Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord

Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord
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  See discussions, stats, and author profiles for this publication at: Functional expression of release-regulatingglycine transporters GLYT1 on GABAergicneurons and GLYT2 on astrocytes in...  Article   in  Neurochemistry International · February 2008 DOI: 10.1016/j.neuint.2007.04.027 · Source: PubMed CITATIONS 38 READS 35 8 authors , including: Some of the authors of this publication are also working on these related projects: Quantitative Microscopy   View projectPublic Dissemination of Science   View projectCesare UsaiItalian National Research Council 144   PUBLICATIONS   2,696   CITATIONS   SEE PROFILE Alberto DiasproIstituto Italiano di Tecnologia 595   PUBLICATIONS   6,317   CITATIONS   SEE PROFILE Marco MilaneseUniversità degli Studi di Genova 59   PUBLICATIONS   869   CITATIONS   SEE PROFILE Giambattista BonannoUniversità degli Studi di Genova 188   PUBLICATIONS   4,250   CITATIONS   SEE PROFILE All content following this page was uploaded by Marco Milanese on 30 December 2013. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  This article was published in an Elsevier journal. The attached copyis furnished to the author for non-commercial research andeducation use, including for instruction at the author’s institution,sharing with colleagues and providing to institution administration.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Functional expression of release-regulating glycine transportersGLYT1 on GABAergic neurons and GLYT2 onastrocytes in mouse spinal cord Luca Raiteri a,1 , Sara Stigliani a,1 , Cesare Usai b , Alberto Diaspro c , Silvio Paluzzi a ,Marco Milanese a , Maurizio Raiteri a,d, *, Giambattista Bonanno a,d a  Department of Experimental Medicine, Pharmacology and Toxicology Section, University of Genoa, Italy b  Institute of Biophysics, National Research Council, Genoa, Italy c  National Institute for the Physics of Matter (INFM) and Department of Physics, University of Genoa, Italy d Center of Excellence for Biomedical Research, University of Genoa, Italy Received 27 February 2007; received in revised form 24 April 2007; accepted 27 April 2007Available online 16 May 2007 Abstract It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocyteswhereas GLYT2 are present on glycinergic neuronalterminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [ 3 H]GABA through GAT1 transporters and, when exposed to glycine insuperfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetrationthrough selective transporters. The glycine-evoked release of [ 3 H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated fromgliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally toevoke [ 3 H]GABA release from GABAergic synaptosomes; even more surprising, the ‘neuronal’ GLYT2 contributed more efficiently than the‘glial’ GLYT1 to mediate the glycine effect in [ 3 H]GABA releasing gliosomes. These functional results were largely confirmed by confocalmicroscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positivesynaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes,indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord. # 2007 Elsevier Ltd. All rights reserved. Keywords:  Glycine transporters; GLYT1; GLYT2; GABA release; Synaptosomes; Gliosomes 1. Introduction GABA and glycine co-exist in the terminal boutons of spinalinterneurons(ToddandSullivan,1990;O¨rnungetal.,1994;Toddet al., 1996) from which they are co-released to activate glycineand GABA A  receptors co-localized on motor neurons (Joneset al., 1998; Keller et al., 2001). However, very little is knownabout possible interactions between the two co-transmitters,particularly at the presynaptic level.There is convincing evidence that transporters for differenttransmitters may co-exist on a same axon terminal, where theirfunction seems not to be limited to co-transmitter reuptake(Bonanno and Raiteri, 1994; Raiteri et al., 2002). It has been clearlyshownthatactivationoftransporterAcanelicitreleaseof the transmitter previously taken up through the co-existingtransporterBorsynthesizedintheterminalcarryingtransportersA and B (Raiteri et al., 2002). In particular, experiments with ratspinal cord synaptosomes had shown that glycine can directlyevoke release of pre-accumulated [ 3 H]GABA in a receptor-independent, external Na + -dependent manner (Raiteri et al.,1992). In that work, the involvement of glycine transporters International 52 (2008) 103–112* Corresponding author at: Department of Experimental Medicine, Pharma-cology and Toxicology Section, University of Genoa, Italy.Tel.: +39 010 3532651; fax: +39 010 3993360. E-mail address: (M. Raiteri). 1 These authors contributed equally to this work.0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.neuint.2007.04.027  Author's personal copy (GLYTs) and their characterization was not determined becauseselective GLYT blockers were not available.The subsequent cloning of two types of GLYTs, termedGLYT1 and GLYT2 (see for reviews, Lo´pez-Corcuera et al.,2001;Arago´nandLo´pez-Corcuera,2005;Eulenburgetal.,2005)and the availability of glycine transporter blockers, such asglycyldodecylamide(GDA),anon-selectiveinhibitor(JavittandFrusciante, 1997; Javitt et al., 1999), sarcosine, an inhibitor/ substrate of GLYT1 (Supplisson and Bergman, 1997) andamoxapine, a GLYT2 blocker (Nu´n˜ez et al., 2000), permitted toextend our srcinal observations. In synaptosomal preparationsfrom mouse spinal cord (Raiteri et al., 2001) glycine-evokedstrychnine-insensitive GABA release which was abolished byGDA, partly but to the same extent prevented by sarcosine oramoxapine and abrogated by a mixture of these two inhibitors,suggesting the presence of both GLYT1 and GLYT2 in oursynaptosomal preparation.This finding was unexpected because GLYT1 and GLYT2have been considered to be essentially glial and neuronaltransporters, respectively (Lo´pez-Corcuera et al., 2001). On the other hand, our study (Raiteri et al., 2001) was carried out withcrude synaptosomal preparations that may contain contaminat-ing gliosomes, fragments of glial cells produced during homo-genization and resealed. Although theviability of gliosomes hasbeen controversial (Henn et al., 1976; Ashton and Dolly, 2000), the possibility existed that gliosomes accumulated [ 3 H]GABAand released it very efficiently following penetration of glycinethrough GLYT1.A purified fraction of gliosomes has recently beenmorphologically and functionally characterized (Stiglianiet al., 2006). The availability of this preparation, its abilityto accumulate [ 3 H]GABA (Nakamura et al., 1993) and theavailability of more potent and selective GLYT1 and GLYT2inhibitors (Atkinson et al., 2001; Caulfield et al., 2001) prompted us to investigate the distribution and the function of GLYT1 and GLYT2 in purified synaptosomes and gliosomes. 2. Experimental procedures 2.1. Materials [ 3 H]GABA (specific activity: 84.0 Ci/mmol) and [ 3 H] D -aspartate (specificactivity: 37.3 Ci/mmol) was purchased from Amersham (Buckinghamshire,UK). Glycine, strychnine, aminooxyacetic acid, Percoll, anti-GFAP (rabbit,G9269)and anti-GFAP (mouse, G3893)primaryantibodies were obtained fromSigma Chemical Co. (St. Louis, MO, USA); 5,7-dichlorokynurenic acid wasfrom Tocris Cookson (Bristol, UK); BAPTA from Fluka Biochemika (Milan,Italy). Anti-GAT1 (rabbit, AB1570W), anti-MAP2 (mouse, MAB378), anti-GLYT1(goat,AB1770),anti-GLYT2(sheep,AB1771)primaryantibodieswereobtained from Chemicon (Temecula, CA, USA). Anti-GLYT1 (rabbit, GLYT1-A) primary antibody was obtained from BioTrend (Ko¨ln, Germany). Thedonkey anti-mouse Alexa Fluor 488-conjugated (A21202), anti rabbit AlexaFluor 488-conjugated (A21206), anti-mouse Alexa Fluor 647-conjugated(A31571), anti rabbit Alexa Fluor 647-conjugated (A31573), anti-sheep AlexaFluor 594-conjugated (A11016) and anti-goat Alexa Fluor 647-conjugated(A21447) secondary antibodies were purchased from Molecular Probes Europe(Leiden, The Netherlands). Glycyldodecylamide was donated by Abel Lajtha(Orangeburg, NY, USA). NFPS (ALX-5407) was a gift from NPS Allelix Corp(Mississauga, Ontario, Canada). ORG 25543B was a gift from Dr. HardySundaram (Organon Laboratories Ltd., Newhouse, Scotland) and SKF89976Awas a gift from Smith Kline & French (Welwyn, UK). 2.2. Animals Adult swiss mice (weighing 20–25 g; Charles River, Calco, Italy) wereused. Animals were housed at constant temperature (22  1  8 C) and relativehumidity(50%)underaregularlight/darkschedule(light7.00 a.mto7.00 p.m).Food and water were freely available. All experiments were carried out inaccordance with the European Community Council Directive of 24 November1986 (86/609/EEC). All efforts were made to minimize animal suffering and touse only the number of animals necessary to produce reliable results. 2.3. Preparation of gliosomes and synaptosomes Animalswere sacrificedandthespinalcordwasquicklyremoved.Purifiedgliosomes and synaptosomes were prepared essentially according to Naka-mura et al. (1993, 1994). The tissue was homogenized in 10 vol. of 0.32 M sucrose, buffered at pH 7.4 with Tris–HCl, using a glass-teflon tissue grinder(clearance 0.25 mm, 12 up-down strokes in about 1 min). In the experimentswith 1,2-bis-(2-aminophenoxy)ethane-  N  ,  N  ,  N  0 ,  N  0 -tetraacetic acid (BAPTA),thetissuewashomogenizedinthepresenceof1 mMofthecalciumchelatorinordertoentrapitintosynaptosomes(Raiterietal.,2000).Thehomogenatewascentrifuged (5 min, 1000  g  at 4  8 C) to remove nuclei and debris and thesupernatantwasgentlystratifiedonadiscontinuousPercoll 1 gradient(2,6,10and 20% (v/v) in Tris-buffered sucrose) and centrifuged at 33,500  g  for5 min. The layers between 2 and 6% Percoll 1 (gliosomal fraction) andbetween10and20%Percoll 1 (synaptosomalfraction)werecollected,washedby centrifugation and resuspended in physiological medium having thefollowing composition: 125 mM NaCl; 3 mM KCl; 1.2 mM MgSO 4 ;1.2 mM CaCl 2 ; 1 mM NaH 2 PO 4 ; 22 mM NaHCO 3 ; 10 mM glucose (aerationwith 95% O 2  and 5% CO 2 ), pH 7.2–7.4. 2.4. Experiments of release Gliosomes and synaptosomes were incubated at 37 8  C for 15 min in thepresence of 0.02  m M [ 3 H]GABA and of 50  m M aminooxyacetic acid (AOAA)to avoid GABA catabolism or in the presence of 0.04  m M [ 3 H] D -aspartate.Aliquotsofthesuspensions(about4or10  m gproteininthecaseofgliosomesorsynaptosomes, respectively) were distributed on microporous filters placed atthe bottom of a set of parallel superfusion chambers maintained at 37  8 C(Superfusion System, Ugo Basile, Comerio, Varese, Italy) and superfused(Raiteri et al., 1974) with standard medium (containing 50  m M AOAA inthe case of [ 3 H]GABA release experiments) at a rate of 0.5 ml/min. After33 min of superfusion with standard medium, five 3 min fractions werecollected. Gliosomes and synaptosomes were exposed to glycine at the endof the second fraction collected ( t   = 39 min). GDA, strychnine, 5,7-dichlor-okynurenic acid (5,7-DCK),  N  [3-(4 0 -fluorophenyl)-3-(4 0 -phenylphenoxy)-pro-pyl]sarcosine (NFPS), 4-benzoyl-3,5-dimethoxy-  N  -[1-(dimethylaminocyclopentyl)-methyl]benzamide (ORG 25543B) or  N  -(4,4-phenyl-3-butenyl)-guvacine (SKF 89976A) was introduced at  t   = 30 min. When appropriate, Ca 2+ wasomittedfrom the superfusionmediumat  t   = 20 min.The Ca 2+ -freemediumcontained 8.8 mM MgCl 2 , substituting for an isoosmotic amount of NaCl. 2.5. Calculations [ 3 H]GABA or [ 3 H] D -aspartate radioactivity wasdeterminedin each fractioncollected and in the superfused filters by liquid scintillation counting. Tritiumreleasedineachfractioncollectedwascalculatedasfractionalrate.Drugeffectswere evaluated by calculating the ratio between the efflux in the fourth fractioncollected (in which the maximum effect of glycine was generally reached) andthat of the second fraction. This ratio was compared to the corresponding ratioobtained under control conditions. Appropriate controls were always run inparallel. The concentration–response curves shown in Fig. 1 were fitted to theexperimental data using the following four parameters logistic equation,provided by the software Sigma Plot version 8.0:  y  =  a  + {( b  a )/[1 + (10 c  / 10  x ) d  ]}, where  a  is the minimum,  b  the maximum value,  c  the EC 50  and  d   is theslope of the curve.The two-tailed Student’s  t  -test was used for statistical comparison of thedata.  L. Raiteri et al./Neurochemistry International 52 (2008) 103–112 104  Author's personal copy 2.6. Confocal microscopy Gliosomes and synaptosomes (70  m g protein) obtained by means of Percollgradients were placed onto coverslips pre-treated with poly- L -ornithine andmaintained 30 min at 37  8 C in a 5% CO 2  atmosphere to allow setting andsticking to the substrate. All the following procedures were conducted at roomtemperature. The preparations were fixed with 2% paraformaldehyde (15 min),washed with PBS (3  5 min) and incubated (5 min) with 0.05% triton X-100.After washing (3   5 min) with PBS containing 0.5% serum albumin, thepreparations were incubated 30 min with the primary antibodies diluted in PBScontaining 3% albumin. The following antibodies were used: rabbit anti-glialfibrillary acidic protein (GFAP, 1:20); mouse anti-GFAP (1:40) mouse anti-microtubule associate protein type 2 (MAP2, 1:50); rabbit anti-GLYT1 (1:50);goat anti-GLYT1 (1:50); sheep anti-GLYT2 (1:50); rabbit anti-GABA trans-porters type 1 (GAT1, 1:50). After washing (3   5 min) with PBS containing0.5% serum albumin, the preparations were incubated 30 min with the appro-priate secondary Alexa Fluor 488-, Alexa Fluor 594-, or Alexa Fluor 647-labeledantibodiesdilutedinPBScontaining3%albuminandwashed3  5 min.Fluorescence image acquisition was performed by a three-channel Leica TCSSP2laser-scanningconfocalmicroscope,equippedwith458,476,488,514,543and 633 nm excitation lines. Images (512  512  8 bit) were taken through aplan-apochromatic oil immersion objective 100   /numeric aperture 1.4. Lightcollection configuration was optimized according to the combination of chosenfluorochromes and sequential channel acquisition was performed to avoidcross-talk phenomena. Leica LCS software package was used for acquisition,storage and visualization. The on-line web service ‘‘Power Up Your Micro-scope’’ (, Department of Physics, University of Genoa, Italy and Re@lityNET, Genova, Italy) was employed to performdeconvolution of images, increasing both image resolution and signal-to-noiseratio. The quantitative estimation of co-localized proteins was performedcalculating the ‘‘co-localization coefficients’’ (Manders et al., 1993). Theyexpress the fraction of co-localizing molecular species in each component of adual-colour image and are based on the Pearson’s correlation coefficient, astandard procedure for matching one imagewith another in pattern recognition.If two molecular species are co-localized, the overlay of their spatial distribu-tions has a correlation value higher then what would be expected by chancealone. Costes et al. (2004) developed an automated procedure to evaluate thecorrelation between the green and red channels with a significance level > 95%.The same procedure automatically determines an intensity threshold for eachcolour channel based on a linear least-square fit of the green and red intensitiesin the image’s 2D correlation cytofluorogram. Costes’ approach was accom-plished by macro routines integrated as plugins (WCIF Colocalization Plugins,Wright Cell Imaging Facility, Toronto Western Research Institute, Canada) inthe ImageJ 1.34f software (Wayne Rasband, National Institutes of Health). 3. Results Purified fractions of gliosomes and synaptosomes preparedfrom various areas of the rodent brain have been obtained andcharacterized in details, as described in very recent works(Pedrazzi et al., 2006; Stigliani et al., 2006). Fig. 1 (upper panel) shows that the mouse spinal cordgliosomes and synaptosomes used in the present investigationrepresent reasonably well purified fractions. Gliosome andsynaptosome preparations were labeled with the neuronalmarker MAP2 (green) and with the astrocytary marker GFAP(red) and analyzed by confocal microscopy. Gliosomes exhibitabundant labeling for GFAP but only a modest positiveness forMAP2; on the contrary, synaptosomes are efficiently stained byMAP2 but not by GFAP. In the two reports mentioned above anumber of differences between gliosomes and synaptosomes Fig. 1. (Upper panel) Immunocytochemical identification of the glial fibrillary acidic protein (GFAP) and the microtubule associated protein type 2 (MAP2) inpurifiedgliosomes(left)andsynaptosomes(right).GliosomesandsynaptosomeswerepurifiedusingadiscontinuousPercollgradientandgluedontocoverslips,fixedwith paraformaldehyde, permeabilized with triton X-100 and incubated with the primary and secondary antibodies. Samples were analyzed by laser confocalmicroscopy. Images show the Alexa Fluor 488-tagged anti-MAP2 (green) and the Alexa Fluor 647-tagged anti-GFAP (red). (Lower panel) Concentration–responsecurve of the glycine-evoked [ 3 H] D -aspartate release (left) or [ 3 H]GABA release (right) from mouse spinal cord gliosomes or synaptosomes. Gliosomes andsynaptosomes were purified using a discontinuous Percoll gradient, labeled with the radioactive tracers and exposed in superfusion to various concentrations of glycine. Glycine was added to the superfusion medium at the end of the second fraction collected and maintained until the end of the experiment. Fractions werecollected and counted for radioactivity. The release of [ 3 H] D -aspartate in the second fraction collected (control basal release) amounted to 5.21  0.24% and to5.95  0.31% of total gliosomal or synaptosomal tritium content ( n  = 10). The release of [ 3 H]GABA in the second fraction collected amounted to 4.18  0.19% andto 6.51  0.35%of total gliosomalor synaptosomal tritiumcontent ( n  = 10). Resultsare expressed aspercent potentiationof the basal release. The data presented aremeans  S.E.M. of five to ten experiments in triplicate (three superfusion chambers for each experimental condition).  L. Raiteri et al./Neurochemistry International 52 (2008) 103–112  105
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