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

: 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 and aspartate levels signiﬁcantly increase in the spinal cord four weeks after a complete spinal cord injury and that glutamine levels decrease 10 weeks after injury as compared to controls. These data might be of interest to those studying the role of neurotransmitters and neuromodulators in recovery from spinal cord injury in vertebrates.


Summary
In contrast to humans, lampreys recover locomotion spontaneously several weeks after a complete spinal cord injury (SCI) [1]. We and others have shown that changes in the functional and anatomical organization of neurotransmitter systems [2][3][4][5][6][7][8][9][10][11] and changes in neurotransmitter levels [12] after a complete SCI could be important contributors to the 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][15][16][17][18]; taurine, [12]). In the course of a recent investigation of the role of taurine during axonal regeneration after SCI in lampreys we used HPLC methods to measure taurine levels in the spinal cord of control and injured animals [12]. We have now used the same samples to measure changes in GABA (and its precursor glutamine) and aspartate levels. We recently showed that endogenous GABA promotes the survival and regeneration of descending neurons of lampreys after a complete SCI by acting through GABAB receptors expressed in those neurons [15]. However, changes in GABA (or its precursor glutamine) levels in the spinal cord of lampreys after SCI have never been measured in Data 2021, 6, 54 2 of 5 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 after SCI. These data can be compared with similar data on other neurotransmitter systems of lampreys and other vertebrates.

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). events that lead to successful recovery from SCI in lampr role of neurotransmitters and neuromodulators in neuro after SCI. These data can be compared with similar d systems of lampreys and other vertebrates.

Data Description
Aspartate, GABA and glutamine levels in the spinal co injured (2, 4, and 10 wpl) mature larval sea lampreys are Table (available also at http://doi.org/10.5281/zenodo.4772 and in Figure 1. As explained in the methods section, as concentrations (ng/mg of protein) were measured in 5 s group. Each sample contained 7 spinal cord pieces (1 mm the injury site at the level of the 5th gill) from 7 different Statistical analyses revealed a significant increase in asp 0.0007; Figure 1A) and GABA (One-way ANOVA p = 0.000 cord at 4 wpl, and a significant decrease in glutamine leve p = 0.0043; Figure 1C).

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 CaCl 2 , 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.

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 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.

Statistical Analyses
Data are presented as mean ± S.E.M in Figure 1. We used Prism 8 (GraphPad Software, 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.  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.