( left ) . the brain parenchyma is equipped with the glymphatic system , which clears interstitial solutes through the exchange of isf and csf along the perivascular space between the wall of the veins and astrocytic endfeet . subsequently , csf is absorbed into the meningeal lymphatic vessels , which are located in the vicinity of the venous sinus . ( a ) schematic representation of microglial development : microglia are derived from c - kit - positive erythromyeloid precursors ( emps ) in the yolk sac at e7.5e8.0 . these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . proliferating a2 cells develop into embryonic microglia . before completion of the blood  brain barrier ( bbb ) , they invade the developing brain in a manner requiring matrix metalloproteinase ( mmp ) activity at e9.5 . unless the bbb is disrupted , microglia and monocytes are independent of each other in the postnatal stage . ( b ) multiple roles of microglia in the postnatal brain : microglia in the postnatal brain are multifunctional . primarily , they are involved in immune surveillance , and when necessary , they initiate an inflammatory response . although this contributes to the elimination of pathogens , an excessive inflammatory response may result in bystander tissue damage . microglia also phagocytose pathogens and apoptotic neurons and exert scavenging actions on tissue debris under pathological conditions . microglia recognizes apoptotic cells by phosphatidylserine and other apoptotic signals . with regard to synaptic refinement in addition , microglia - derived tnf can exert neuroprotective as well as neurodestructive action within different contexts . microglia also play a role in angiogenesis through by synthesizing vascular endothelial growth factor ( vegf ) . in the postnatal brain , microglia have multiple functions ( figure 2(b ) ) . as the powerhouse of local innate immunity , microglia protect the postnatal brain from infection . in response to pathogens invading the cns , microglia act as a front - line defense system by phagocytosing pathogens , secreting humoral substances , such as proinflammatory cytokines , and producing reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) . microglia - producing proinflammatory cytokines include interleukin-1  ( il-1 ) , il-6 , and tumor necrosis factor  ( tnf ) . the activation pattern of microglia in response to invading pathogens is typical of the classical proinflammatory phenotype of macrophages ( m1 polarization ) . for instance , cerebral ischemia and amyloid  ( a ) stimulate il-1 synthesis in microglia through the activation of the inflammasome . they tend to retract their processes and under extreme conditions , assume an amoeboid shape . dead neurons expose phosphatidylserine and other apoptotic  eat - me  signals on their cell surface , which prompts microglia to carry out phagocytosis . such microglia - mediated clearance of unwanted neurons is an important process of proper brain tissue remodeling . with regard to microglia function under the physiological condition , recent in  vivo imaging experiments have uncovered that these resident microglia constantly survey their surrounding microenvironment with their motile processes , which is likely relevant to immune surveillance . however , such motion has been observed even in the absence of offending microorganisms . increasing evidence has revealed that microglia engulf presynaptic and postsynaptic elements by extending their processes to contact dendritic spines , axons , and synapses as observed in the developing brain . microglia - mediated synaptic pruning is dependent on regional neuronal activity . in the visual cortex , visual experience causes microglial processes to change their morphology and motility ; these processes alter distributions of extracellular space , display phagocytic structures , appose synaptic clefts more frequently , and envelope synapse - associated elements more extensively . intriguingly , synaptic pruning occurs preferentially in response to less active inputs , which apparently contributes to activity - dependent synaptic plasticity and learning . microglia express cr3 , the high - affinity receptor for c3 , which is required for the precise execution of synaptic pruning . moreover , microglia are involved in synapse formation by secreting brain - derived neurotrophic factor ( bdnf ) . consistent with these data , genetic inhibition of microglia results in derangement of both synapse formation and elimination of dendritic spines . cortical spreading depression ( csd ) is defined as a slowly propagating ( 25  mm / min ) wave of rapid , near - complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons ( figure 3 ) . by a strict definition , csd refers only to the electrical silence of brain electrical activity following spreading depolarizations . csd is accompanied by secondary changes of cerebral blood flow ( cbf ) , which comprised the following four distinct phases : ( i ) an initial , brief hypoperfusion , ( ii ) a marked , transient hyperemia , ( iii ) a later , smaller hyperemia , and ( iv ) a long - lasting oligemia . experimentally , csd can be elicited by chemical stimulation ( high potassium exposure , atpase inhibitors , endothelin-1 , n - methyl - d - aspartate ( nmda ) receptor agonists ) , pinprick stimulation , and electrical stimulation of the intact cerebral cortex . the most clinically convincing evidence for this came from an mri study by hadjikhani et  al . , which demonstrated the clinico - radiological correlation of visual percept of aura symptoms and propagation of cortical blood oxygenation level - dependent ( bold ) signals in the visual cortex of a patient experiencing a migraine . in addition , csd has been recorded by electrocorticography in patients with ischemic and hemorrhagic stroke and brain trauma . in most cases , however , electrical activity of brain tissue is already compromised before the development of spreading depolarizations . as a result , the characteristic temporal profile of csd , in which rapidly evolving depolarizations of neural cells were followed by suppression of electrical activity , may not necessarily be observed . because of this ,  spreading depolarization  is now regarded as a more precise and preferred term to describe such conditions than csd . spreading depolarization can be elicited by cerebral ischemia or traumatic brain injury experimentally . detailed electrophysiological analysis using hippocampal slices revealed that the apical dendrites of neurons were depolarized earlier than the somata during csd . as compared to action potentials , csd induces a greater magnitude of extracellular potential shift ( typically 1020 millivolts ) which continues for much longer ( at least several minutes ) . the initiation of csd entails a rapid increase in [ k]e from 4  mm to 3060  mm and a rapid decline in [ na]e and [ cl]e from 140  mm to 5070  mm and [ ca]e from 1.52.0  mm to 0.20.8  mm . for example , glutamate , the excitatory amino acid , is released into the extracellular space and binds to nmda - type glutamate receptors to further promote the influx of ca and na . in response to these environmental changes , consistent with these results , a loss - of - function mutation in the gene encoding the 2 isoform of the glial na , k - atpase ( atp1a2 ) causes familial hemiplegic migraine type 2 ( fhm2 ) , which is clinically characterized by prolonged aura episodes . hence , clearance of potassium and glutamate from the extracellular space appears to be a critical event that determines predisposition to csd inductions . activation of the glial na , k - atpase is required to establish neuronal resting membrane potential and correct the csd - induced derangement of the ionic environment . since this process is accompanied by elevated atp consumption . as expected , multiple consecutive csd episodes robustly increase the cerebral metabolic rate of glucose ( cmrglu ) , as compared to a single csd episode . csd - associated depolarizations occur first in the dendrites and then propagate to the soma in cortical neurons . the initiation of csd entails a rapid increase in [ k]e from 4  mm to 3060 mm and a rapid decline in [ na]e from 140  mm to 5070  mm ( black arrows ) . na , k - atpase activity then rectifies the resultant abnormal ion distribution ( red arrows ) . however , csd - associated oligemia may have a negative impact on na , k - atpase activity . csd - associated depolarizations occur first in the dendrites and then propagate to the soma in cortical neurons . the initiation of csd entails a rapid increase in [ k]e from 4  mm to 3060 mm and a rapid decline in [ na]e from 140  mm to 5070  mm ( black arrows ) . na , k - atpase activity then rectifies the resultant abnormal ion distribution ( red arrows ) . however , csd - associated oligemia may have a negative impact on na , k - atpase activity . microglia express voltage - sensitive ion channels , including nav1.1 , kv1.3 , and kv1.5 , and are thought to sense electrical activity pertaining to csd . csd initially elevates extracellular ph , which subsequently leads to a gradual decrease in tissue ph . accumulation of protons may be detected by microglial transient receptor potential cation channel subfamily v , member 1 ( trpv1 ) . a recent study revealed that microglia exhibit increased nmda - dependent inward rectifying potassium conductance after csd , which can be interpreted as a compensatory mechanism for elevated extracellular k concentration . reported that the number of major histocompatibility complex ( mhc ) class ii antigen - positive microglia significantly increased in the rat cerebral cortex between 16 and 24  h after csd . moreover , it has been shown that csd can increase the proliferation and migration of microglia , both of which are well - recognized essential features of the immune response . biochemically , there is evidence that csd induces ros production in microglia that are located in the affected brain tissues . in addition , csd stimulates microglial secretion of il-1 and tnf. collectively , these data suggest that csd can initiate inflammatory activation of microglia . interestingly , ros and tnf have been shown to lower the threshold for csd induction , thus forming a positive feedback mechanism that favors the perpetuation of csd induction . on the other hand , tnf has been shown to reduce csd amplitudes in a dose - dependent manner . in addition , il-1 is also known to attenuate csd amplitudes via the gabaa receptor activity at lower concentrations . however , at a high dose , il-1 did not alter the magnitude of csd amplitudes . since these cytokines promote vascular permeability , as a whole , they are likely to have a deleterious effect on cns tissue in csd pathophysiology . moreover , in hippocampal slice culture studies , selective depletion of microglial cells with clodronate conferred resistance to csd induction . conversely , restoration of microglial cells to previously depleted cultures restored the susceptibility to csd . a similar phenomenon has been reported in an in  vivo neuroimaging study . an in  vivo calcium imaging technique demonstrated that microglia depletion led to a perturbation of neuronal calcium response . hence , it is inferred that a certain microglia - regulated neuronal calcium response may be required for csd occurrence . furthermore , evidence shows that microglial csd - generating activity can be inhibited by insulin - like growth factor-1 ( igf-1 ) treatment and environmental enrichment , the latter of which promotes m2-polarization of microglia ( figure 4 ) . it has been demonstrated that igf-1 antagonizes tnf. these data were obtained in experimental settings where csd was induced in cluster . clinically , successive csd / spreading depolarizations have been demonstrated in patients with stroke and traumatic brain injury . experimental evidence demonstrates that csd / spreading depolarizations contribute to the expansion of infarct volume by several mechanisms . first , a limited supply of atp due to ischemia renders neurons unable to reestablish the resting membrane potential , which may lead up to the occurrence of terminal depolarization . igf-1 treatment and other therapeutic interventions that promote m2-polarization of microglia may ameliorate secondary brain damage resulting from cerebral ischemia . resultant production of tnf and ros enhances the susceptibility of brain tissue to csd development . in addition , microglia polarized to the m2 activation mode by environment enrichment exhibit much less csd susceptibility . the action of environmental enrichment can be mimicked by nasal administration of interleukin-11 ( il-11 ) . resultant production of tnf and ros enhances the susceptibility of brain tissue to csd development . in addition , microglia polarized to the m2 activation mode by environment enrichment exhibit much less csd susceptibility . the action of environmental enrichment can be mimicked by nasal administration of interleukin-11 ( il-11 ) . as mentioned above , multiple csd inductions are likely to activate microglia . consequently , to determine if microglial activation occurs after a single csd event , as seen in the usual migraine aura , we compared morphological changes of microglia in response to single and multiple csd episodes . we found that a single csd induction led to only subtle morphological changes in microglia , whereas multiple csd inductions caused marked enlargement of microglia after 24  h. this morphological change normalized by 72  h after csd inductions . our data suggest that a single episode of csd is  innocuous  to the brain in terms of microglial activation . we did not detect activation of caspase-3 or dna fragmentation detectable by the tunel assay ( unpublished data ) . nedergaard and hansen reported that csd was not associated with neuronal injury in the normal brain . this is also consistent with the traditionally held belief that a single attack of migraine with aura is a benign condition that does not cause any clinically relevant structural brain damage . we sought to clarify the events upstream of the multiple csd - induced microglial enlargements . reported that csd causes the release of hmgb1 ( high - mobility group box 1 ) from neurons via pannexin 1 channels . although hmgb1 is primarily located in the nucleus , upon injurious stimuli , it is released into the extracellular space , where it serves as a damage - associated molecular pattern ( damp ) . we found that neuronal hmgb1 release was dependent on the number of csd inductions such that only multiple csd events were able to cause significant hmgb1 release from neurons ( figure 5 ) . in addition , transcriptional activity of the hmgb1 gene in cortical neurons was enhanced in response to multiple csd events , which may reflect an attempt to replenish the cellular pool of the hmgb1 protein . in general , damp molecules bind to their corresponding receptors , thus transmitting a danger signal to surrounding cells . the ligation of hmgb1 with these receptors initiates intracellular signaling cascades that involve myd88 and irak4 . we found that multiple csd inductions enhanced the transcriptional activity of the tlr2 , tlr4 , myd88 , and irak4 genes in brain tissue . moreover , application of anti - hmgb1 antibody to the cortical surface attenuated the morphological alterations of microglia caused by multiple csd inductions . these data indicate that the hmgb1tlr2/4 axis plays a crucial role in the microglial activation caused by multiple csd events . we observed that the majority of hypertrophic microglia displayed prominent immunoreactivity for cathepsin d , a lysosomal acid hydrolase ( figure 6(a ) ) . nevertheless , we did not observe any apoptotic changes in the brain tissue subjected to multiple csd episodes , making it unlikely that activated microglia are involved in the execution of apoptosis or phagocytosis of dead neurons . an alternative concept is that activated microglial phagocytotic activity is involved in synaptic pruning ( figure 6(b ) ) . a recent in  vivo multiphoton microscopy study disclosed that csd causes dramatic structural alterations of synapses between axons and the dendritic spines of cortical neurons . several minutes after csd , axonal bouton density increased by 20% and bouton size decreased by 25 to 40% compared to the resting state . concomitantly , there was a morphological shift from predominantly stubby spines to thin or mushroom spines after csd , which implies enhancement of synaptic excitability . although single csd events cause abnormalities in synaptic morphology and functionality , these alterations are short - lived and reversible . however , successive csd episodes prevent the recovery of synaptic abnormalities , indicating that the accumulated stress of prior csd events lead to irreversible dendritic injury . thus , it is plausible that activated microglia engage in the repair of damaged synapses through synaptic pruning and formation after multiple csd episodes . figure 5.microglial activation is influenced by the number of csd episodes . a single csd event , as seen in the usual aura of migraine patients , does not cause significant release of hmgb1 from cortical neurons . conversely , clustering of csd , as seen in stroke and brain trauma , induces a robust hmgb1 release from cortical neurons . subsequently , hmgb1 acts on the tlr2/4 on the surface of microglia , which is followed by microglial activation , as demonstrated by their hypertrophic morphological alterations . they may contribute to neuroprotection , for example , through the synaptic repair on the dendritic spines of multiple csd - affected cortical neurons . figure 6.upregulation of cathepsin d , a representative lysosomal acid hydrolase , in activated microglia subjected to multiple csd episodes . ( a ) as compared to cortical microglia in untreated mice ( upper row ) , those subjected to multiple ( five times ) csd inductions ( lower row ) exhibit increased cathepsin d immunoreactivity ( arrow ) . bar  =  20  m . ( b ) microglia activated by multiple csd events may be recruited to damaged dendritic spines for synaptic repair . microglial activation is influenced by the number of csd episodes . a single csd event , as seen in the usual aura of migraine patients , does not cause significant release of hmgb1 from cortical neurons . conversely , clustering of csd , as seen in stroke and brain trauma , induces a robust hmgb1 release from cortical neurons . subsequently , hmgb1 acts on the tlr2/4 on the surface of microglia , which is followed by microglial activation , as demonstrated by their hypertrophic morphological alterations . they may contribute to neuroprotection , for example , through the synaptic repair on the dendritic spines of multiple csd - affected cortical neurons . upregulation of cathepsin d , a representative lysosomal acid hydrolase , in activated microglia subjected to multiple csd episodes . ( a ) as compared to cortical microglia in untreated mice ( upper row ) , those subjected to multiple ( five times ) csd inductions ( lower row ) exhibit increased cathepsin d immunoreactivity ( arrow ) . bar  =  20  m . ( b ) microglia activated by multiple csd events may be recruited to damaged dendritic spines for synaptic repair . although aforementioned data suggest that microglia are required for the occurrence of csd , the mechanisms by which microglia induce csd remain unknown . provided that microglia raise extracellular k concentration before csd occurrence , they would have to follow through in a voltage - independent manner . microglia have been shown to express potassium intermediate / small conductance calcium - activated channels , subfamily n , member 4 ( kcnn4/kca3.1 ) . this voltage - insensitive and calcium / calmodulin - regulated potassium channel has been implicated in lipopolysaccharide- and ischemia - induced brain damage . therefore , the involvement of kcnn4/kca3.1 in the initiation of csd may be worthy of investigation . another unresolved issue is whether multiple csd - induced microglial activation is an adaptive phenomenon or a harmful event to brain tissue . in - depth analysis of the molecules produced in these cells would be required to solve this problem . if microglia are indeed involved in synaptic pruning , the identification of synaptic components , like psd95 , within enlarged microglia should be an important confirmatory finding . lastly , it remains uncertain whether immunological and inflammatory activation of multiple csd - subjected microglia can influence systemic immunological activity . as mentioned above , the recently discovered lymphatic system makes possible immunological communication between the brain parenchyma and the peripheral lymphatic system . in this paradigm , fhm is characterized by prolonged migraine aura , and neuroimaging data support repeated inductions of csd . it has been reported that fhm attacks often cause high fever and in a fewer cases , csf pleocytosis . it may , therefore , be interesting to explore whether a potential crosstalk between activated microglia and the csf / lymphatic system is really relevant to csd pathophysiology . the publication of this article was supported by a jsps kakenhi ( grant number 26460706 to ms ) and a grant from nippon zoki pharmaceutical co. , ltd . the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article .
microglia play a pivotal role in innate immunity in the brain . during development , 
 they mature from myeloerythroid progenitor cells in the yolk sac and colonize the brain to establish a resident population of tissue macrophages . in the postnatal brain , they exert phagocytosis and induce inflammatory response against invading pathogens 
 . microglia also act as guardians of brain homeostasis by surveying the microenvironment using motile processes . 
 cortical spreading depression ( csd ) is a slowly propagating ( 25  mm / min ) wave of rapid , near - complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons . 
 not only has csd been implicated in brain migraine aura , but csd - like events have also been detected in stroke and traumatic injury . 
 csd causes a considerable perturbation of the ionic environment in the brain , which may be readily detected by microglia . 
 although csd is known to activate microglia , the role of microglial activation in csd - related neurological disorders remains poorly understood . in this article , we first provide an overview of microglial development and the multiple functions of microglia . 
 then , we review existing data on the relationship between microglia and csd and discuss the relevance of csd - induced microglial activation in neurological disease .