Data on the effect of a muscimol treatment in caspase activation in descending neurons of lampreys after a complete spinal cord injury

Da a A icle Da a on he e ec o a muscimol ea men in caspase ac i a ion in descending neu ons o lamp eys a e a comple e spinal co d inju y Daniel Sob ido-Cameán, Ma ía Celina Rodicio 1 , An ón Ba ei o-Iglesias n ,1 Depa men o Func ional Biology, CIBUS, Facul y o Biology, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain a icle in o A icle his o y: Recei ed 26 Sep embe 2018 Recei ed in e ised o m 31 Oc obe 2018 Accep ed 1 No embe 2018 A ailable online 6 No embe 2018 abs ac In his a icle, caspase ac i a ion in iden ifiable e iculospinal neu ons o lamp eys was inhibi ed a e a comple e spinal co d inju y using a specific agonis o he GABAA ecep o (muscimol). The da a p esen ed in his a icle a e quan ifica ions o fluo escen labelling o iden ifiable descending neu ons o la al lamp eys a e a comple e spinal co d inju y using fluo och ome-labelled inhibi o s o caspases (FLICA) and he co esponding s a is ical analysis. A single dose o muscimol dec eased he in ensi y o FLICA labelling in gian iden ifiable e iculospinal neu ons ollowing spinal co d inju y in lamp eys. &2018 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Specifica ions able Subjec a ea Neu oscience Mo e specific subjec a ea Regene a i e biology Type o da a G aph, Figu e, Table Con en s lis s a ailable a ScienceDi ec jou nal homepage: www.else ie .com/loca e/dib Da a in B ie h ps://doi.o g/10.1016/j.dib.2018.11.003 2352-3409/&2018 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). n Co espondence o: Depa amen o de Biología Funcional, Edificio CIBUS, Campus Vida, Uni e sidade de San iago de Compos ela, CP. 15782 San iago de Compos ela, A Co uña, Spain. E-mail add ess: [email p o ec ed] (A. Ba ei o-Iglesias). 1 Equal con ibu o s. Da a in B ie 21 (2018) 2037–2041 How da a was acqui ed Con ocal mic oscope (TCS-SP2; Leica, We zla , Ge many) Da a o ma Analysed da a, p ocessed da a. Expe imen al ac o s La al sea lamp eys we e ea ed wi h muscimol a e a comple e spinal co d inju y. Caspase ac i a ion was analysed in iden ifiable descending neu ons using fluo och ome-labelled inhibi o s o cas- pases (FLICA). Expe imen al ea u es The e ec o muscimol on caspase ac i a ion a e a comple e spinal co d inju y was analysed using fluo escence mic oscopy. Da a sou ce loca ion Depa men o Func ional Biology, Facul y o Biology, CIBUS, Uni- e sidade de San iago de Compos ela Da a accessibili y The da a a e a ailable wi hin his a icle. Rela ed esea ch a icle Romaus-Sanju jo e al. [1] Value o he da a  Fluo och ome-labelled inhibi o s o caspases allow he de ec ion o changes in caspase ac i a ion.  This da ase is o in e es o hose s udying signalling pa hways modula ing neu onal su i al a e spinal co d inju y in fishes.  This da ase is o in e es o he de elopmen o neu op o ec an s as a he apy o spinal co d inju y. 1. Da a Recen ly, ou g oup epo ed ha endogenous GABA p omo es axonal egene a ion o iden ifiable e iculospinal neu ons a e a comple e spinal co d ansec ion in lamp eys [1]. The beneficial e ec o GABA appea s o be caused by a educ ion in he ac i a ion o caspases h ough he ac i a ion o GABAB ecep o s [1]. O he au ho s also epo ed ha inc eased GABAe gic inhibi ion ough GABAA ecep o s is ela ed o a be e eco e y o unc ion ollowing spinal co d inju y in lamp eys [2]. He e, we show ha he ac i a ion o GABAA ecep o s a e a comple e spinal co d ansec ion also inhibi s caspase ac i a ion in iden ifiable e iculospinal neu ons o lamp eys. A ea men wi h a single dose o muscimol, which is a specific agonis o he GABAA ecep o , ollowing a comple e spinal co d ansec ion educed he ac i a ion o caspases in gian e iculospinal neu ons o lamp eys as e ealed by a dec eased fluo escen in ensi y o FLICA labelling (Fig. 1). Table 1 shows he mean 7S.E.M. o fluo escence in ensi y o each neu onal ype and he numbe o iden ifiable neu ons used o he s a is ical analysis. The da a om each indi idual neu on is gi en in Table S1. 2. Expe imen al design, ma e ials, and me hods 2.1. Animals All expe imen s in ol ing animals we e app o ed by he Bioe hics Commi ee a he Uni e si y o San iago de Compos ela and he Conselle ía do Medio Ru al e do Ma o he Xun a de Galicia (License e e ence JLPV/IId; Galicia, Spain) and we e pe o med in acco dance o Eu opean Union and Spanish guidelines on animal ca e and expe imen a ion. Animals we e deeply anaes he ized wi h 0.1% MS-222 (Sigma, S . Louis, MO) in lamp ey Ringe solu ion (137 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl 2 ,2 mM HEPES; pH 7.4) be o e all expe imen al p ocedu es and eu hanized by decapi a ion a he end o he expe imen s. Ma u e and de elopmen ally s able la al sea lamp eys, Pe omyzon ma inus L. (n¼17; be ween 95 and 120 mm in body leng h, 5–7 yea s o age), we e used in he s udy. La al lamp eys we e collec ed om he i e Ulla (Galicia, Spain), wi h pe mission om he Xun a de Galicia) and D. Sob ido-Cameán e al. / Da a in B ie 21 (2018) 2037–20412038 main ained in ae a ed esh wa e aqua ia a 14–20 °C wi h a bed o i e sedimen un il hei use in expe imen al p ocedu es. Lamp eys we e andomly dis ibu ed be ween he di e en expe imen al g oups. 2.2. Spinal co d inju y su gical p ocedu es Comple e spinal co d ansec ions we e pe o med as p e iously desc ibed [3]. The os al spinal co d was exposed om he do sal midline a he le el o he 5 h gill by making a longi udinal incision wi h a scalpel (#11). A comple e spinal co d ansec ion was pe o med wi h Cas o iejo scisso s and he spinal co d cu ends we e isualized unde he s e eomic oscope. Animals wi h a comple e spinal co d ansec ion we e assigned o ei he a ehicle ea ed con ol g oup (n¼8) o o muscimol ea ed g oup (n¼9). A e spinal ansec ions, he animals we e e u ned o esh wa e anks. The animals we e allowed o eco e in indi idual esh wa e anks a 19.5 °C. Animals we e analysed 2-weeks pos -lesion (wpl). The expe imen was ca ied ou in 3 di e en ba ches o animals. Fig. 1. Muscimol ea men inhibi s caspase ac i a ion in iden ifiable descending neu ons. A: Pho omic og aph o a whole-moun ed b ain showing iden ifiable descending neu ons wi h in ense FLICA labelling in con ol animals. B: Pho omic og aph o a whole- moun ed b ain showing iden ifiable descending neu ons wi h a educ ion in he in ensi y o FLICA labelling in muscimol ea ed animals. C: G aph showing a significan change (Mann Whi ney U- es , p¼0.0054; as e isks) in he le el o caspase ac i a ion (in ensi y o fluo escen FLICA labelling; Yaxis) a e he muscimol ea men in iden ifiable descending neu ons (Xaxis). Ros al is up and he en icle o he le in all pho omic og aphs. Scale ba s: 150 mm. D. Sob ido-Cameán e al. / Da a in B ie 21 (2018) 2037–2041 2039 2.3. D ug ea men s Muscimol was dissol ed in dis illed wa e a a concen a ion o 25 mM, soaked in a small piece o Gel oam (Pfize ; New Yo k, NY) and placed on op o he si e o inju y a he ime o ansec ion as p e iously desc ibed [4]. Gel oam soaked in dis illed wa e se ed as a con ol. 2.4. De ec ion o ac i a ed caspases in whole-moun ed b ain p epa a ions The Image-iT LIVE G een Poly Caspases De ec ion Ki (Ca . No. I35104, In i ogen, USA) was used o de ec ac i a ed caspases in iden ifiable descending neu ons ( he M1, M2, M3, I1, I2, I3, I4, I5, I6, B1, B2, B3, B4, B5, B6, M h and M h’neu ons) o la al sea lamp eys 2 weeks a e he comple e spinal co d ansec ion. This ki con ains 1 ial (componen A o he ki ) o he lyophilized FLICA eagen (FAM-VAD-FMK). The eagen associa es a fluo ome hyl ke one (FMK) moie y, which can eac co alen ly wi h a cys eine, wi h a caspase-specific aminoacid sequence [ aline-alanine-aspa ic acid (VAD)]. A ca boxyfluo escein g oup (FAM) is a ached as a fluo escen epo e . The FLICA eagen in e ac s wi h he enzyme ac i e cen e o an ac i a ed caspase ia he ecogni ion sequence, and hen a aches co alen ly h ough he FMK moie y. Expe imen s o he de ec ion o ac i a ed caspases in whole-moun ed b ain p epa a ions using FLICA labelling we e done as p e iously desc ibed [5]. 2.5. Imaging and quan ifica ions The quan ifica ion o he in ensi y o FLICA labelling was done as p e iously desc ibed [6]. B iefly, pho omic og aphs we e acqui ed wi h a spec al con ocal mic oscope (model TCS-SP2; Leica, We zla , Ge many). Images we e always acqui ed unde he same mic oscope condi ions o con ol and ea ed animals. Quan ifica ion o mean fluo escen in ensi y (mean g ey alue) o each iden ifiable neu on was done using he Fiji so wa e [7]. The mean o fluo escence in ensi y o each ype o iden ifiable descending neu on was used o s a is ical analyses. Figu e pla es we e gene a ed using Adobe Pho oshop CS6 (Adobe Sys ems). Table 1 Table showing he o al numbe o iden ifiable e iculospinal neu ons ha we e included in he analyses and he mean 7S.E. M. o fluo escence in ensi y o FLICA labelling o each iden ifiable neu on. The da a om each indi idual neu on is gi en in Table S1. Con ol Muscimol Mean S.E.M. NMean S.E.M. N M1 96.53958 712.3778 12 75.67378 713.63257 14 M2 101.704 710.53632 12 58.14621 77.223279 14 M3 89.88461 710.92918 13 79.14021 712.03266 14 I1 109.5599 715.80085 12 88.42269 711.18598 16 I2 68.21775 710.45724 12 57.74719 77.989043 16 I3 75.28416 79.889297 13 71.58913 79.475986 16 I4 88.61723 712.39366 13 64.75288 79.115862 16 I5 65.69509 79.503008 11 50.27394 76.789322 16 I6 82.24782 717.39615 11 49.91087 75.125011 16 B1 91.59154 712.3856 15 85.62478 710.22685 18 B2 72.6452 79.034819 15 71.90128 79.277125 18 B3 105.5087 714.23046 15 87.06628 711.55049 18 B4 89.14333 710.9955 15 78.09394 77.780739 18 B5 74.33907 79.773049 15 73.49995 712.7075 18 B6 93.76653 714.91869 15 72.91844 711.33416 18 M h 89.1006 713.2872 15 90.32294 713.61253 18 M h' 61.15147 77.887609 15 56.45844 76.828169 18 D. Sob ido-Cameán e al. / Da a in B ie 21 (2018) 2037–20412040 2.6. S a is ical analyses S a is ical analysis was ca ied ou using P ism 6 (G aphPad so wa e, La Jolla, CA). Da a a e p e- sen ed as mean 7S.E.M. No mali y o he da a was de e mined by he Shapi o-Wilk, D'Agos ino and Pea son omnibus and Kolmogo o –Smi no no mali y es s. All da a passed all he no mali y es s. The esul s o con ol e sus ea men g oups we e analysed by Mann-Whi ney U- es . Acknowledgmen s G an sponso s: Spanish Minis y o Economy and Compe i i eness and he Eu opean Regional De elopmen Fund 2007–2013 (G an s numbe : BFU2014-56300-P and BFU2017-87079-P) o M.C.R. A.B.-I. was suppo ed by a g an om he Xun a de Galicia (G an numbe : 2016-PG008) and a g an om he c owd unding pla o m P ecipi a (FECYT; Spanish Minis y o Economy and Compe i i e- ness; g an numbe 2017-CP081). The au ho s would like o acknowledge he ollowing indi idual dono s o he c owd unding campaign in P ecipi a: Blanca Fe nández, Emilio Río, Guille mo Vi a , Pablo Pé ez, Jo ge Fé nandez, Ignacio Valino, Pago de los Cen ena ios, E a Candal, Ma ía del Pila Balsa, Jo ge Fa aldo, Isabel Rod íguez-Moldes, José Manuel López, Juan José Pi a, Ma ía E. Cameán, Jesús To es, José Puma es, Ve ónica Rod íguez, Sa a López, Tania Villa es Balsa, Rocío Lizcano, José Ga cía, Ana M. Ce eijo, Ma ía Pa do, Ne ea San ama ía, Ca olina He nández, Jesús López and Ma ía Manei o. The au ho s hank he s a o Ximonde Biological S a ion o p o iding lamp eys used in his s udy, and he Mic oscopy Se ice (Uni e si y o San iago de Compos ela) and D . Me cedes Ri as Cascalla o con ocal mic oscope acili ies and help. T anspa ency documen . Suppo ing in o ma ion T anspa ency da a associa ed wi h his a icle can be ound in he online e sion a h ps://doi.o g/ 10.1016/j.dib.2018.11.003. Appendix A. Suppo ing in o ma ion Supplemen a y da a associa ed wi h his a icle can be ound in he online e sion a h ps://doi. o g/10.1016/j.dib.2018.11.003. Re e ences [1] D. Romaus-Sanju jo, R. Ledo-Ga cía, B. Fe nández-López, K. Hanslik, J.R. Mo gan, A. Ba ei o-Iglesias, M.C. Rodicio, GABA p omo es su i al and axonal egene a ion in iden ifiable descending neu ons a e spinal co d inju y in la al lamp eys, Cell Dea h Dis. 9 (2018) 663. h ps://doi.o g/10.1038/s41419-018-0704-9. [2] E. S ensson, O. Kim, D. Pa ke , Al e ed GABA and soma os a in modula ion o p op iocep i e eedback a e spinal co d inju y in lamp ey, Neu oscience 3 (2013) 109–118. h ps://doi.o g/10.1016/j.neu oscience.2013.01.017. [3] A. Ba ei o-Iglesias, G. Zhang, M.E. Selze , M.I. Shi man, Comple e spinal co d inju y and b ain dissec ion p o ocol o subsequen wholemoun in si u hyb idiza ion in la al sea lamp ey, J. Vis. Exp. 92 (2014) e51494. h ps://doi.o g/10.3791/51494. [4] D.Sob ido-Camean,D.Robledo,L.Sanchez,M.C.Rodicio,A.Ba ei o-Iglesias, Se o onin inhibi s axonal egene a ion o iden ifiable descending neu ons a e a comple e spinal co d inju y in lamp eys, bioRxi (2018) 335844. h ps://doi.o g/10.1101/335844. [5] A. Ba ei o-Iglesias, A, M.I. Shi man, Use o fluo och ome-labeled inhibi o s o caspases o de ec neu onal apop osis in he whole-moun ed lamp ey b ain a e spinal co d inju y, Enzym. Res 2012 (2012) 835731. h ps://doi.o g/10.1155/2012/835731. [6] A. Ba ei o-Iglesias, D. Sob ido-Cameán, M.I. Shi man, Re og ade ac i a ion o he ex insic apop o ic pa hway in spinal- p ojec ing neu ons a e a comple e spinal co d inju y in lamp eys, Biomed. Res. In . 2017 (2017) 5953674. h ps://doi.o g/ 10.1155/2017/5953674. [7] J. Schindelin, I. A ganda-Ca e as, E. F ise, V. Kaynig, M. Longai , T. Pie zsch, S. P eibisch, C. Rueden, S. Saal eld, B. Schmid, J.Y. Tine ez, D.J. Whi e, V. Ha ens ein, K. Elicei i, P. Tomancak, A. 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