Data on the Quantification of Aspartate, GABA and Glutamine Levels In

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Data Descriptor Data on the Quantiﬁcation of Aspartate, GABA and Glutamine Levels in the Spinal Cord of Larval Sea Lampreys after a Complete Spinal Cord Injury

Blanca Fernández-López 1 , Natividad Pereiro 2, Anunciación Lafuente 3,†, María Celina Rodicio 1 and Antón Barreiro-Iglesias 1,*

1 Department of Functional Biology, CIBUS, Faculty of Biology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] (B.F.-L.); [email protected] (M.C.R.) 2 Ministry of Health, Paseo del Prado, 18, 28014 Madrid, Spain; [email protected] 3 Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas s/n, 32004 Ourense, Spain * Correspondence: [email protected] † Deceased.

Abstract: We used high-performance liquid chromatography (HPLC) methods to quantify aspartate, GABA, and glutamine levels in the spinal cord of larval sea lampreys following a complete spinal cord injury. Mature larval sea lampreys recover spontaneously from a complete spinal cord transection and the changes in neurotransmitter systems after spinal cord injury might be related to their amazing regenerative capabilities. The data presented here show the concentration of the aminoacidergic neurotransmitters GABA (and its precursor glutamine) and aspartate in the spinal cord of control (non-injured) and 2-, 4-, and 10-week post-lesion animals. Statistical analyses showed that GABA Citation: Fernández-López, B.; and aspartate levels signiﬁcantly increase in the spinal cord four weeks after a complete spinal cord Pereiro, N.; Lafuente, A.; Rodicio, injury and that glutamine levels decrease 10 weeks after injury as compared to controls. These data M.C.; Barreiro-Iglesias, A. Data on the might be of interest to those studying the role of neurotransmitters and neuromodulators in recovery Quantiﬁcation of Aspartate, GABA from spinal cord injury in vertebrates. and Glutamine Levels in the Spinal Cord of Larval Sea Lampreys after a Dataset: http://doi.org/10.5281/zenodo.4772417 Complete Spinal Cord Injury. Data 2021, 6, 54. https://doi.org/10.3390/ Dataset License: CC-BY-NC. data6060054

Keywords: aspartate; GABA; glutamine; HPLC; spinal cord injury; lampreys Academic Editor: Juraj Gregáˇn

Received: 4 May 2021 Accepted: 23 May 2021 Published: 24 May 2021 1. Summary In contrast to humans, lampreys recover locomotion spontaneously several weeks Publisher’s Note: MDPI stays neutral after a complete spinal cord injury (SCI) [1]. We and others have shown that changes in the with regard to jurisdictional claims in functional and anatomical organization of neurotransmitter systems [2–11] and changes in published maps and institutional afﬁl- neurotransmitter levels [12] after a complete SCI could be important contributors to the iations. recovery process in lampreys. Interestingly, recent work from our group has also shown that different neurotransmitters control the regeneration of descending axons and the survival of descending brainstem neurons after a complete SCI in lampreys (serotonin, [13]; GABA, [14–18]; taurine, [12]). In the course of a recent investigation of the role of taurine Copyright: © 2021 by the authors. during axonal regeneration after SCI in lampreys we used HPLC methods to measure Licensee MDPI, Basel, Switzerland. taurine levels in the spinal cord of control and injured animals [12]. We have now used the This article is an open access article same samples to measure changes in GABA (and its precursor glutamine) and aspartate distributed under the terms and levels. We recently showed that endogenous GABA promotes the survival and regenera- conditions of the Creative Commons tion of descending neurons of lampreys after a complete SCI by acting through GABAB Attribution (CC BY) license (https:// receptors expressed in those neurons [15]. However, changes in GABA (or its precursor creativecommons.org/licenses/by/ glutamine) levels in the spinal cord of lampreys after SCI have never been measured in 4.0/).

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However, changes in GABA (or its precursor glutamine) levels in the spinal cord of lampreys after SCI have never been measured in the past. Aspartate acts as an excitatory neurotransmitter in the central nervous system of vertebrates [19,20] and lampreys contain a complex aspartatergic system in the spinal cord [21]. However, changes in the organization of the aspartatergic system or in aspartate levels has not been analyzed yet after SCI in lampreys. Here, we provide a dataset with measurements of aspartate, GABA and glutamine levels in the spinal cord of control (non‐injured) and 2‐, 4‐, and 10‐week post‐lesion (wpl) larval sea lampreys. These data might be of interest to understand the events that lead to successful recovery from SCI in lampreys and for those studying the role of neurotransmitters and neuromodulators in neuronal regeneration and plasticity Data 2021, 6, 54 after SCI. These data can be compared with similar2 of 5 data on other neurotransmitter systems of lampreys and other vertebrates. the past. Aspartate acts as an excitatory neurotransmitter in the central nervous system of vertebrates [19,20] and lampreys contain a complex aspartatergic system in the spinal 2.cord Data [21]. However, Description changes in the organization of the aspartatergic system or in aspartate levels has not been analyzed yet after SCI in lampreys. Here, we provide a dataset with measurementsAspartate, of aspartate, GABA GABA and and glutamineglutamine levels in levels the spinal in cord the of control spinal cord of control (un‐lesioned) and (non-injured) and 2-, 4-, and 10-week post-lesion (wpl) larval sea lampreys. These data injuredmight be of interest(2, 4, to and understand 10 thewpl) events mature that lead to successfullarval recoverysea lampreys from SCI in are shown in the Supplementary Tablelampreys (available and for those studying also theat role http://doi.org/10.5281/zenodo.4772417, of neurotransmitters and neuromodulators in accessed on 20 May 2021) neuronal regeneration and plasticity after SCI. These data can be compared with similar anddata on in other Figure neurotransmitter 1. As systems explained of lampreys in and the other vertebrates.methods section, aspartate, GABA, and glutamine concentrations2. Data Description (ng/mg of protein) were measured in 5 samples for each experimental Aspartate, GABA and glutamine levels in the spinal cord of control (un-lesioned) and group.injured (2, 4,Each and 10 sample wpl) mature contained larval sea lampreys 7 spinal are shown cord in the Supplementary pieces (1 mm rostral and 1 mm caudal from theTable injury (available alsosite at http://doi.org/10.5281/zenodo.4772417at the level of the 5th gill), accessed from on 20 May7 different 2021) animals (140 animals in total). and in Figure1. As explained in the methods section, aspartate, GABA, and glutamine Statisticalconcentrations (ng/mganalyses of protein) revealed were measured a significant in 5 samples for each increase experimental in aspartate (One‐way ANOVA p = group. Each sample contained 7 spinal cord pieces (1 mm rostral and 1 mm caudal from 0.0007;the injury siteFigure at the level 1A) of theand 5th gill)GABA from 7 different(One‐ animalsway (140ANOVA animals in p total). = 0.0004; Figure 1B) levels in the spinal cordStatistical at analyses 4 wpl, revealed and a signiﬁcant a significant increase in aspartate decrease (One-way in ANOVA glutaminep = 0.0007; levels at 10 wpl (One‐way ANOVA Figure1A ) and GABA (One-way ANOVA p = 0.0004; Figure1B) levels in the spinal cord pat = 4 0.0043; wpl, and a signiﬁcantFigure decrease1C). in glutamine levels at 10 wpl (One-way ANOVA p = 0.0043; Figure1C).

Figure 1. Cont.

Figure 1. (A) Graph showing aspartate concentration in the spinal cord of control and injured animals (control: 0.001288 ± 0.0002611 ng aspartate/mg protein; 2 wpl: 0.002647 ± 0.0005217 ng

Data 2021, 6, 54 2 of 4

2. Data Description Aspartate, GABA and glutamine levels in the spinal cord of control (un‐lesioned) and injured (2, 4, and 10 wpl) mature larval sea lampreys are shown in the Supplementary Table (available also at http://doi.org/10.5281/zenodo.4772417, accessed on 20 May 2021) and in Figure 1. As explained in the methods section, aspartate, GABA, and glutamine concentrations (ng/mg of protein) were measured in 5 samples for each experimental group. Each sample contained 7 spinal cord pieces (1 mm rostral and 1 mm caudal from the injury site at the level of the 5th gill) from 7 different animals (140 animals in total). Statistical analyses revealed a significant increase in aspartate (One‐way ANOVA p = 0.0007; Figure 1A) and GABA (One‐way ANOVA p = 0.0004; Figure 1B) levels in the spinal cord at 4 wpl, and a significant decrease in glutamine levels at 10 wpl (One‐way ANOVA p = 0.0043; Figure 1C).

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Figure 1. (A) Graph showing aspartate concentration in the spinal cord of control and injured animals Figure(control: 0.001288 1. (A±)0.0002611 Graph ng aspartate/mgshowing protein; aspartate 2 wpl: 0.002647 concentration± 0.0005217 ng aspartate/mg in the spinal cord of control and injured protein; 4 wpl: 0.00422 ± 0.00079 ng aspartate/mg protein; 10 wpl: 0.0008227 ± 6.944 × 10−5 ng animalsaspartate/mg (control: protein). Note 0.001288 the significant ± increase 0.0002611 (asterisks) ng in aspartate aspartate/mg concentration at protein; 4 wpl 2 wpl: 0.002647 ± 0.0005217 ng (Dunnett’s multiple comparisons test; p = 0.0018). (B) Graph showing GABA concentration in the spinal cord of control and injured animals (control: 0.002721 ± 0.0004307 ng GABA/mg protein; 2 wpl: 0.002958 ± 0.0006070 ng GABA/mg protein; 4 wpl: 0.006030 ± 0.0009828 ng GABA/mg protein; 10 wpl: 0.001109 ± 7.763 × 10−5 ng GABA/mg protein). Note the significant increase (asterisks) in GABA concentration at 4 wpl (Dunnett’s multiple comparisons test; p = 0.0044). (C) Graph showing glutamine concentration in the spinal cord of control and injured animals (control: 0.003933 ± 0.0008003 ng glutamine/mg protein; 2 wpl: 0.002228 ± 0.0004552 ng glutamine/mg protein; 4 wpl: 0.003610 ± 0.0006883 ng glutamine/mg protein; 10 wpl: 0.0007130 ± 7.339 × 10−5 ng glutamine/mg protein). Note the significant decrease (asterisks) in glutamine concentration at 10 wpl (Dunnett’s multiple comparisons test; p = 0.0031). 3. Methods 3.1. Animals and Complete SCI Mature and developmentally stable larval sea lampreys, Petromyzon marinus L. (140 animals in total; between 95 and 120 mm in body length, 5 to 7 years of age), were used in this study. Animals were anaesthetized with 0.1% MS-222 (Sigma, St. Louis, MO, USA) in lamprey Ringer solution (137 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl2, 2 mM HEPES; pH 7.4) before all experimental procedures and euthanized by decapitation at the end of the experiments. All experiments 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 (Galicia, Spain) and were performed in accordance with European Union and Spanish guidelines on animal care and experimentation. Complete spinal cord transections were performed as previously described [22]. Briefly, 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. The animals were allowed to recover in individual fresh-water tanks at 19.5 ◦C for 2, 4, or 10 wpl.

3.2. High-Performance Liquid Chromatography The high-performance liquid chromatography (HPLC) method was used to measure the concentration of aspartate, GABA, and glutamine in the spinal cord of control and injured (2, 4, and 10 wpl) animals (n = 5 samples per experimental group with 7 animals per sample). These time points were chosen because in lampreys axon retraction predominates in the ﬁrst 2 weeks after a complete SCI, axon re-growth starts to predominate at 4 wpl [23], and at 10 wpl descending axons have already regenerated through the site of injury and animals show normal appearing locomotion [7]. Each HPLC sample contained a pool of Data 2021, 6, 54 4 of 5

7 pieces (from 7 animals) of spinal cord from 1 mm rostral to the site of injury to 1 mm caudal to the site of injury. In control animals 2 mm of spinal cord at the same spinal cord level were collected. We analyzed 5 samples for each of the 4 experimental groups (controls and 2, 4, and 10 wpl animals). The samples were sonicated in acetic acid and stored at –80 ◦C until their use for HPLC. Aspartate, GABA, and glutamine were separated and analyzed using HPLC with fluorescence detection after precolumn derivatization with o-phthalaldehyde (OPA), as previously described [24]. An aliquot of 20 µL of the tissue supernatant containing homoserine as internal standard was neutralized with OPA reagent (4 mM OPA, 10% methanol, 2.56 mM 2-mercaptoethanol, in 1.6 M potassium borate buffer, pH 9.5) for 1 min. After this period, the reaction was stopped by adding acetic acid (0.5% v/v). Samples were immediately loaded through a Rheodyne (model 7125) injector system with a 50 µL loop sample to reach a C18 reverse-phase column (4.6 mm i.d. × 150 mm, Nucleosil 5, 100 A) eluted with a mobile phase consisting of 0.1 M sodium acetate buffer (pH 5.5) containing 35% methanol, at a flow rate of 1 mL/min and at a pressure of 140 bar. The column was subsequently washed with the same buffer containing 70% methanol and re-equilibrated with the elution buffer before use. The HPLC system consisted of a solvent delivery system coupled to a filter fluorometer (excitation 340 nm, emission 455 nm). Aspartate, GABA, and glutamine content were calculated from the chromatographic peak areas using standard curves and the internal standard. The total protein content was measured in a NanoSpectrophotometer.

3.3. Statistical Analyses Data are presented as mean ± S.E.M in Figure1. We used Prism 8 (GraphPad Soft- ware, La Jolla, CA) to perform the statistical analyses. Normality of the HPLC data was determined by the Kolmogorov–Smirnov normality test. All data passed the normality test. The results of control versus 2, 4, and 10 wpl groups were analyzed by One-way ANOVA and Dunnett’s multiple comparisons test to compare each post-lesion group with the control group. p < 0.05 was considered statistically signiﬁcant.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/data6060054/s1, Table S1: Aspartate, GABA, and glutamine concentration (ng/mg protein) in each HPLC sample (7 spinal cord pieces/sample). The dataset can also be found at http://doi.org/ 10.5281/zenodo.4772417, accessed on 20 May 2021. Author Contributions: Conceptualization, B.F.-L., M.C.R., and A.B.-I.; obtaining data and methodol- ogy, B.F.-L., N.P., and A.L.; writing—original draft preparation, B.F.-L.; writing—review and editing, M.C.R. and A.B.-I.; funding acquisition, M.C.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by FEDER/Ministerio de Ciencia, Innovación y Universidades— Agencia Estatal de Investigación (Grant number: BFU2017-87079 P). Institutional Review Board Statement: All experiments 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 (Galicia, Spain) and were performed in accordance with European Union and Spanish guidelines on animal care and experimentation. Informed Consent Statement: Not applicable. Data Availability Statement: All data can be found as supplementary material within the article. Acknowledgments: The authors would like to thank the staff of the Ximonde Biological Station for providing the lampreys used in this study. The authors would like to dedicate this article to the memory of Anunciación Lafuente. Conﬂicts of Interest: The authors declare no conﬂict of interest. Data 2021, 6, 54 5 of 5

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