Direct contactsof Microgliaon myelin sheath and Ranvier'snode in the corpus callosuMin rats

Jingdong Zhang,Xinglong Yang,You Zhou,Howard Fox,Huangui Xiong Department of Pharmacology and Experimental Neuroscience,University of Nebraska Medical Center,Omaha,NE 6898,USA;

2 Department of Clinical and Scienti fi c Training,Af fi liated Hospital to Academy of Military Medicine Sciences,Beijing 100071,China;

3 Center for Biotechnology,University of Nebraska at Lincoln,School of Veterinary Medicine and Biomedical Sciences,Lincoln,NE 68588,USA.

AbstractOver the recent years,it has been found that microglia pseudopodia contact synapses,detect sick ones and prune them,even in adult animals.Myelinated nervesalso carry out plasticity in which microglia removemyelin debrisby phagocytosis.However,it remainsunknown whether Microgliaexplore structureson nerve fi bers,such as Ranvier’s node(RN)or myelin sheath,before they become debris.By double or triple staining RNs or myelin sheathes and microglia in healthy rat corpus callosum,this study unveiled direct contacts of Microglia pseudopodia with RNs and with para-and inter-nodal myelin sheathes,which was then veri fi ed by electron microscopic observations.Our data indicated that microglia also explore unmyelinated nerve fi bers.Furthermore,we used the animals with matured white matter;therefore,Microglia may be actively involved in plasticity of matured white matter tracts as it does for synapse pruning,instead of only passively phagocytize myelin debris.

Keywords:Ranvier’s node,myelin sheath,microglia,contact,matured white matter tract

Microglia,the brain resident cells that can sense pathological tissue alterations,are believed to be quiescentin physiological circumstances[1-2].However,in recent decades,Microglia are found to be more than passive responders.They show high levels of activity with their pseudopodia exploring the surrounding environment,which is known as“dynamic surveillance of brain parenchyma”[3-5].One of the new ly observed physiological functions of Microglia is their involvement in synaptic stripping or pruning[4-5].Under a twophoton live-image Microscope,fi ne microglia pseudopodia are viewed to extend to pre-and post-synaptic structures at a certain rate.Further,direct contacts of labeled Microglia processes onto synapses and/or engulfed post-synaptic protein in Microglia cytoplasMhave been envisioned under the electron microscope[4-6].During development,microglia are responsible for removing the less-used synapses[5,7].

Introduction

This was veri fi ed by comparing Microglia activity in neonatal visual cortex with normal visual experience to that activity in the visual cortex with monocular deprived visual experience[8].In addition,possible participation of microglia in shaping white matter tracts in visual callosuMhas been reported in a siMilarly designed study[9].

Like synapses in the neuropil,nerve fi bers,especially the myelinated nerve fi bers,also undergo plasticity in both developing and matured white matter tracts[10-11].It is rational to contemplate that Microglia may also surveil and maintain the integrity of whitematter tracts.Myelinated nerve fi bers among white matter tracts play a pivotal role in the brain communication between neurons or nuclei in a manner of saltatory conduction[12].The Ranvier’s node(RN)and the internodal myelin sheath are key structures for the generation of saltatory conduction because they concentrate voltage gated sodium(Na+v)channelson the nodeand insulate internode axons with myelin sheath[13-14].The node area is protected by Microvilli of Schwan cells in myelinated peripheral nerve fi bers as this area is extremely important for the rapid transMission of neuronal signals[15-16].In addition,Microvilli may be involved in stabilizing Na+v channels at the node through transinteractionof Microvillidystroglycanwith nodal axolemmal molecules,since selective knockout of Schwan cell’s dystroglycan can result in nodal Na+v channel density reduction and nerve conduction slowdown[16].

However,which structure in the brain white matter tracts can play the same role as microvilli does in peripheral nerves is still not clear.Fine process of astrocyte has been known to contact the RNs in the central nervesystem(CNS),butthe functional pro fi leof these contacts are still equivocal[17-18].A function proposed by some scholars is that astrocyte nodal processesbuffer fluctuation ofionsaround thenode,but the molecular details and the mechanism are still unknown[17-18].Moreover,electron Microscopy studies on contacts of labeled astrocyte processes with RNs in the corpuscallosum(CC)and optic nervehave revealed that a considerable number of RNs therein are still uncovered or only partially covered[17,19],leaving a broad rooMon nodal axolemma for other cellular components to reach.In light of the recent fi ndings aforementioned,we hypothesized that Microglia pseudopodiuMmight extend to both CNS RN and myelin sheath,during developing myelination or post white matter tract maturation.Of particular interest was the role of pseudopodia in adult white matter tracts,as that might be related to some white matter injuries in adulthood.The concentration of Na+v channels at the nodal area[13]allowed us to apply double and triple immuno fluorescent staining of the node and microglia.In combination of labeling para-or inter-nodal domains by pertained markers,we showed in this work the potential contacts of Microglia pseudopodia with the RNs and with para-and inter-nodal myelin sheathes in the rat CC.Finally,electron Microscopy was conducted to verify contactsbetween microglia pseudopodiuMand RN.A ll experimentswere performed using young adult ratsof 45-55 days,when major developing myelination fi nished and white matter had developed into a matured stage[20-21].

Materials and methods

Animals

Thirteen adult Sprague-Daw ley rats(45-55 daysold;175-250 g;7 male and 6 female),purchased froMCharles River Laboratories(Wilmington,MA),were used in this study.Samples froMten rats(5 male,5 female)underwent double and triple labeling,and then were observed under confocal Microscopy.Samples froMthree animals(2 male,1 female)were used for electron Microscopy.All experimental protocols and animal care were carried out in accordance with the National Institutes of Health Guide for the Care ofLaboratory Animals in Research and approved by the Institutional Animal Careand Use Committeeof the University of Nebraska Medical Center.All effortswere made to Minimize animal suffering and the number of animals used in this study.

Immuno fluorescent staining for confocal Microscopy

Double labeling of myelin sheath and microglia

Animals were euthanized with iso flurane and transcardially perfused with saline followed by 4%paraformaldehyde in phosphate buffer(PB;0.1 mol/L,pH 7.2-7.4).The brain containing the CC was cut into 10-μMcoronal or sagittal frozen sections and directly mounted on plus-coated slides.The sections with the CC were incubated with either rabbit anti-myelin basic protein(MBP;1:200-1:500,Abcam,Cambridge,MA)plus goat anti-Ionized calciuMbinding adaptor-1(Iba-1)or rat anti-MBP(1:100,Millipore,Temecula CA)with rabbit anti-Iba-1(1:500,Wako USA,Richmond,VA)overnight at rooMtemperature(RT).Alex Flour conjugated secondary antibodies(all diluted as 1:200-1:500;Molecular Probes,Eugene,OR)against primary antibodies were applied to display fluorescent labeling.Control staining for different antigens was carried out without primary antibody.

Double labeling of RN and microglia

Brain tissues were processed in the same way as aforementioned.The coronal sections were incubated with either rabbit anti-pan Voltage-Gated sodiuMchannels(Pan Na+v;1:200,Alomone,Jerusalem,Israel)with goat anti-Iba-1(1:200-1:500,Abcam),or mouse anti-Na+v subunit 1.6(Clone K87A/10;1:100-1:200,UC Davis/NINDS/NIH NeuroMab,Davis CA)with rabbit anti-Iba-1(1:300-1:500,Wako USA)for revealing RN and microglia with rami fi ed pseudopodia.Alex Flour conjugated secondary antibodies(all diluted as 1:200-1:500;Molecular Probes)against primary antibodies were applied to display fluorescent labeling.

Triple labeling of RN,paranodal/internodal myelin sheath and microglia

Brain tissues were processed in the same way as aforementioned except that 7.5%saturated picric acid(75%)was added to the fi xative.The CC sections were immunostained with a cocktail of rabbit anti-MBP(1:300,Abcam),mouseanti-Na+v 1.6(1:200,UCDavis NeuroMeb)and goat anti-Iba-1(1:200,Abcam).Then,A lexa Flour 488 conjugated donkey anti-rabbit,Alexa Flour 568 attached donkey anti-mouseand ByLight 405 linked donkey anti goat(1:200;kindly provided by Microscopy Facility at University of Nebraska at Lincoln)wereapplied to visualize the structures labeled by primary antibodies.

Triple labeling of RN,paranodal domain and microglia

Brain tissues were processed in the same way as aforementioned.Ashasbeen known,a characteristic of the RN in myelinated nerve fi ber is that the node is always nested in-between two paranodal domains that are contactin-associated protein 1(Caspr1)positive[22].Thus,antibody against Caspr1 was added in triple labeling of RNs,paranodal domains and Microglia.Then,the sections were incubated in cockatiel of rabbit anti-pan Na+v(1:300,A lomone),mouse anti-Caspr1(1:200,UC Davis NeuroMeb)and goat anti-Iba-1(1:200,Abcam)overnight at RT.Next,Alexa Flour 568 conjugated donkey anti-rabbit,fluorescein conjugated donkey anti-mouseand ByLight405 conjugated donkey anti goat(provided by UNL)were applied to reveal the node,paranodal domain and Microglia.

Preparation of tissues for electron Microscopy

Immunohistochemistry staining

Animals were euthanized by iso flurane and transcardially perfused with saline followed by PB,asmentioned above,containing 2%paraformaldehyde and 0.5%glutaraldehyde with 7.5%saturated picric acid(75%).The front brain containing the CC was extracted and cut into 50-μMcoronal sectionsby a vibratome and saved in PB.Thesectionswere immunohistochemically stained as previously described[23].

Osmi fi cation and plate embedding

The sections then underwent osMi fi cation and plate embedding as described previously[23].The labeled microglia in the embedded sections were identi fi ed under light Microscope(Fig.4A-B),and then trimmed off for ultrathin sectioning.Ultrathin sections were cut with Leica EMUC7 Ultracut Microtome,stained with lead citrate and observed with Hitachi H7500 TEM(at Core Research facility,Center of Biotechnology,University of Nebraska at Lincoln).And the work continued afterward at Computer Imaging and Microscopy Core Facility,A f fi liated Hospital of Academy of Military Medicine Sciences,Beijing,China.Ultrathin sections were cut with LKB ultra-microtome,stained with lead citrateand observed with FEITecnai G2Spirit transMission electron Microscope.

I maging acquisition and processing

Digital images of fluorescent labeling were collected using Nikon A1 confocal system(Japan)equipped with Nikon 90iupright fluorescencemicroscopeand BioRad Laser Sharp 2000 imaging program(Digital BioRad Center,Pleasanton,CA,USA).A lexa Fluor 568 labeling was viewed through 561 excitation laser line with 10 nMresolution of spectra.Alexa Fluor 488 and ByLight 405 were viewed through 488 and 405 excitation laser line,also through 10 nMresolution spectra.Confocal images were captured with×20 and×40 objective at iris of 2.0-2.5 in box size of 1,024×1,024.All Z-scan was set up at 1μMlayer of laser scan step and saved as“avi”video fi les and the clearestdoubleor triple labeling imagewasselected and converted to Photoshop 7.0.1(Adobe,CA,USA)through Bio-Rad Plug-In software and stored as“tiff”fi le at 1,024×1,024 pixels.The electron microscopy microphotographs presented here were taken with FEI Tecnai G2Spirit electron Microscope.

Results

Double labeling of Myelin sheath and Microglia

Fig.1 Direct contacts of Microglia pseudopodia with inter-,para-nodal Myelin sheath and probably Ranvier'snode(RN).A-C:Iba-1 positivemicrogliapseudopodium(arrowheads)directly contactsontomyelinbasic protein(MBP)labeled myelin sheath(opened arrowheads)in the corpus callosum(CC,paired arrowheads).D:contacts(paired arrowheads)between microglia pseudopodia and myelin sheathes viewed in crosssection.E-H:a singlemicrogliawith itspseudopodia(arrowheads)in closeapposition upon a RN(opened arrows)and itspara-and internodal myelin sheath(opened arrowheads).Merged panel shows Iba-1 positive processcontactson a MBP labeled internodal(paired down-point arrowheads)myelin sheath.Meanwhile,the other pseudopodiuMseems to contact with a RN and its paranodal apparatus(upper-point opened arrow and arrowhead,plusa left-point arrowhead).This image isconstructed froM3 scanned layers to show an entiresinglemicrogliawith both soma and processes.

Using MBP to label myelin sheath and Iba-1 to mark microglia,weobserved in both coronal(Fig.1A-C)and sagittal sections(Fig.1B)that Microglia pseudopodia(arrowheads)explore the inter-nodal myelin sheath(opened arrowheads)frequently(paired arrowheadsand opened arrowheads in Fig.1C and D).A ll control staining without primary antibody resulted no labeling.

Fig.4 Ultrastructural view of Microglia pseudopodia contacts on RNs.A and B:Iba-1 immunohistochemically labeled,osmicated and plate-embedded sections observed under bright-fi eld microscope,on which the labeled microglia and pseudopodia(arrowheads)could be seen clearly.Scalebar=15µm.C and D:ultrathin sections cut froMA or B,exhibiting ends of microglia pseudopodia(opened arrowheads)direct contact on naked axolemma of RN area.Apposition of microglia pseudopodiuMupon paranodal myelin sheath is also viewed(D),but there seems to be a tiny gap(about 5-10 nm)between them.A ligned arrows point towards spiral loops of paranodal apparatus.

Trip le labeling of RN,para-/inter-nodal Myelin sheath and Microglia

Unlike Caspr1,which isexpressed only on paranodal domains,MBPcan mark both paranodal and internodal myelin sheath.We encountered a paranode-RNmicroglia complex,characterized by MBP positive paranodal apparatus immediate abut against a Na+v 1.6 labeled RN(opened arrows)that was touched by a microglia process marked by Iba-1(Fig.1E-H).Fig.1H showed that asingleMicrogliawith itspseudopodia(arrowhead)appeared to explore a RN and its nearby paranodal(a pair of opened arrows and arrowhead pointing up),and a continued internodal(a pair of arrowheads and opened arrowhead pointing down)myelin sheath simultaneously.

Double labeling of RN and Microglia

Under laser scan of 1-µMlayer of the CC section,it was explicitly observed that microglia pseudopodia(arrowheads)immediately contact the possible RNs or clusters of Na+v channel(opened arrows in Fig.2).However,in Fig.2A-C,a nerve fi ber lining and connecting with the labeled node(opened arrow)was also stained by anti-Na+v antibody,inferring thisnerve fi ber was an unmyelinated fi ber,since the Na+v channels appears to evenly distribute along the unmyelinated nerve fi bers in addition to aggregating in the node-like area[24].Besides,clusters of Na+v channels on unmyelinated nerve fi ber is suggested by a recent fi nding that Na+v channels piled fragmentally upon lipid rafts on axolemma of node like area in unmyelinated fi bers[25].

Trip le labeling of RN,paranodal domains and Microglia

As the RN in myelinated nerve fi ber is always situated in-between of paranodal domains that express Caspr1[22],we attempted to show the node and paranodal domain simultaneously to clarify whether Microglia pseudopodia indeed explore the RN in myelinated nerve fi bers.Then,by means of triple labeling,we unveiled that Microglia pseudopodia(arrowheads)stretched to contact RNs labeled by Na+v channels antibody(opened arrows)and/or paranodal domains marked by Caspr 1(arrows in Fig.3A).In somecases,the Iba-1 positivemicrogliapseudopodia are closely apposite upon both RN and paranodal domains simultaneously(Fig.3B,arrowhead,opened and fi lled arrow together in Merged),although we did not know whether these contacts were instant or constant.

Electron Microscopic observations

With plate-embedding,labeled Microglia and their pseudopodia could be visualized under conventional light Microscope(Fig.4A and B,arrowheads).The tissue block containing labeled Microglia(Fig.4A,B)was marked and extracted froMplate-embedded sections under anatoMical Microscope for further preparation of ultrathin section.Ultrathin sections were cut along with longitudinal axis of nerve fi bers in the CC asshown in Fig.4C and D.Endsof microglia pseudopodia(opened arrowheads in C and D)wereseen to contact axolemma on RNs.These contacts appeared between the naked axolemma on the node and the ends of pseudopodiawithout any gap(C and D).The contact between paranodal myelin sheath and Microglia pseudopodia was also observed(Fig.4D).That paranodal domain was termed as axoglial apparatus ultrastructurally characterized by spiral loops formed by paranodal myelin sheath(pointed by aligned small arrows in C and D).There appeared to be a tiny gap(about 5-10 nm)between plasMmembrane of microglia and paranodal myelin sheath;however,the other end of pseudopodia seemed to have direct contact with the RN and another two myelin sheathes of myelinated fi bers(Fig.4D,opened arrowheads).

Discussion

To the best of our know ledge,the fi ndings presented here,demonstrate for the fi rst time that Microglia pseudopodia directly contact with RN and paranodal or internodal myelin sheath in healthy young adult CNS white matter.It has been reported that in rodents,the maturation of white matter tracts in the CC is between 40-45 postnatal days[20-21]when about 30%of axons have been myelinated and nerve fi ber compound action potentials become stable[20-21].Therefore,those direct contacts of microglia pseudopodia upon RNs and myelin sheath are probably associated with plasticity of myelinated nerve fi bers[10-11]in adulthood rather than developing myelination during peak myelination.On the other hand,there may be contacts between microglia pseudopodia and clusters of Na+v channels on unmyelinated nerve fi bers[24-26],becauseabout 70%of nerve fi bers in matured CC are unmyelinated fi bers[20-21].Considering that microglia dynaMically survey thesurrounding environment in grey matter[3],it isnot surprising that microgliawould exploreboth RNs in myelinated fi bers and node-like regions on unmyelinated nerves[25-26],playing asiMilar roleasin thegrey matter[3].However,whether Microglia play any special role for myelinated nerve fi bers by close contact with RNs is not clear,but that should not be overlooked.

Ample evidences have shown that CNSwhite matter tracts persist on myelination and/or remyelination after the developing myelination period,and indeed,are likely to do so throughout the lifespan[10,27].For example,further myelinating unmyelinated nerve fi bers and/or remodeling or replacing established myelin sheath continue when organism’s task performance changes,i.e.a new skill-related function is established or via theaging process[10,27].Physiological remodeling or replacement of myelin sheathes should not compromise their normal function,but old myelin sheaths need to be removed before new oligodendrocyte processes begin to w rap axons[28-29].Thus,the timew indow froMthe identi fi cation and breakdown of sick myelin,to the rebuilding of new sheath could not be too long.The direct contacts of Microglia pseudopodia onto myelin sheath might provide more precise data about the condition of myelin sheathes.Microglia can actively removemyelin debrisduring demyelination;however,it remains unknown whether the Microglia can recognize and remove sick or degrading myelin sheathes before they break into debris.It is also unclear whether those microglia pseudopodia are periodically or constantly contacting the node[3],which could be further studied under two-photon live-image microscopy.

Furthermore,the RN isa key structure to accomplish saltatory conduction as aforementioned.It is rational to presume if thenerve fi bersmalfunction,activitiesof the RNs w ill be altered and vice versa.Thus,the best approach to monitor physiological states of a myelinated nerve fi ber is to monitor its RN activity.The ratio ofions around a RN is absolutely important for the generation of saltatory conduction[30];whereas,the concentration fluctuation of some key cations re flects the RNs functional states[12-13].It has been demonstrated that microglia sense density changes of some cations;for instance,the increment of extracellular potassium (K+)concentration can elicit microglia activation[31].A few of studies uncovered that a reduction of extracellular Ca2+concentration around neuronal somataor dendritesevoked theconvergenceof Microglia pseudopodia which then moved toward dendrites[32-33].This fi nding suggests microglia may beable to sense Ca2+surrounding the RNs.Commonly,Ca2+around RN is stable and Ca2+in flux occurs only when axolemma or paranodal apparatus are disrupted[34-35].However,physiological Ca2+in flux through the RN axolemma was found recently in cases where the axons discharged at a remarkably high frequency[36-37].Hence,monitoring the K+and/or Ca2+concentration around RNsMightbeoneof theduties for thoseMicroglia to have closeapposition or contactupon the RNs.

If Microglia monitor and maintain the function of RNsby meansof close apposition of their pseudopodia upon RNs,it is intriguing for us to know what w ill happen when thoseprocessesareshortened or shrunken upon Microgliaactivation.Our recentstudy showed that the activation of microglia per se would induce white matter tract function changes,especially declination of axon compound action potentials[38].The length and thickness of Microglia pseudopodia dramatically changed froMdelicate soma with thin raMi fi ed pseudopodia to amoeboid cells with short hyper-rami fi ed morphology when microglia were activated.The number of contacts between Microglia and RNs or myelin sheath might change in this circumstance.More interestingly,the extent of declination of compound action potentials is correlated with the states of microglia activation[38].However,the functional signi fi cance of those contacts of microglia pseudopodia with RNs and para-or internodal myelin sheathes is still an enigma.Nonetheless,this fi nding w ill open a new avenue for exploration into theeffectof microglia,in addition to overtphagocytosis and/or invisible cytokines secretion,on white matter tract integrity.

Acknow ledgments

We warMly thank Dr.Reker Ashlie for her critical reading and scienti fi c commentson thismanuscript.We greatly thank colleagues in 307 Hospital,Computer Imaging and Microscopy Core Facility for their helps.This study was supported by R01 NS063878 and P30 MH062261(to H.X.,H.S.F.).