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

Data Article Data on the effect of a muscimol treatment in caspase activation in descending neurons of lampreys after a complete spinal cord injury Daniel Sobrido-Cameán, María Celina Rodicio 1 , Antón Barreiro-Iglesias n ,1 Department of Functional Biology, CIBUS, Faculty of Biology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain article info Article history: Received 26 September 2018 Received in revised form 31 October 2018 Accepted 1 November 2018 Available online 6 November 2018 abstract In this article, caspase activation in identifiable reticulospinal neurons of lampreys was inhibited after a complete spinal cord injury using a specific agonist of the GABAA receptor (muscimol). The data presented in this article are quantifications of fluorescent labelling of identifiable descending neurons of larval lampreys after a complete spinal cord injury using fluorochrome-labelled inhibitors of caspases (FLICA) and the corresponding statistical analysis. A single dose of muscimol decreased the intensity of FLICA labelling in giant identifiable reticulospinal neurons following spinal cord injury in lampreys. &2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Specifications table Subject area Neuroscience More specific subject area Regenerative biology Type of data Graph, Figure, Table Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/dib Data in Brief https://doi.org/10.1016/j.dib.2018.11.003 2352-3409/&2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). n Correspondence to: Departamento de Biología Funcional, Edificio CIBUS, Campus Vida, Universidade de Santiago de Compostela, CP. 15782 Santiago de Compostela, A Coruña, Spain. E-mail address: [email protected] (A. Barreiro-Iglesias). 1 Equal contributors. Data in Brief 21 (2018) 2037–2041 How data was acquired Confocal microscope (TCS-SP2; Leica, Wetzlar, Germany) Data format Analysed data, processed data. Experimental factors Larval sea lampreys were treated with muscimol after a complete spinal cord injury. Caspase activation was analysed in identifiable descending neurons using fluorochrome-labelled inhibitors of caspases (FLICA). Experimental features The effect of muscimol on caspase activation after a complete spinal cord injury was analysed using fluorescence microscopy. Data source location Department of Functional Biology, Faculty of Biology, CIBUS, Universidade de Santiago de Compostela Data accessibility The data are available within this article. Related research article Romaus-Sanjurjo et al. [1] Value of the data  Fluorochrome-labelled inhibitors of caspases allow the detection of changes in caspase activation.  This dataset is of interest for those studying signalling pathways modulating neuronal survival after spinal cord injury in fishes.  This dataset is of interest for the development of neuroprotectants as a therapy for spinal cord injury. 1. Data Recently, our group reported that endogenous GABA promotes axonal regeneration of identifiable reticulospinal neurons after a complete spinal cord transection in lampreys [1]. The beneficial effect of GABA appears to be caused by a reduction in the activation of caspases through the activation of GABAB receptors [1]. Other authors also reported that increased GABAergic inhibition trough GABAA receptors is related to a better recovery of function following spinal cord injury in lampreys [2]. Here, we show that the activation of GABAA receptors after a complete spinal cord transection also inhibits caspase activation in identifiable reticulospinal neurons of lampreys. A treatment with a single dose of muscimol, which is a specific agonist of the GABAA receptor, following a complete spinal cord transection reduced the activation of caspases in giant reticulospinal neurons of lampreys as revealed by a decreased fluorescent intensity of FLICA labelling (Fig. 1). Table 1 shows the mean 7S.E.M. of fluorescence intensity of each neuronal type and the number of identifiable neurons used for the statistical analysis. The data from each individual neuron is given in Table S1. 2. Experimental design, materials, and methods 2.1. Animals All experiments involving animals were approved by the Bioethics Committee at the University of Santiago de Compostela and the Consellería do Medio Rural e do Mar of the Xunta de Galicia (License reference JLPV/IId; Galicia, Spain) and were performed in accordance to European Union and Spanish guidelines on animal care and experimentation. Animals were deeply anaesthetized with 0.1% MS-222 (Sigma, St. Louis, MO) in lamprey Ringer solution (137 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl 2 ,2 mM HEPES; pH 7.4) before all experimental procedures and euthanized by decapitation at the end of the experiments. Mature and developmentally stable larval sea lampreys, Petromyzon marinus L. (n¼17; between 95 and 120 mm in body length, 5–7 years of age), were used in the study. Larval lampreys were collected from the river Ulla (Galicia, Spain), with permission from the Xunta de Galicia) and D. Sobrido-Cameán et al. / Data in Brief 21 (2018) 2037–20412038 maintained in aerated fresh water aquaria at 14–20 °C with a bed of river sediment until their use in experimental procedures. Lampreys were randomly distributed between the different experimental groups. 2.2. Spinal cord injury surgical procedures Complete spinal cord transections were performed as previously described [3]. The rostral spinal cord was exposed from the dorsal midline at the level of the 5th gill by making a longitudinal incision with a scalpel (#11). A complete spinal cord transection was performed with Castroviejo scissors and the spinal cord cut ends were visualized under the stereomicroscope. Animals with a complete spinal cord transection were assigned to either a vehicle treated control group (n¼8) or to muscimol treated group (n¼9). After spinal transections, the animals were returned to fresh water tanks. The animals were allowed to recover in individual fresh water tanks at 19.5 °C. Animals were analysed 2-weeks post-lesion (wpl). The experiment was carried out in 3 different batches of animals. Fig. 1. Muscimol treatment inhibits caspase activation in identifiable descending neurons. A: Photomicrograph of a whole-mounted brain showing identifiable descending neurons with intense FLICA labelling in control animals. B: Photomicrograph of a wholemounted brain showing identifiable descending neurons with a reduction in the intensity of FLICA labelling in muscimol treated animals. C: Graph showing a significant change (Mann Whitney U-test, p¼0.0054; asterisks) in the level of caspase activation (intensity of fluorescent FLICA labelling; Yaxis) after the muscimol treatment in identifiable descending neurons (Xaxis). Rostral is up and the ventricle to the left in all photomicrographs. Scale bars: 150 mm. D. Sobrido-Cameán et al. / Data in Brief 21 (2018) 2037–2041 2039 2.3. Drug treatments Muscimol was dissolved in distilled water at a concentration of 25 mM, soaked in a small piece of Gelfoam (Pfizer; New York, NY) and placed on top of the site of injury at the time of transection as previously described [4]. Gelfoam soaked in distilled water served as a control. 2.4. Detection of activated caspases in whole-mounted brain preparations The Image-iT LIVE Green Poly Caspases Detection Kit (Cat. No. I35104, Invitrogen, USA) was used to detect activated caspases in identifiable descending neurons (the M1, M2, M3, I1, I2, I3, I4, I5, I6, B1, B2, B3, B4, B5, B6, Mth and Mth’neurons) of larval sea lampreys 2 weeks after the complete spinal cord transection. This kit contains 1 vial (component A of the kit) of the lyophilized FLICA reagent (FAM-VAD-FMK). The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific aminoacid sequence [valine-alanine-aspartic acid (VAD)]. A carboxyfluorescein group (FAM) is attached as a fluorescent reporter. The FLICA reagent interacts with the enzyme active centre of an activated caspase via the recognition sequence, and then attaches covalently through the FMK moiety. Experiments for the detection of activated caspases in whole-mounted brain preparations using FLICA labelling were done as previously described [5]. 2.5. Imaging and quantifications The quantification of the intensity of FLICA labelling was done as previously described [6]. Briefly, photomicrographs were acquired with a spectral confocal microscope (model TCS-SP2; Leica, Wetzlar, Germany). Images were always acquired under the same microscope conditions for control and treated animals. Quantification of mean fluorescent intensity (mean grey value) of each identifiable neuron was done using the Fiji software [7]. The mean of fluorescence intensity of each type of identifiable descending neuron was used for statistical analyses. Figure plates were generated using Adobe Photoshop CS6 (Adobe Systems). Table 1 Table showing the total number of identifiable reticulospinal neurons that were included in the analyses and the mean 7S.E. M. of fluorescence intensity of FLICA labelling of each identifiable neuron. The data from each individual neuron is given in Table S1. Control 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 Mth 89.1006 713.2872 15 90.32294 713.61253 18 Mth' 61.15147 77.887609 15 56.45844 76.828169 18 D. Sobrido-Cameán et al. / Data in Brief 21 (2018) 2037–20412040 2.6. Statistical analyses Statistical analysis was carried out using Prism 6 (GraphPad software, La Jolla, CA). Data are presented as mean 7S.E.M. Normality of the data was determined by the Shapiro-Wilk, D'Agostino and Pearson omnibus and Kolmogorov–Smirnov normality tests. All data passed all the normality tests. The results of control versus treatment groups were analysed by Mann-Whitney U-test. Acknowledgments Grant sponsors: Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund 2007–2013 (Grants number: BFU2014-56300-P and BFU2017-87079-P) to M.C.R. A.B.-I. was supported by a grant from the Xunta de Galicia (Grant number: 2016-PG008) and a grant from the crowdfunding platform Precipita (FECYT; Spanish Ministry of Economy and Competitiveness; grant number 2017-CP081). The authors would like to acknowledge the following individual donors of the crowdfunding campaign in Precipita: Blanca Fernández, Emilio Río, Guillermo Vivar, Pablo Pérez, Jorge Férnandez, Ignacio Valino, Pago de los Centenarios, Eva Candal, María del Pilar Balsa, Jorge Faraldo, Isabel Rodríguez-Moldes, José Manuel López, Juan José Pita, María E. Cameán, Jesús Torres, José Pumares, Verónica Rodríguez, Sara López, Tania Villares Balsa, Rocío Lizcano, José García, Ana M. Cereijo, María Pardo, Nerea Santamaría, Carolina Hernández, Jesús López and María Maneiro. The authors thank the staff of Ximonde Biological Station for providing lampreys used in this study, and the Microscopy Service (University of Santiago de Compostela) and Dr. Mercedes Rivas Cascallar for confocal microscope facilities and help. Transparency document. Supporting information Transparency data associated with this article can be found in the online version at https://doi.org/ 10.1016/j.dib.2018.11.003. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at https://doi. org/10.1016/j.dib.2018.11.003. References [1] D. Romaus-Sanjurjo, R. Ledo-García, B. Fernández-López, K. Hanslik, J.R. Morgan, A. Barreiro-Iglesias, M.C. Rodicio, GABA promotes survival and axonal regeneration in identifiable descending neurons after spinal cord injury in larval lampreys, Cell Death Dis. 9 (2018) 663. https://doi.org/10.1038/s41419-018-0704-9. [2] E. Svensson, O. Kim, D. Parker, Altered GABA and somatostatin modulation of proprioceptive feedback after spinal cord injury in lamprey, Neuroscience 3 (2013) 109–118. https://doi.org/10.1016/j.neuroscience.2013.01.017. [3] A. Barreiro-Iglesias, G. Zhang, M.E. Selzer, M.I. Shifman, Complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey, J. Vis. Exp. 92 (2014) e51494. https://doi.org/10.3791/51494. [4] D.Sobrido-Camean,D.Robledo,L.Sanchez,M.C.Rodicio,A.Barreiro-Iglesias, Serotonin inhibits axonal regeneration of identifiable descending neurons after a complete spinal cord injury in lampreys, bioRxiv (2018) 335844. https://doi.org/10.1101/335844. [5] A. Barreiro-Iglesias, A, M.I. Shifman, Use of fluorochrome-labeled inhibitors of caspases to detect neuronal apoptosis in the whole-mounted lamprey brain after spinal cord injury, Enzym. Res 2012 (2012) 835731. https://doi.org/10.1155/2012/835731. [6] A. Barreiro-Iglesias, D. Sobrido-Cameán, M.I. Shifman, Retrograde activation of the extrinsic apoptotic pathway in spinalprojecting neurons after a complete spinal cord injury in lampreys, Biomed. Res. Int. 2017 (2017) 5953674. https://doi.org/ 10.1155/2017/5953674. [7] J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.Y. Tinevez, D.J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, A. Cardona, Fiji: an open-source platform for biologicalimage analysis, Nat. Methods. 9 (2012) 676–682. https://doi.org/10.1038/nmeth.2019. D. Sobrido-Cameán et al. / Data in Brief 21 (2018) 2037–2041 2041