glutamate reuptake represents the principal mechanism for inactivation of synaptically released glutamate [ 1 , 2 ] . in the rodent hippocampus , it is mainly accomplished by astrocytic glutamate transporters ( eaats : excitatory amino acid transporters ) , namely , glast ( glutamate / aspartate transporter ) and glt-1 ( glutamate - transporter-1 ; rodent analogues of eaat1 and eaat2 , resp . ; [ 37 ] ) . glutamate uptake is energized by the concomitant inward transport of three sodium ions and a proton , while one potassium ion is transported outward . consequently , its activation is accompanied by an increase in the intracellular sodium concentration of astrocytes [ 8 , 9 ] . under pathological conditions ,
astrocytes undergo a complex reaction referred to as reactive astrogliosis , which is seen in diverse preparations and conditions ranging from primary cell culture to the intact brain [ 10 , 11 ] .
the hallmarks of reactive gliosis are a massive upregulation of the expression of the intermediate filament glial fibrillary acidic protein ( gfap ) and a cellular hypertrophy [ 12 , 13 ] .
reactive astrocytes display several features of immature astrocytes ( e.g. , [ 14 , 15 ] ) and can partially reenter the cell cycle [ 16 , 17 ] .
following traumatic brain injury , a dense glial scar forms at the lesion site due to strong astrocytic reorganization and proliferation [ 10 , 11 , 18 ] . at some distance to the lesion and scar , astrocytes usually do not divide , and their morphological reorganization and hypertrophy is less severe .
the establishment of a glial scar around injured tissue is regarded as hindrance for the growth and regeneration of axons [ 19 , 20 ] .
notwithstanding , reactive astrocytes might also exert a protective role and support regeneration [ 11 , 13 , 21 ] .
there is evidence that reactive gliosis is also accompanied by an alteration in the expression level of glial glutamate transporters .
most studies performed in the rodent and human brain reported an overall downregulation of protein levels of both glast and glt-1 and/or a reduction in functional glutamate uptake in response to brain injury and astrogliosis , respectively ( e.g. , [ 2227 ] ) .
the reduction in the expression of glial glutamate transporters has been suggested to contribute to the elevation of extracellular glutamate concentrations and to glutamate - mediated excitotoxicity which is observed under many pathological conditions .
injury - induced changes in the expression of the glutamate transporter subtypes might , however , also depend on the degree of astrogliosis
. this might be especially relevant and visible upon a mechanical lesion , where reactivity of astrocytes , as judged for example based on their gfap expression and morphology , ranges from severe astrogliosis in the scar region to only moderate - mild astrogliosis distant from the lesion [ 10 , 11 ] . to study this question , we analyzed expression of glast and glt-1 following a mechanical lesion in organotypic tissue slice cultures of the mouse hippocampus . furthermore , we employed ratiometric sodium imaging as functional assay for glutamate uptake in astrocytes .
our results show that mechanical tissue injury generates subsets of reactive astrocytes depending on the distance from the lesion site , which differ in both morphological features and their ability to take up sr101 .
furthermore , these subsets show discrete changes in glutamate transporter expression and glutamate uptake capacity , indicating that glutamate clearance might be largely functional in the periphery of the lesion , but strongly hampered in the scar region .
this study was carried out in strict accordance with the institutional guidelines of the heinrich heine university duesseldorf , germany , as well as the european community council directive ( 86/609/eec ) .
all experiments were communicated to and approved by the animal welfare office at the animal care and use facility of the heinrich heine university duesseldorf , germany ( institutional act number : o52/05 ) . in accordance with the german animal welfare act ( tierschutzgesetz , articles 4 and 7 ) , no formal additional approval for the postmortem removal of brain tissue was necessary . for generation of acute slices , mice were quickly decapitated ( following the recommendation of the european commission published in : euthanasia of experimental animals , luxembourg : office for official publications of the european communities , 1997 ; isbn 92827 - 9694 - 9 ) .
acute tissue slices of the hippocampus were prepared from balb / c mice ( mus musculus ) of both genders at postnatal days 7 to 8 ( p7 - 8 ) using standard procedures . for some experiments , transgenic animals ( fvb / n - tg[gfapgfp]14mes /
j ) expressing green fluorescence protein ( gfp ) under the gfap promoter were used ( obtained from jackson laboratory ; harbor , usa ) .
after decapitation of the animals , brains were quickly excised and hemisected in ice - cold artificial cerebrospinal fluid ( acsf ) composed of ( in mm ) 125 nacl , 2.5 kcl , 2 cacl2 , 1 mgcl2 , 1.25 nah2po4 , 26 nahco3 , and 20 glucose , bubbled with 95% o2 and 5% co2 , and adjusted to a ph of 7.4 .
hemisections were trimmed , and transverse slices ( 200  m ) comprising the entorhinal cortex , hippocampus , fimbria , and thalamus were prepared using a vibratome ( microm hm650v , thermo fischer scientific , walldorf , germany ) .
organotypic hippocampal slice cultures were prepared and cultured according to the protocol introduced by stoppini et al .   with minor modifications .
to this end , acute slices were transferred to a millicell culture insert ( picm org 50 , hydrophilized ptfe , pore size 0.4  m ; merck millipore , darmstadt , germany ) and maintained at the interface of a serum - based culture medium free of antibiotics in a humidified incubator atmosphere of 5% co2 at 37c .
the culture medium was composed of 30% sterile filtered normal horse serum ( nhs ; gibco / life technologies , darmstadt , germany ) , 30% dulbecco 's modified eagle medium ( dmem ; gibco / life technologies , darmstadt , germany ) , and 40% hank 's balanced salt solution ( hbss ; gibco / life technologies , darmstadt , germany ) supplemented with 38  mm glucose ( ph adjusted to 7.3 - 7.4 ) . the medium was changed three times a week , and the insert surface was washed with medium once a week .
after at least 12 days in culture , a mechanical lesion was performed using a sterile scalpel blade .
the lesion was positioned in the ca1 area perpendicular to the stratum pyramidale , included the strata oriens , pyramidale , radiatum , and lacunosum moleculare , and spanned the entire depth of the slice ( cf .   figure 2 ) .
after lesioning , slice cultures were maintained for another 6 - 7 days ( designated as 6 - 7 days postlesion ) .
unlesioned control slices were cultured in parallel for a corresponding number of days . for visualization of cell death , acsf containing 0.5 
g / ml propidium iodide ( pi ) was applied to the slice surface and incubated for 3 hours at 37c and 5% co2 , followed by a wash with acsf .
documentation was either accomplished with an epifluorescence microscope ( nikon eclipse 90i ; nikon instruments , dsseldorf , germany ) or at an olympus fluoview300 laser scanning microscope ( olympus , hamburg , germany ) .
astrocyte soma size in organotypic control and lesioned slices was calculated from images of gfp - fluorescence derived from gfp / gfap mice which were obtained at a confocal microscope ( olympus fluoview300 ; olympus , hamburg , germany ; see also below ) . to this end
, a semiquantitative approach was used , in which somata of single cells were manually encircled ( cf .   figure 4(b ) ) , and the resulting area was calculated using imagej software ( nih , bethesda , usa ) . all chemicals were purchased from sigma - aldrich chemical ( munich , germany ) unless stated otherwise . for imaging experiments ,
organotypic slices were excised from the millicell inserts and incubated in acsf containing 2.5  m sr101 for 30  min at 35c to stain astrocytes ( see above ) .
sr101 is a highly specific and widely used tool for the identification of mature astrocytes in many brain regions including the hippocampus [ 29 , 30 ] .
it has recently been shown to be taken up into mature hippocampal astrocytes via an active transport mechanism involving organic anion transport polypeptides .
wide - field fluorescence imaging was performed using a variable scan digital imaging system ( till photonics , martinsried , germany ) attached to an upright microscope ( bx51wi , olympus europe , hamburg , germany ) and a ccd camera ( till imago vga , till photonics , martinsried , germany ) .
sr101 was excited at 575  nm , and emission was collected above 590  nm .
excitation wavelength for detection of gfp was 488  nm , and emission was collected above 510  nm . for intracellular sodium imaging
, slices were additionally incubated with the membrane permeable form of the sodium - sensitive fluorescent dye sbfi ( sbfi - am ; sodium - binding benzofuran isophthalate - acetoxymethyl ester ; molecular probes / life technologies , darmstadt , germany ) as described earlier [ 3234 ] .
ratiometric sodium imaging was performed by alternate excitation of sbfi at 340  nm ( weakly sodium - sensitive wavelength ) and at 380  nm ( sodium - sensitive wavelength ) at 4  hz . emission ( > 440  nm ) was collected in defined regions of interest ( roi ) representing cell bodies .
after background correction , the fluorescence ratio ( f340/f380 ) was calculated for the individual rois and analyzed offline using originpro 8 g software ( originlab corporation , northampton , ma , usa ) .
changes in sbfi fluorescence ratio were expressed as changes in sodium concentration based on in situ calibrations as reported before [ 32 , 33 , 35 ] . to equilibrate extra- and intracellular na - concentrations ,
sbfi - loaded slices were perfused with saline containing ionophores ( 3  m gramicidin d , 100  m monensin ) and the na / k - atpase blocker ouabain ( 100  m ) , as well as different concentrations of na .
the glutamate transporter agonist d - aspartate was applied by a pressure application device ( pdes-02d , npi electronic gmbh , tamm , germany ) coupled to standard micropipettes ( hilgenberg , waldkappel , germany ) placed 20100  m from cell bodies of selected cells .
the following antisera were utilized , which represent well - established , commercially available standard markers : guinea - pig glt-1 antiserum directed against the c - terminus of rat glt-1 ( chemicon international / millipore corp . ,
carrigtwohill , ireland ; e.g. , ) and guinea - pig glast antiserum directed against the c - terminus of rat glast ( chemicon international / millipore corp . , carrigtwohill , ireland ; e.g. , ) . validation and a detailed characterization of both antibodies were provided before [ 3840 ] . for identification of astrocytes , polyclonal rabbit antibodies against gfap ( gfap - pab ; dako cytomation , denmark ; [ 40 , 41 ] ) and s100 ( abcam , united kingdom ; ) were employed .
when double staining with the latter antibody was performed , a monoclonal mouse antibody detecting gfap was used ( gfap - mab ; sigma aldrich , germany ; [ 40 , 43 ] ) .
standard fluorochrome - conjugated antibodies ( alexafluor , invitrogen / life technologies , darmstadt , germany ) were employed as secondary antibodies for immunohistochemistry . prior to immunohistochemical processing , organotypic slices were immersion fixed for 30  min at rt in 4% paraformaldehyde ( pfa ) in phosphate - buffered saline ( pbs ) following three washes every 30  min with pbs .
cell membranes were permeabilized , and unspecific binding sites were blocked in pbs containing 0.25% triton - x100 ( tx ) and 2% normal goat serum ( ngs ; gibco / life technologies , darmstadt , germany ) for 90  min at 4c followed by incubation with the primary antibody gfap - pab ( 1  :  1000 ; dako cytomation , glostrup , denmark ) , diluted in the same solution over night at 4c . in case of s100/gfap double stainings , slices were incubated with a mixture of the primary antibodies gfap - mab ( 1  :  1000 , dako cytomation , glostrup , denmark ) and rabbit - s100 ( 1  :  100 , abcam , united kingdom ) . after five washes in pbs containing 0.25% tx and 2% ngs
, slices were incubated either with guinea pig - anti - glast or with guinea pig - anti - glt-1 ( both 1  :  1000 , diluted in 0.25% tx/2% ngs / pbs ) for 4  hrs at rt .
antirabbit - alexafluor594 and antiguinea pig - alexafluor488 or antimouse - alexafluor488 ( 1  :  100 in blocking solution ) were used for visualization of antibody binding and incubated for 2  hrs at rt .
the slices were subjected to dapi staining ( 4,6-diamidino-2-phenylindole ; 0.5  m ; invitrogen ) , washed three times , and mounted on glass slides with mowiol / dabco ( calbiochem , fluka , distributed by sigma - aldrich chemical , munich , germany ) .
negative controls were run in parallel to each staining by either omitting all or just one of the primary antibodies .
control stainings in which one of the primary antibodies was omitted showed the identical labeling pattern for the remaining antibody as in the double stainings . omitting
documentation of immunofluorescence was either performed with an epifluorescence microscope ( nikon eclipse 90i ; nikon instruments , dsseldorf , germany ) or a confocal laser scanning microscope ( olympus fluoview300 ; olympus , hamburg , germany ) . the epifluorescence microscope was equipped with a standard dapi ( ex 340380 ; dm 400 ; ba 435485 ) , fitc ( ex 465495 ; dm 505 ; ba 515555 ) , and tritc ( ex 540/25 ; dm 365 ; ba 605/55 ) filter set .
illumination was provided by an intensilight fiber lamp ( c - ghfi ; nikon instruments , dsseldorf , germany ) , and emission was detected with a monochrome digital camera ( ds - qi1mc ; nikon instruments , dsseldorf , germany ) .
images were collected with either a 20x/0.75 ( planapovc , nikon instruments , dsseldorf , germany ) air objective or a 60x/1.40 oil immersion objective ( planapovc , nikon instruments , dsseldorf , germany ) .
nis - elements software ( nikon instruments , dsseldorf , germany ) was used for image acquisition . for confocal microscopy ,
an olympus bx51wi microscope coupled to a confocal laser scanning system ( fv300 ) equipped with a multiline argon ( 488  nm ) and a helium - neon laser ( 543  nm , both melles griot , bensheim , germany ) was used .
images were collected with either a 20x/0.50 ( umplanfl , olympus , hamburg , germany ) , a 40x/0.80 water immersion ( lumplan , olympus , hamburg , germany ) , or a 60x/1.40 oil immersion objective ( planapovc , nikon instruments , dsseldorf , germany ) .
a kalman filter 4 was employed at every scan . simultaneous or sequential scanning of both fluorophores revealed no difference in their staining patterns , indicating absence of cross - excitation or spectral bleed through .
the thickness of z - plane sections was 1  m , and the number of optical sections varied depending on the preparation .
figures show extended focus images as specified in the figure legends , which were calculated from z - stacks of optical sections using imagej software ( nih , bethesda , usa ) .
images were overlaid employing adobe photoshop cs2 ( adobe systems , cologne , germany ) .
data were statistically analyzed by student 's t - test employing the procedures implemented in originpro 8 g software ( originlab corporation , northampton , ma , usa ) .
each set of experiments was performed on at least three tissue slices obtained from different animals .
the aim of the present study was to analyze changes in glutamate transporter expression and function in astrocytes in response to a mechanical injury .
to this end , we prepared organotypic tissue slice cultures of the mouse hippocampus , a well - established model system in which the layering and basic cellular organization of the neural network are maintained over time .
after 1925 days in culture , organotypic tissue slices had flattened to a thickness of 4050  m and were composed of 3 to 4 cell layers . at this stage , propidium iodide assays detected only few dead cells dispersed throughout the slices ( 3  0.6 cells per ca1 subfield ; 18  3.4 in entire preparation ; n = 6 ; data not shown ) , indicating that the preparation was viable and in a stable condition . to visualize astrocytes in cultured slices , we used hippocampi of transgenic mice in which green fluorescent protein ( gfp ) is expressed under the control of the promoter of the astrocyte - specific intermediate filament glial fibrillary acidic protein ( gfap ; fvb / n - tg[gfapgfp ] ;  gfp / gfap mice  ) .
a visualization of the gfp fluorescence demonstrated that the general distribution and organization of astrocytes were preserved in the strata pyramidale and radiatum of the ca1 region of organotypic cultures , confirming earlier reports ( n = 3 ; figure 1(a ) ; [ 44 , 45 ] ) .
the vast majority of gfp - expressing cells ( ~95% ) also labeled with the astrocyte - specific vital marker sr101 in organotypic slices ( n = 3 ; figures 1(a ) and 1(b ) ) .
furthermore , immunohistochemical stainings for gfap in slice cultures derived from wildtype animals showed that the majority of gfap - positive cells were also positive for s100 ( n = 12 ; figure 1(c ) ) , a marker for mature astrocytes .
taken together , the nearly complete overlap in the cellular staining pattern for the vital dye sr101 with the expression of gfp in gfp / gfap mice , as well as the overlap in the immunofluorescence for gfap and s100 in organotypic slices indicates that these markers are well suited to identify astrocytes in this preparation . to induce astrogliosis in organotypic slice cultures
, we used a scalpel blade and performed a scratch under semisterile conditions through the entire ca1 region oriented perpendicular to the stratum pyramidale ( figure 2(a ) ) .
6 - 7 days after the lesion , a general enhancement of gfap immunoreactivity was observed , indicating astrogliosis ( figure 2(b ) ) . the greatest enhancement of gfap immunoreactivity occurred within a distance of less than 100  m along the lesion ( n = 26 ; figures 2(b ) and 2(c ) ) . here , a dense meshwork of thick , gfap - positive labels was present , indicative of the formation of a glial scar .
furthermore , the region close to the lesion site exhibited a discernible increase in s100 immunoreactivity ( n = 12 ; figure 2(c ) ) .
both the prominent expressions of gfap and s100 are characteristics of strongly reactive astrocytes , which represent the major cellular component of the glial scar forming after mechanical tissue injury .
more distal to the lesion ( > 100350  m ) , a less dramatic rearrangement of gfap immunoreactivity was observed . a clear difference in astrocyte properties between the scar region and its periphery
was also seen following sr101 labeling . in the periphery of the scar ( 100350  m distance from the lesion ) , sr101 resulted in a reliable and bright cellular staining , similar to what had been observed in control , unlesioned slices ( n = 12 ; figure 3(a ) , cf .   figure 1 ) .
in contrast to this , cellular sr101 staining was detectable but only faint within a distance of less than 100  m on both sides of the lesion ( figure 3(a ) ; mean width of sr101-free area 111  3  m ; n = 5 ) . in slices from gfp / gfap transgenic animals
( n = 5 ; figure 3(b ) ) , gfap - positive , weakly sr101-positive scar cells also stained with the sodium - sensitive fluorescence indicator sbfi - am ( acetoxymethyl ester of sodium binding benzofuran isophthalate ; see below ; n = 3 ) , indicating that they were able to take up and de - esterify this dye ( figure 3(b ) ) .
in addition to gfap - positive cells , the scar region hosted microglial cells , identified by their vital staining with texas red - coupled lectin from lycopersicon esculentum ( tomato lectin ; n = 4 ; not shown ) .
tomato lectin - positive microglia , however , did not take up sbfi - am ( n = 4 ; not shown ) , confirming earlier reports that microglial cells do not incorporate am - ester dyes in situ . to further characterize reactive gliosis in response to the scratch wound , we determined the area of astrocyte somata in confocal images of the gfp fluorescence in control and lesioned slices obtained from gfp / gfap transgenic animals .
these measurements revealed that , compared to astrocytes in control slices , the soma area of astrocytes in the periphery of a lesion was increased by 18% and that of astrocytes in the scar region was increased by 24% ( control : 163  2  m ; lesion periphery : 193  3  m ; scar tissue : 201  4  m ; p < 0.001 ; n = 945 , 496 , and 439 in 7 control and 9 lesioned slices ; figures 4(a ) and 4(b ) ) .
thus , astrocytes show a second hallmark of reactive gliosis after lesion , namely , a hypertrophy of their cell bodies , which is observed both in the scar tissue as well as in the periphery of the lesion . taken together
, these data show that 6 - 7 days after the mechanical lesion , a glial scar has formed along the lesion site .
this scar encompasses strongly activated astrocytes , characterized by a robust increase in gfap and s100 expression .
these proximal reactive astrocytes , located within a distance of less than 100  m from the lesion , exhibit long gfap - positive processes , have significantly swollen somata , and only weakly stain with sr101 .
astrocytes in the periphery of the scar ( 100350  m from the lesion ) display swollen cell bodies as well , but show only moderate upregulation of gfap and maintain their ability to efficiently take up sr101 . to determine the spatial distribution of the glutamate transporters glast and glt-1 in organotypic slices and changes therein in response to the lesion , immunohistochemistry was employed . in control slices ,
glast ( n = 13 ; figure 5(a ) ) as well as glt-1 immunoreactivity ( n = 11 ; figure 5(b ) ) appeared as punctate labeling throughout the entire ca1 area .
the staining patterns for both transporters in the tissue slice cultures were thus similar to those reported from acute tissue slice preparations of the hippocampus [ 36 , 48 , 49 ] .
the relatively homogeneous distribution of immunoreactivity throughout the preparation found in control slice cultures was abandoned following a lesion . here , distinct accumulations of glast ( n = 17 ; figure 5(a ) ) and glt-1 immunoreactivity ( n = 12 ; figure 5(b ) ) were found in the scar region . in both cases , these preferentially colocalized with thick gfap - positive bundles running in parallel to the lesion ( figure 5 ) . apart from the marked accumulation of glast reactivity in parallel to the scar , glast immunoreactivity appeared generally increased in some lesioned slice preparations ( figure 5(a ) ) but unaltered in others both within the scar as well as in its periphery .
for glt-1 , a decline in the intensity of immunoreactivity was observed with increasing distance from the lesion ( figure 5(b ) ) . in summary , these results reveal distinct alterations in the immunoreactivity of glast and glt-1 following a mechanical lesion . in the scar region ,
clusters of glt-1 and glast immunoreactivity are present , primarily colocalized with strongly gfap - positive fibers . apart from these distinct clusters
, glt-1 immunoreactivity appears to decline with distance from the scar , whereas glast immunoreactivity seems largely uniform throughout the preparation .
glutamate uptake is accompanied by the inward flux of sodium , resulting in increase in the intracellular sodium concentration of astrocytes . to monitor its function
, we assayed sodium increases induced by d - aspartate , a nonmetabolized substrate of sodium - dependent glutamate uptake which is also transported   in astrocytes in the stratum radiatum of organotypic slices . to this end
, sr101-stained slices were additionally loaded with the sodium - sensitive fluorophore sbfi - am ( figure 6(a ) , cf .
the sbfi fluorescence revealed a linear increase in its fluorescence ratio at intracellular sodium concentrations between 10 and 40  mm ( n = 107 ; figure 6(b ) ) as reported before . repetitive pressure application of d - aspartate for 500  ms induced reliable increases in the intracellular sodium concentration in astrocytes , indicating activation of sodium - dependent glutamate uptake ( n = 488 ; figure 6(c ) ) .
the experiments were performed in the presence of the sodium - channel blocker ttx ( tetrodotoxin , 0.5  m ) to suppress action potential generation .
the amplitude of d - aspartate - induced sodium increases was dependent on the concentration applied ( figure 6(c ) ) and saturated at about 2  mm ( n = 19 ; figure 6(d ) ) , as reported before for mouse cortical astrocytes in culture . at a concentration of 1  mm , the amplitude of d - aspartate - induced sodium transients was near maximum and amounted to 3.8  0.2  mm ( n = 161 ; figures 6(c ) and 6(d ) ) , a value comparable to that in astrocytes in acute slices obtained in our lab . to probe for changes in glutamate uptake , we compared sodium signals induced by 1  mm d - aspartate between control and lesioned slices ( figure 7 ) . in lesioned slices ,
astrocytes located in the periphery of the lesion ( 100350  m distance ) responded with a sodium transient to application of 1  mm d - aspartate ( see cell 2 in figures 7(a ) and 7(b ) ) .
the amplitude of d - aspartate - induced sodium signals in cells in the lesion periphery was similar to that observed in control slices ( 3.8  0.3  mm ; n = 111 ; see cell 1 in figures 7(a ) , 7(b ) , and 7(c ) ) .
application of d - aspartate also evoked sodium transients in weakly sr101-positive , sbfi - filled astrocytes located in the glial scar area ( < 100  m from the lesion ; see cells 35 in figures 7(a ) and 7(b ) ) .
these cells , however , displayed significantly smaller peak amplitudes ( 1.6  0.21  mm , n = 26 ; p < 0.001 ; figures 7(b ) and 7(c ) ) than astrocytes in the periphery and in control slices .
the kinetics of d - aspartate - induced sodium transients differed between all three groups .
this was true for the slope of the sodium increase ( 10%90% change ) , which was significantly reduced in astrocytes from lesioned slices versus astrocytes from control slices ( 0.9  0.05  mm / s in control versus 0.6  0.06  mm / s in the periphery and 0.2  0.04  mm / s in the scar region ; n = 161 , 111 , and 26 , resp . ;
in addition , the decay back to baseline was significantly slower following lesion ( figures 7(b ) and 7(c ) ) .
the time needed for the signal to decay by 67% was approximately doubled ( decay time  was 44  2  s in control ,  = 81  4  s in periphery of the lesion , and  = 95  10  s in scar cells ; figures 7(b ) and 7(c ) ) .
these results show that mechanical injury results in distinct alterations in the kinetics and/or amplitudes of d - aspartate - induced intracellular sodium transients in reactive astrocytes , depending on their distance to the lesion .
our results show that within 6 - 7 days after setting a scratch wound in organotypic hippocampal slices , a dense glial scar had formed along the lesion site . up to approximately 100  m distance from the lesion , the scar tissue was characterized by a substantial elevation of gfap expression , a pronounced cellular hypertrophy , and reduced uptake of sr101 by astrocytes .
while prominent clusters of glast and glt-1 immunoreactivity were observed along gfap - positive structures in scar cells , d - aspartate - induced intracellular sodium signals were strongly dampened , indicating a significant reduction in glutamate uptake capacity close to the lesion . in the periphery of the lesion ( 100350  m ) , astrocytes showed less pronounced reactivity , maintained their ability to efficiently take up sr101 , and displayed only minor changes in glutamate transporter immunoreactivity and function .
thus , depending on their distance from the lesion , astrocytes showed different grades of reactivity and displayed discrete changes in glutamate transporter immunoreactivity and uptake capacity .
organotypic tissue slice cultures of the hippocampus represent a well - established model system , in which the basic tissue architecture is maintained , but which still hosts the major advantages of cell cultures such as good accessibility and control of experimental conditions .
they have been used extensively for the analysis of neuronal properties and developing neuronal networks [ 28 , 51 , 52 ] or the study of excitotoxic neuronal damage ( e.g. , [ 53 , 54 ] ) .
recent studies also demonstrated that basic morphological characteristics of astrocytes and the typical glia - synapse organization are well preserved in organotypic slice cultures ( e.g. , [ 44 , 45 , 55 , 56 ] ) .
in addition , we found a nearly complete overlap of sr101 staining with the expression of gfp in slice cultures obtained from gfp / gfap mice , demonstrating that sr101 is well suited to identify astrocytes in this preparation as described before for acute tissue slices and in vivo [ 29 , 30 ] . performing a scratch through
the ca1 region of organotypic slices resulted in the formation of a glial scar along the lesion site , as judged by the prominent increase in expression of gfap and s100. in addition , astrocyte cell bodies showed a significant hypertrophy .
furthermore , astrocytes in the periphery of the scar ( 100350  m from the lesion ) showed mild to moderate reactivity , displaying swollen cell bodies , and only moderate upregulation of gfap . these changes are hallmarks of reactive gliosis observed after tissue injury [ 10 , 13 , 57 ] . generally , the degree of injury and the distance of the astrocytes from the site of injury define the degree of their activation [ 11 , 13 ] .
our study also revealed that strongly reactive astrocytes in the scar region only weakly stain with sr101 .
the reduced ability of strongly activated astrocytes to accumulate sr101 indicates that strong glial activation is accompanied by a downregulation of the organic anion transporter responsible for uptake of sr101 .
this is reminiscent of immature astrocytes in the early postnatal hippocampus , which are sr101-negative   and in line with a wealth of experimental evidence suggesting that reactive gliosis represents a process comprising a dedifferentiation of astrocytes .
glutamate uptake in the hippocampus is mainly achieved by the glial glutamate transporters glt-1 and glast [ 1 , 2 ] .
a central factor in many brain pathologies , including traumatic brain injury , is an increase in extracellular glutamate and excitotoxicity ; it has been suggested that a change in the expression levels of glial glutamate transporters might play a critical role in the failure of glutamate clearance [ 6 , 5860 ] .
earlier work has found an overall downregulation of protein levels of both glast and glt-1 following astrocyte activation [ 2227 ] , indicating that this is causal to the elevation of extracellular glutamate .
in contrast to this notion , other studies reported increased glutamate transport capacity of reactive astrocytes and suggested a protective influence [ 61 , 62 ] . to visualize possible differences in the spatial expression profile of glt-1 and glast in strongly reactive astrocytes along the scar as compared to moderately reactive astrocytes in its periphery , we performed immunohistochemical stainings .
these revealed an accumulation of glt-1 and glast immunoreactivity along thick gfap - positive fibers , which was particularly pronounced for glast . besides these clusters , overall glast immunoreactivity seemed unaltered as compared to unlesioned slices , whereas glt-1 immunoreactivity seemed weaker and declined with increasing distance from the scar .
clustering of glutamate transporters has been described by several reports [ 48 , 63 , 64 ] . in developing hippocampal astrocytes ,
cluster formation was preferentially found in branches opposed to synapses and was increased with increased neuronal activity , indicating that it is necessary to cope with synaptic release of glutamate . along the same lines ,
it was observed that sustained astroglial activation by ciliary neurotrophic factor ( cntf ) in the rat striatum induced a concentration of glast and glt-1 into raft microdomains and improved glutamate clearance , indicating that cluster formation increased the cellular capacity for glutamate uptake .
however , another study found glutamate - induced clustering of glt-1 that induced its endocytosis and intracellular trafficking without changing the total expression levels as detected by western blots , arguing for a decrease in functional glial glutamate uptake capacity . in the present study ,
antibodies against glt-1 and glast were employed for immunohistochemistry after permeabilization of the plasma membrane .
the clusters of glast and glt-1 along gfap - positive fibers in the scar region ( as glast and glt-1 immunoreactivity in general ) could thus represent glutamate transporters in the plasma membrane or in intracellular compartments or both .
consequently , the observed clustering does not allow a prediction about possible functional changes in glutamate uptake .
we probed for the functional activation of glutamate transport by application of the transportable agonist d - aspartate , which results in an increase in the intracellular sodium concentration of astrocytes [ 8 , 9 ] .
the amplitudes of d - aspartate - induced sodium transients in astrocytes in control organotypic slices were similar to those reported from astrocytes in acute slices obtained in our lab , indicating similar cellular glutamate transport capacity in both preparations .
following a lesion , d - aspartate - induced intracellular sodium signals were clearly dampened in scar cells .
moreover , the slope of the increase in sodium was significantly reduced , and the decay back to baseline was slowed . because d - aspartate was applied at nearly saturating concentration , the reduction in peak amplitudes suggests a reduction in functional glutamate uptake capacity in cells close to the lesion , which might be mediated by a decrease in the overall number of glutamate transporters available at the plasma membrane .
in contrast , the alteration in the kinetics of the d - aspartate - induced sodium signals can be explained by the cellular hypertrophy that was observed in reactive astrocytes . at an equal transport - mediated sodium influx across the membrane
, the slope of resulting changes in the sodium concentration will be decreased in cells with a larger volume .
furthermore , the decay of intracellular sodium transients back to baseline is mainly governed by the activity of the na / k - atpase as well as by diffusion .
the slower recovery is in line with earlier studies reporting a downregulation of the sodium pump following reactive gliosis [ 65 , 66 ] .
moreover , sodium is not buffered in the cell and travels solely by means of diffusion in the cytoplasm .
hindered diffusion , resulting from increased cytoplasmic protein content or impaired gap junction coupling which both occur upon astrocytic activation , might thus also partially contribute to delayed sodium recovery [ 12 , 34 , 67 ] .
in contrast to scar cells , the amplitude of d - aspartate - induced intracellular sodium signals was not significantly altered in moderately activated astrocytes in the periphery of the lesion , indicating that the overall number of functional glutamate transporters was not significantly altered . because these cells showed hypertrophy as well ,
the slowed kinetics of the sodium transients are in line with an increased cellular volume as well as a possible downregulation of na / k - atpase and/or a slowed diffusion as argued above .
taken together , our results indicate a significant reduction in glutamate uptake capacity in strongly activated astrocytes in the scar region .
thus , the prominent clustering of glast and glt-1 immunoreactivity along gfap - positive structures close to the lesion is likely to reflect a loss of glutamate transporters from the plasma membrane upon transporter internalization as reported by nakagawa et al . .
the authors of the latter study speculated that large and prolonged increases in glutamate concentrations are necessary to induce such cluster formation and endocytosis in the tissue , as they would possibly only occur under pathological conditions .
indeed , ambient glutamate concentrations were shown to be significantly increased after traumatic brain injury [ 6870 ] .
along these lines , it can be assumed that in our model system , extracellular glutamate concentrations rose less severely in the periphery of the lesion , preventing a comparable clustering and loss of functional glutamate transport activity in moderately activated astrocytes .
our results show complex changes in glutamate transporter expression and function during astrocyte activation following mechanical injury .
they confirm that the degree of astrocyte activation depends on the distance from the insult and comprises different endpoints regarding cellular morphology and physiology .
furthermore , our results highlight that astrocytes which show different grades of reactivity also display discrete changes in glutamate transporter expression and function .
while immunohistochemistry revealed a prominent clustering of glt-1 and glast immunoreactivity in the scar region , our functional assay clearly showed that glutamate uptake capacity is strongly reduced in scar cells , while it is largely maintained in moderately activated astrocytes in the periphery .
thus , mild - to - moderate astrogliosis in the periphery of a mechanical lesion does not necessarily seem to be accompanied by a significant change in glial glutamate uptake capacity . at the glial scar itself , in strongly reactive astrocytes
, a clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in astroglial glutamate uptake , which may contribute to glutamate - mediated excitotoxicity in this region .
 astrocytes express the sodium - dependent glutamate transporters glast and glt-1 , which are critical to maintain low extracellular glutamate concentrations . here , we analyzed changes in their expression and function following a mechanical lesion in the ca1 area of organotypic hippocampal slices . 
 6 - 7 days after lesion , a glial scar had formed along the injury site , containing strongly activated astrocytes with increased gfap and s100 immunoreactivity , enlarged somata , and reduced capability for uptake of sr101 . astrocytes in the scar 's periphery were swollen as well , but showed only moderate upregulation of gfap and s100 and efficiently took up sr101 . in the scar , clusters of glt-1 and glast immunoreactivity colocalized with gfap - positive fibers . apart from these , 
 glt-1 immunoreactivity declined with increasing distance from the scar , whereas glast expression appeared largely uniform . 
 sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery . 
 our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function . 
 strongly reactive astrocytes in the scar , however , display clusters of glast and glt-1 immunoreactivity together with reduced glutamate transport activity . 
 this reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions