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Neuroscience 182 (2011) 1–10
DIFFERENTIAL INVOLVEMENT OF PERINEURONAL ASTROCYTESAND MICROGLIA IN SYNAPTIC STRIPPING AFTER HYPOGLOSSAL
AXOTOMYJ. YAMADA,a,b H. NAKANISHIa* AND S. JINNOb*aKyushu University, Laboratory of Aging Science and Pharmacology,
aculty of Dental Sciences, Fukuoka 812-8582, JapanbKyushu University, Department of Anatomy and Neurobiology, Grad-ate School of Medical Sciences, Fukuoka 812-8582, Japan
Abstract—Following peripheral axotomy, the presynaptic ter-minals are removed from lesioned neurons, that is synapticstripping. To elucidate involvement of astrocytes and micro-glia in synaptic stripping, we herein examined the motoneu-ron perineuronal circumference after hypoglossal nerve tran-section. As reported previously, axotomy-induced slow celldeath occurred in C57BL/6 mice but not in Wistar rats. Syn-aptophysin labeling in the hypoglossal nucleus exhibited aminor reduction in both species after axotomy. Slice patchrecording showed that the mean frequency of miniature post-synaptic currents in axotomized motoneurons was signifi-cantly lower in rats than in mice. We then estimated therelative coverage of motoneuron perineuronal circumferenceby line profile analysis. In the synaptic environment, axo-tomy-induced intrusion of astrocytic processes was signifi-cantly more extensive in rats than in mice, whereas microglialintrusion into the synaptic space was significantly more se-vere in mice than in rats. Interestingly, in the extrasynapticenvironment, the prevalence of contact between astrocyticprocesses and lesioned motoneurons was significantly in-creased in rats, while no significant axotomy-induced altera-tions in astrocytic contact were observed in mice. Thesefindings indicate that astrocytic, but not microglial, reactionmay primarily mediate some anti-apoptotic effects throughsynaptic stripping after hypoglossal nerve axotomy. In addi-tion, enlargement of astrocytic processes in the extrasynap-tic environment may also be involved in neuronal protectionvia the increased uptake of excessive glutamate. © 2011IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: astrocyte, axotomy, microglia, motoneurondeath, synapse.
It has been suggested that astrocytes and microglia playpivotal roles in maintaining brain homeostasis (Halassaand Haydon, 2010; Graeber and Streit, 2010). Both astro-cytes and microglia secrete myriad of bioactive sub-
*Corresponding author. Tel: �81-92-642-6413 or �81-92-642-6053;fax: �81-92-642-6415 or �81-92-642-6059.E-mail address: [email protected] (H. Nakanishi) or [email protected] (S. Jinno).Abbreviations: ANOVA, analysis of variance; APV, 2-amino-5-phos-honovalerate; CLSM, confocal laser scanning microscope; DNQX,,7-dinitroquinoxaline-2,3-dione; GFAP, glial fibrillary acidic protein;LAST, glutamate-aspartate transporter; Iba1, ionized calcium bind-
ng adaptor protein 1; mEPSC, miniature excitatory postsynaptic cur-
ents; mIPSC, miniature inhibitory postsynaptic currents; NMDA, N-ethyl D-aspartate.0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All righdoi:10.1016/j.neuroscience.2011.03.030
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stances, and regulate the activity and functions of neurons.(Banati et al., 1993; Raivich et al., 1999; Schwartz et al.,2006). For instance, astrocytes are involved in metabolicsupport of neurons (Haydon, 2001; Horner and Palmer,2003). Microglial cells continuously survey the local mi-croenvironment and work as housekeepers in the brain(Davalos et al., 2005; Nimmerjahn et al., 2005). In additionto these beneficial functions, glial cells also mediate detri-mental effects on neurons under specific pathological con-ditions. Activated astrocytes impede neuronal survival andregeneration by forming scar tissue and synthesizing neu-rotoxic molecules, that is, nitric oxide and arachidonic acidmetabolites (Marchetti and Abbracchio, 2005). Reactivemicroglial cells kill and phagocytose target neuronal cellsfollowing excessive and uncontrolled adverse stimulation(van Rossum and Hanisch, 2004; Cardona et al., 2006).Altogether, the evidence to date suggests that glial cellsmay act as a double-edged sword with neuroprotectivefeatures predominating in the healthy nervous system andneurodestructive properties in various pathological condi-tions (Biber et al., 2007).
Experimental axotomy such as the hypoglossal nervetransection paradigm in rodents allows systematic anddetailed study of neuronal cell death and glial reactions(Koliatsos and Price, 1996). Seminal work by Blinzingerand Kreutzberg (1968) has demonstrated that perineuro-nal microglia actively engage in the displacement of syn-aptic boutons from the surface of regenerating motoneu-rons. This phenomenon is referred to as synaptic stripping,and is implicated in the survival of axotomized motoneu-rons (Graeber et al., 1993). Experiments using axotomymodels provide important clues to better understandingcellular/molecular events associated with neuronal survivaland with apoptotic cell death (Kiryu-Seo et al., 2006; Du-puis et al., 2008).
In this study, we focused on perineuronal glial reac-tions in Wistar rats and C57BL/6 mice after hypoglossalaxotomy. Because it is known that axotomy induces apo-ptotic cell death in mice but not in rats (Kiryu-Seo et al.,2006), we considered that comparative analysis might givesome key to understanding the mechanisms underlyingcell fate decision. Particularly, we investigated the axo-tomy-induced changes in the astrocytic and microglial cov-erage of motoneuron synaptic circumference in mice andrats. The data presented here indicate that astrocytic butnot microglial reaction may contribute to motoneuron pro-tection via synaptic stripping. In addition, enlargement of
astrocytic processes in the extrasynaptic environment mayts reserved.
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also be involved in neuronal survival through the increaseof uptake of excessive glutamate.
EXPERIMENTAL PROCEDURES
Animals
Adult male C57BL/6N mice (22–25 g body weight, 8–11 weeksold) and adult male Wistar rats (250–350 g body weight, 8–11weeks old) were used for histological analysis. Juvenile C57BL/6Nmice (20–30 days old) and juvenile Wistar rats (20–30 days old)were also used for electrophysiological analysis. Every procedurewas approved by the Committee of the Ethics on Animal Experi-ment in the Graduate School of Dental Sciences, Kyushu Univer-sity, and was conducted in accordance with the National Instituteof Health Guide for the Care and Use of Laboratory Animals (NIHPublications No. 80-23, Rev. 1996). All efforts were made tominimize the number of animals used and their suffering.
Hypoglossal axotomy
Animals were anaesthetized with pentobarbital (45 mg/kg bodyweight, i.p.; Dainippon Sumitomo Pharma, Osaka, Japan), and werepositioned supine. The right hypoglossal nerve was carefully ex-posed under the digastric muscle. At the proximal side of the hypo-glossal nerve bifurcation, the nerve was transected with a pair ofscissors and the nerve stumps were placed not to touch with eachother. Samples from sham control animals were taken from animalssubjected to identical surgical exposure without axotomy.
Tissue processing
Animals were deeply anesthetized with sodium pentobarbital (100mg/kg body weight, i.p.) and perfused transcardially with phos-phate buffered saline (PBS, pH 7.4) followed by fixative: a mixtureof 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde in 0.1 Mphosphate buffer (PB) for immunostaining. The brains were left insitu for 1–3 h at room temperature, and then were cut transverselyinto 50-�m-thick sections on a vibrating microtome (VT1000S;Leica Microsystems, Wetzlar, Germany).
Immunofluorescence procedure
Sections were incubated with 1.0% bovine serum albumin in PBScontaining 0.3% Triton-X 100 and 0.05% sodium azide for 3 h at 4 °C.Then, they were incubated for 5 days at 4 °C with following primaryantibodies raised in different species; rabbit polyclonal anti-ionizedcalcium binding adaptor protein 1 (Iba1) antibody (1:10,000; WakoPure Chemical Industries, Osaka, Japan), goat polyclonal anti-Olig2antibody (1:25,000; R&D systems, Minneapolis, MN, USA), guineapig polyclonal anti-glutamate-aspartate transporter (GLAST) anti-body (1:5,000; Frontier Science, Sapporo, Japan), mouse monoclo-nal anti-glial fibrillary acidic protein (GFAP) antibody (1:10,000; Dako,Carpinteria, CA, USA) and mouse monoclonal anti-synaptophysinantibody (1:25,000; Sigma-Aldrich, St. Louis, MO, USA). They werethen incubated with a mixture of 7-amino-4-methyl coumarin-3-aceticacid (AMCA)-conjugated donkey anti-rabbit IgG antibody (1:100;Jackson ImmunoResearch Laboratories, West Grove, PA, USA),fluorescein isothiocyanate (FITC)-conjugated donkey anti-guinea pigIgG antibody (1:250; Jackson ImmunoResearch Laboratories), Cy3-conjugated donkey anti-mouse IgG antibody (1:250; Jackson Immu-noResearch Laboratories), Cy5-conjugated donkey anti-goat IgG an-tibody (1:250; Jackson ImmunoResearch Laboratories), and DNA/RNA markers TOTO-3 (1:2,000; Invitrogen, Karlsruhe, Germany) orYOYO-1 (1:5,000; Invitrogen) for 3 h. For fluorescent Nissl staining,selected sections were incubated for 3 h with Neurotrace 500/525(Invitrogen). The sections were then mounted in Vectashield (VectorLaboratories, Peterborough, UK) and examined under a confocal
laser scanning microscope (CLSM; TCS-SP2; Leica Microsystems). 4Estimation of cell survival after axotomy in rats andmice
Twelve sections from three animals were examined from eachgroup (a total of six mice and six rats). Nissl-stained hypoglossalmotoneurons in injured and control sides were counted separatelyby using the Cell Counter plugin of ImageJ 1.38 (NIMH; Bethesda,MD, USA). The survival ratios were calculated by dividing thenumbers of motoneurons in the lesioned side by those in thecontrol side.
Estimation of neuronal atrophy in mice
Six sections from three mice were examined from axotomized andsham-operated groups, respectively (a total of six mice). YOYO-1(Invitrogen) was used for morphometric measurements of cellbody size. Vascular endothelial cells were excluded from theanalysis. The analyses were carried out by using ImageJ 1.38(NIMH). The maximum diameters of individual cell bodies weremeasured on the optical section which showed the maximumarea. The peaks of histograms for soma size were determined byfitting of Gaussian function by using the statistical environment Rversion 2.11 (http://www.R-project.org). Statistical significances ofhistogram distributions were examined with Kolmogorov–Smirnovtest.
Electrophysiological analysis of synaptictransmission
Seven to twelve cells from five animals were examined from eachgroup (a total of 10 mice and 10 rats). Animals were killed bydecapitation. Coronal brain slices (250-�m-thick) containing theypoglossal nucleus were cut on a vibrating microtome (VT1000S;eica Microsystems). Miniature excitatory postsynaptic currentsmEPSCs) and miniature inhibitory postsynaptic currents (mIP-Cs) were recorded with an Axopatch 200B patch-clamp amplifier
Axon instruments, Foster city, CA, USA). The current signalsere filtered at 3 kHz and digitized at 20 kHz. All electrophysiolog-
cal recordings were performed at 25 °C. Patch pipettes (4–7 M�)ere filled with an internal solution, as described previously (Yamadat al., 2008). mEPSCs were recorded in the presence of 10 �M
bicuculline methiodide (Sigma-Aldrich) and 1 �M strychnine (Sig-a-Aldrich). mIPSCs were recorded in the presence of 20 �M
6,7-dinitroquinoxaline-2,3-dione (DNQX; Sigma-Aldrich) and 50�M 2-amino-5-phosphonovalerate (APV; Sigma-Aldrich). The re-orded membrane current was analyzed using Mini Analysis Pro-ram version 6 (Synaptosoft, Fort Lee, NJ, USA). Each event ofiniature synaptic currents was detected, and its amplitude and
iming of occurrence were measured. All detected events wereonitored and checked visually to ensure that all detected eventsad typical PSC waveforms.
Quantification of synaptophysin-positive area
Eight sections from four animals were selected from each group (atotal of eight mice and eight rats). The area of synaptophysin-positive puncta was estimated from single representative opticalsections in each stack using ImageJ 1.38 (NIMH). The lowerthreshold value was set at 33% of maximum intensity of the grayscale, and the puncta smaller than 0.1 �m2 were eliminated fromhe results considering the resolution limit of CLSM.
Line profile analysis of cellular componentssurrounding somata
Twelve sections from three animals were selected from eachgroup (a total of six mice and six rats). Lines perpendicular to thesoma plasma membrane were made at 3 �m intervals (see Fig.
). Then, the pixel intensities within 2 �m from the cell surface
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J. Yamada et al. / Neuroscience 182 (2011) 1–10 3
were measured using the image analysis software ImageJ 1.38(NIMH). The maximum pixel intensity values of each channel arearbitrarily fixed at 100%. TOTO-3 signals less than 20% werejudged as outside the soma. Line profiles including synaptophy-sin-positive puncta were defined to represent synaptic environ-ment, and those avoiding synaptophysin-positive puncta wereclassified as extrasynaptic environment. Approximately 200 lineswere examined in each animal.
Data analysis and illustration preparations
The statistical analysis was conducted with Welch’s t-test or one-way analysis of variance (ANOVA) with post hoc Tukey’s test byKareida Graph 3.6J (Hulinks, Tokyo, Japan). Significant differencewas considered to occur when a P-value of �0.05 was obtained.Three-dimensional (3D) illustrations were made using Recon-struct 1.1 (http://synapses.clm.utexas.edu). Selected imageswere processed by Adobe Photoshop CS (Adobe Systems, Moun-tain View, CA, USA). Only brightness and contrast were adjustedfor the whole frame, and no part of a frame was enhanced ormodified in any way.
RESULTS
Species differences in motoneuron survival afteraxotomy between rats and mice
At the beginning of the experiment, we examined the spe-cies differences in survival of hypoglossal motoneuronsafter axotomy between rats and mice. There were nosignificant reduction in hypoglossal motoneuron numbersin rats until 28 days after nerve transection (D28; Fig. 1A,
). Even 56 days after the operation, the survival ratio ofat motoneurons was 98% (not shown). By contrast, thereas a progressive decline in numbers of hypoglossal mo-
oneurons in mice (Fig. 1B, D). These results indicate thatypoglossal axotomy induces slow cell death in mice butot in rats, as reported previously (Kiryu-Seo et al., 2006).
To investigate the possible occurrence of motoneurontrophy following axotomy in mice, we estimated the sizeistribution of neuronal cell bodies in the mouse hypoglos-al nucleus. Here we discriminated neurons from glial cellssing three immunocytochemical markers for the majorypes of glial cells in the brain: Iba1 was used to detecticroglia, GFAP was used to detect astrocytes, and Olig2as used to detect oligodendrocyte lineage cells (Fig.C1–C5). The diameter of individual cells was examinedsing cytoplasmic staining with YOYO-1. The distributionsf soma size were significantly different between glialarker-negative putative neurons and glial marker-posi-
ive cells in the mouse hypoglossal nucleus after axotomyFig. 1E; Kolmogorov–Smirnov test, P�0.0001). On thether hand, there was no significant size difference in glialarker-negative putative neuronal cells between sham-oper-ted mice and axotomized mice (Fig. 1E; Kolmogorov–Smir-ov test, P�0.9436). These results indicate that extensiveeuronal atrophy does not occur in the mouse hypoglossalucleus after axotomy.
uppression of synaptic transmission after axotomy
o estimate the influence of synaptic inputs on survival ofnjured motoneurons, we first examined the patterns of
xpression of presynaptic terminal marker, synaptophysin, tn the hypoglossal nucleus (Fig. 2A–C). In both species,he area of synaptophysin-positive puncta in the hypoglos-al nucleus showed a minor decline on D7 (30.8% reduc-ion in rats and 10.6% reduction in mice; Fig. 2D). Atatistically significant decrease was detected in ratsP�0.05), and a trend toward decreased synaptophysin-ositive area was seen in mice (P�0.11).
We then examined the possible alteration in synapticransmission to hypoglossal motoneurons following axo-omy by patch clamp recording. The frequency of mEPSCshowed significant axotomy-induced suppression in bothpecies on D7 (Fig. 2E, G). The rates of decrease inrequency of mEPSCs were significantly larger in rats95% decrease) than in mice (60% decrease). By contrast,he amplitude of mEPSCs showed no significant changesfter axotomy (Fig. 2H). Similar trends were observed inuppression of mIPSCs after axotomy (Fig. 2F, I, J).
lterations in coverage patterns of hypoglossalotoneuron perineuronal circumference after
xotomy
uadruple labeling images for Iba1, GLAST, synapto-hysin, and TOTO-3 were obtained in the hypoglossalucleus (Fig. 3A–L), and the pixel intensities within 2 �m
from the cell surface were measured (Fig. 4A, B). 3Dreconstructions demonstrated the representative glialand synaptic coverage of hypoglossal motoneurons(Fig. 3M–O). In the present study, the lines crossingperineuronal (within 2 �m) synaptophysin-positive
uncta were categorized to represent the synaptic envi-onment as follows: S-type, the profile closest to theoma was a synaptophysin-positive puncta (Fig. 3P);-type, the closest profile was a GLAST-positive astro-ytic process (Fig. 3Q); M-type, the closest profile wasn Iba1-positive microglial process (Fig. 3R).
We first quantitatively examined relative synapticoverage of hypoglossal motoneurons (Fig. 5A). Therevalence of synaptophysin-crossing lines exhibitedignificant axotomy-induced decline from D3 to D28 inats, but only on D14 in mice (Fig. 5B). There were noignificant species differences in the prevalence of aynaptic environment. A significant decline in S-typeas observed after axotomy in both species (D7, 78.8%
eduction in rats and 52.8% reduction in mice; Fig. 5C).he percentages of S-type were significantly higher inice than in rats. A significant increase in A-type wasbserved after axotomy in both species (D7, 276.3%
ncrease in rats and 198.5% increase in mice; Fig. 5D).he fractions of A-type were significantly higher in rats
han in mice from D3 to D14, but no significant differenceas detected on D28. The fractions of M-type alsohowed significant increase in both species (D7, 423%
ncrease in rats and 1880% increase in mice; Fig. 5E).he percentages of M-type were significantly higher inice than in rats from D3 to D14. Relative ratios of-type to M-type showed transient decline in both spe-ies (Fig. 5F). The ratios were significantly higher in rats
han in mice from D3 to D28.
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J. Yamada et al. / Neuroscience 182 (2011) 1–104
We then examined the lines avoiding synaptophysin-ositive puncta, which were defined as the representa-ion of the extrasynaptic environment (Fig. 6A). Theatterns of coverage of somata in the extrasynapticnvironment were classified into three categories (Fig.A): EA-type, the profile closest to the soma was anstrocytic process; EM-type, the closest profile was aicroglial process; unclassified, no apparent profilesere detected. The prevalence of extrasynaptic environ-ent defined by synaptophysin-avoiding lines showed a
ignificant increase from D3 to D28 in rats, but only on14 in mice (Fig. 6B). There were no significant species
Fig. 1. Species differences in motoneuron survival after hypoglossal a(B) hypoglossal nuclei 28 d after the axotomy (D28). The dashed lineYOYO-1 (C1), Iba1 (C2), GFAP (C3), Olig2 (C4) and merged (C5) in theutline of glial marker-negative putative neuronal cells. (D) Survival raE) Histograms showing the soma size distributions of glial marker-posypoglossal nuclei (Axo; red), and glial marker-negative putative neuroith Gaussian functions. Scale bars in (A)�130 �m, (B)�100 �m, (etween rats and mice: * P�0.05, ** P�0.01. For interpretation of the rf this article.
ifferences in the fraction of extrasynaptic environment.
he fractions of unclassified type were very low in bothpecies (Fig. 6C). A significant increase in EA-type wasbserved after axotomy in rats (D7, 125.5% increase),ut no alterations were observed in mice (Fig. 6D). The
ractions of EA-type were significantly higher in rats thann mice. A significant increase in EM-type after axotomyas seen in both species (D7, 2750% increase in ratsnd 15750% increase in mice; Fig. 6E). The fractions of
EM-type were significantly higher in mice than in rats(Fig. 6E). Relative ratios of EA-type to EM-type showedtransient decline in both species (Fig. 6F). Although theratios in rats gradually recovered after D3, those in mice
A, B) Representative florescent Nissl staining of the rat (A) and mousee outline of the hypoglossal nucleus. (C) Pseudo-colored images forssal nucleus of axotomized mice on D28. The dashed line shows thesioned motoneurons in rats (open circles) and mice (shaded circles).(green) and glial marker-negative putative neurons in the axotomizedcontrol hypoglossal nuclei (Co; blue). Lines indicate the curves fittedm (applies to C1–C5). Asterisks in (D) show statistical significancesto color in this figure legend, the reader is referred to the Web version
xotomy. (shows thhypoglo
tios of leitive cellsns in the
C5)�30 �
showed no recovery until D28.
(C)�20 �ed side a
J. Yamada et al. / Neuroscience 182 (2011) 1–10 5
DISCUSSION
The current study describes a significant decline in mo-toneuron numbers following axotomy in the mouse hy-poglossal nucleus. However, it should be noted thatperipheral nerve axotomy induces not only neuronaldeath but also neuronal atrophy (McPhail et al., 2004b).Namely, some neurons may remain in an atrophied stateafter axotomy, and it cannot be denied that these atro-phic cells lead to an underestimate of neuron counts(Kwon et al., 2002). Considering the difficulty in detect-ing atrophied motoneurons by neuronal markers, suchas NeuN (McPhail et al., 2004a), here we examined theaxotomy-induced alterations in soma size distributions
Fig. 2. Species differences in alterations of synaptophysin immunorephysin (SYP) immunoreactivity in the hypoglossal nucleus of sham-optransection (D7). (D) Axotomy-induced alterations in synaptophysin-ptraces of mEPSC (E) and mIPSC (F) in sham-operated rats, axotommEPSCs frequency (G) and amplitude (H) in rats (open bars) and miceamplitude (J) in rats (open bars) and mice (shaded bars). Scale bar inshown by solid lines, and statistical significance between sham-operat
of putative neuronal cells that were negative for glial
markers. To label glial cells as much as possible, weused three markers for the major types of glial cells inthe brain, that is, Iba1 for microglia, GFAP for astro-cytes, and Olig2 for oligodendrocyte lineage cells (Lu etal., 2000). Then, we found that the soma size distribu-tions were significantly different between glial marker-positive cells and glial marker-negative putative neuronalcells. Furthermore, there was no significant difference in thesoma size distributions of glial marker-negative putativeneuronal cells between axotomized mice and sham-operated mice. Taken together, we consider that thereduction of motoneurons in the mouse hypoglossal nu-cleus following axotomy is not attributable to extensive
nd synaptic transmission after hypoglossal axotomy. (A–C) Synapto-ts (A), axotomized rats (B) and axotomized mice (C) 7 d after nerve
ea in rats (open bars) and mice (shaded bars). (E, F) Representativeand axotomized mice on D7. (G, H) Axotomy-induced alterations inbars). (I, J) Axotomy-induced alterations in mIPSCs frequency (I) andm (applies to A–C). Statistical significance between rats and mice arend axotomized side are shown by dashed lines: * P�0.05, ** P�0.01.
activity aerated ra
ositive arized rats(shaded
atrophy, but to neuronal loss.
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J. Yamada et al. / Neuroscience 182 (2011) 1–106
A characteristic feature of the peripheral nerve axo-omy is the broad detachment of presynaptic terminalsrom dendrites and cell bodies of motoneurons. This reac-ion is usually referred to as synaptic stripping (Moran andraeber, 2004). The original model for synaptic stripping is
he facial nerve axotomy, but a similar phenomenon haseen reported in other neurons as well (Graeber et al.,
Fig. 3. Immunocytochemical analysis of glial and synaptic coverage oE, I), GLAST (B, F, J), synaptophysin (SYP; C, G, K) and TOTO-3 (D, H(I–L) on D7. Insets show the higher magnification images of representimages of three categories of coverage pattern: S-type (M), A-type (Nin A–D; N�insets in E–H; O�insets in I–L). (P–R) Line profile analysisthe results obtained from the white lines in (M), (N), and (R), respectiv100%. Scale bars in (L)�20 �m (applies to A–L), in inset of (L)�1 �mof the references to color in this figure legend, the reader is referred
993; Oliveira et al., 2004; Thams et al., 2008). Synaptic m
tripping is widely believed to have neuroprotective effectshrough contact-dependent signaling or suppression ofynaptic inputs (Raivich et al., 1999; Moran and Graeber,004; Cullheim and Thams, 2007). Here we confirmed thatypoglossal axotomy induced slow cell death in mice butot in rats. Our electrophysiological and anatomical anal-ses also showed that axotomy-induced synaptic detach-
ssal motoneurons. (A–L) Fluorescent quadruple labeling for Iba1 (A,am-operated rats (A–D), axotomized rats (E–H) and axotomized miceerage patterns indicated by arrows in (A–L). (M–O) 3D reconstructiontype (O). Each image represents the area shown in insets (M�insetsge patterns of motoneurons. Pixel intensities in (P), (Q), and (R) show
maximum pixel intensity values of each channel are arbitrarily fixed atto insets of A–L), in (O)�0.5 �m (applies to M–O). For interpretationb version of this article.
f hypoglo, L) in sh
ative cov) and M-of coveraely. The(applies
ent was significantly more extensive in rats than in mice.
i er is refe
J. Yamada et al. / Neuroscience 182 (2011) 1–10 7
Taken together, these experiments suggest that the spe-cies differences in the extent of synaptic stripping might berelated to the differences in cell survival of injured mo-toneurons between mice and rats.
Fig. 4. Sampling procedure for line profile analysis of coverageperpendicular to the soma membrane are drawn at 3 �m intervals.nterpretation of the references to color in this figure legend, the read
Fig. 5. Differential responses of astrocytes and microglia in the synapinto S-type, A-type and M-type. (B) Axotomy-induced alterations in t(shaded bars). (C–E) Axotomy-induced alterations in the fractions o(dark-colored bars). (F) Relative ratios of A-type to M-type in the synapstatistical significances between rats and mice: * P�0.05, ** P�0.01.between sham-operated and axotomized animals. For interpretation o
version of this article.The notion that reactive microglia are mainly involvedin the removal of synaptic boutons from axotomized mo-toneurons has been maintained since the seminal work onthis model (Blinzinger and Kreutzberg, 1968). Earlier ultra-
of hypoglossal motoneuron perineuronal circumference. (A) Linesprofile analysis showing the pixel intensity of orange line in (A). Forrred to the Web version of this article.
nment after axotomy. (A) Synaptophysin-crossing lines are classifiedlence of synaptophysin-crossing lines in rats (white bars) and mice(C), A-type (D) and M-type (E) in rats (light-colored bars) and micenment in rats (open circles) and mice (shaded circles). Asterisks showtal bar at the upper-level of each graph shows statistical significancerences to color in this figure legend, the reader is referred to the Web
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J. Yamada et al. / Neuroscience 182 (2011) 1–108
structural studies indicated that processes of activatedmicroglia established close contact with morphologicallyintact boutons prior to their removal. Microglia becomeactivated in response to neuronal injury and can engage insynaptic stripping without turning into phagocytosis(Kreutzberg, 1996; Cullheim and Thams, 2007). Astro-ytes have also been implicated in the displacement ofynaptic terminals (Sumner, 1975, 1979; Emirandetti et al.,006). The tips of astrocytic process become thin lamellarell extensions after axotomy, which intervene around theerineuronal environment. Ensheathment of the axoto-ized motoneuron by astrocytes has been suggested toartially depress the synaptic drive from selected inputs,s well as to provide metabolic support (Aldskogius et al.,999). Here we found that there were substantial speciesifferences in glial reactions in the synaptic environment:strocytic intrusion was more extensive in rats than inice, whereas microglial invasion was more severe inice than in rats. An important point to note is that celleath is not likely the primary cause of microglial activa-ion, because the occurrence of activated microglia pre-eded motoneuron death in both species. These results
Fig. 6. Differential responses of astrocytes and microglia in the extclassified into EA-type, EM-type, and unclassified. (B) Axotomy-inducbars) and mice (shaded bars). (C–E) Axotomy-induced alterations in(light-colored bars) and mice (dark-colored bars). (F) Relative ratios ofmice (shaded circles). Asterisks show statistical significances betweenraph shows statistical significance between sham-operated and axotohe reader is referred to the Web version of this article.
ndicate that astrocytic, but not microglial, reaction may
mediate some anti-apoptotic effects through suppressionof synaptic inputs after hypoglossal axotomy.
Interestingly, prevalence of contact between astrocyticprocesses and lesioned motoneurons in the extrasynapticsites was significantly increased in rats after axotomy (D7,125.5% increase in EA-type), whereas there were no sig-nificant alterations in mice (D7, 6.1% decrease in EA-type).Astrocytes regulate extrasynaptic glutamate in two oppo-site manners: by importing glutamate through transporters(Huang and Bergles, 2004) or by releasing glutamate(Agulhon et al., 2008). Extrasynaptic glutamate dynamicshas been implicated in the activation of extrasynaptic glu-tamate receptors via volume transmission, which regulatessynaptic plasticity and neuronal survival (Hardingham etal., 2002). Previous studies reported prevention of mo-toneuron death after facial nerve transection by N-methylD-aspartate (NMDA) glutamate receptor blockers (Casano-vas et al., 1996). It has recently been suggested thatglutamate exocytosis from astrocytes critically controlsneuronal activity through activation of extrasynaptic NMDAreceptors (Fellin et al., 2004; Nie and Weng, 2010). Sim-ulation experiments indicate that astrocytic sheaths are
ic environment after axotomy. (A) Synaptophysin-avoiding lines aretions in the prevalence of synaptophysin-avoiding lines in rats (whitetions of unclassified type (C), EA-type (D) and EM-type (E) in ratsto EM-type in the extrasynaptic environment in rats (open circles) andmice: * P�0.05, ** P�0.01. Horizontal bar at the upper-level of each
imals. For interpretation of the references to color in this figure legend,
rasynapted altera
the fracEA-typerats and
important in determining glutamate spillover and synaptic
J. Yamada et al. / Neuroscience 182 (2011) 1–10 9
transmission (Rusakov, 2001; Lehre and Rusakov, 2002).It should also be noted that increased expression of astro-cytic glutamate transporter rescued degradation of mo-toneurons in amyotrophic lateral sclerosis animals (Leporeet al., 2008; Blackburn et al., 2009). Taken together, en-largement astrocytic processes in the extrasynaptic envi-ronment seen in rats might be involved in neuronal protec-tion via the increased uptake of excessive glutamate. Dif-ferently from the astrocytic reaction, the prevalence ofIba1-positive microglial processes in the extrasynaptic en-vironment increased in both species, while the microglialfraction was significantly larger in mice than in rats.
It might be worth pointing out that there were poten-tially significant species differences in immune system be-tween mice and rats. For instance, peripheral nerve tran-section leads to infiltration of lymphocyte in mice, but not inrats (Graeber et al., 1990; Raivich et al., 1998). Similar tothe hypoglossal axotomy model, the facial motoneuronsexhibited very little degeneration after nerve transection inrats (Streit and Kreutzberg, 1988), while pronounced neu-ronal death occurred in the facial motor nucleus followingaxotomy in mice (Ferri et al., 1998). These findings indi-cate the presence of additional factors that might influencethe survival of hypoglossal motoneurons after axotomy.
Although the present research was purely descriptiveand correlative in nature, the results provide an essentialstructural basis for elucidating mechanisms of synapticstripping after axotomy. In addition, our findings lend somesupport for the hypothesis regarding the double-edgedsword nature of glial cells: the astrocytic activation maypotentially exert some protective effects on axotomizedmotoneurons, whereas the microglial response might beinvolved in slow apoptotic cell death of motoneurons. Ulti-mately, identifying molecular mechanisms regulating dif-ferential reactions of perineuronal astrocytes and microgliain cell survival will be critical to establishing therapeuticstrategies for motoneuron diseases.
Acknowledgments—The authors thank Dr. Eric Bushong for hisconstructive comments and grammatical corrections on earlierversions of the manuscript. The authors also thank Mrs. C.Tanaka for her secretarial assistance. This study was partiallysupported by Grants-in-Aid for Scientific Research B (20390472 toH.N.) and C (21500328 to S.J.) from the Ministry of Education,Science and Culture, Japan.
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