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  • br Conclusion In the present study we found

    2022-08-10


    Conclusion In the present study, we found that the majority of commercially available antibodies and antibody-based tools against galanin and its receptors that we tested do not meet validation requirements. Therefore, critical validation of these tools by the user is essential before being used in research, and the validation results should be included in publications reporting data generated with these tools. Here, we validated one commercial antibody that is specific against human GAL1-R (GTX108207, GeneTex), one customized antibody that is specific against human GAL2-R (S4510-1, PTG), and one commercial antibody that is specific against human GAL3-R (GTX108163, GeneTex). We also identified two commercial antibodies that are specific against human galanin (T-4325, Peninsula/Bachem; Ab00523-1.1, Absolute Antibody). Furthermore, we successfully developed and validated a specific galanin ELISA that uses commercially available materials. We used this ELISA to detect galanin peptide levels in plasma of migraine patients.
    Author contributions SMB and AK designed and performed experiments, analyzed data and wrote the manuscript. JS and FL designed and performed experiments, analyzed data and contributed to drafting the manuscript. BB, SE, BSH, IR and KG designed and performed experiments and analyzed data. SL, FE and PS collected and/or provided human patient samples and co-edited the manuscript. RL designed experiments, critically discussed the data and co-edited the manuscript. BK obtained resources for the study, designed experiments, critically discussed the data and co-edited the manuscript. All authors approved the final version of the manuscript.
    Declaration of interest
    Acknowledgments This research was supported by the Austrian Research Promotion Agency (FFG) (822782/THERAPEP) and the Paracelsus Medical University Salzburg, Austria (R-11/05/030-ERN). The authors thank Heike Münzberg for providing formalin-fixed tissues from GAL1-R-KO mice.
    Introduction Galanin (GAL) is a multifunctional neuropeptide that has a widespread distribution in the peripheral and central nervous system (CNS). Galanin consists of 29–30 amino rotenone sequences and was first isolated from porcine intestine [23]. In mammals, galanin has been implicated in the modulation of several different physiological processes including nociception [4], cognition [21], feeding behavior [1], neuronal growth [11] and reproduction [20]. Galanin has also been shown to play a role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress, as well as in the pathogenesis of pituitary adenomas [25], [27]. In mammals, the physiological effects of galanin are mediated through its interaction with three galanin receptor subtypes GALR1, GALR2, and GALR3, which are G protein-coupled receptors (GPCRs). GALR1 expression is largely restricted to the nervous system including ventral hippocampus, thalamic nuclei, amygdala, brainstem nuclei, hypothalamus, posterior horns of the spinal cord, and dorsal root ganglia [2], [8]. Unlike GALR1, GALR2 is widely distributed in almost all tissues, including the brain, such as the olfactory system, preoptic area, hypothalamus, hippocampus, amygdala, pituitary gland, and brainstem [2], [3], [5], [6], [14]. GALR3 showed limited expression in CNS but is relatively abundant in peripheral tissues [12]. In zebrafish, immunohistochemical analysis using the anti-Galanin antibody—a marker for galanin neuropeptide—revealed that galanin-immunoreactive (galanin-IR) nerve cells are located in the preoptic region, hypothalamus, midbrain tegmentum, and rostral hindbrain [16]. In addition, nerve fibers of galanin-IR neurons have been detected primarily in the ventral region of the brain, including the subpallium, anterior commissure, postoptic commissure, and commissure of the posterior tuberculum [16]. A recent study has shown that galanin is the neuropeptide most frequently colocalized with histaminergic neurons and that galanin-IR fibers surround the histaminergic neurons in the ventrocaudal hypothalamus of the adult zebrafish brain [22]. It plays a role in regulating blood glucose level, indicating that galanin function is evolutionarily conserved [15], [16]. BLAST survey and phylogenetic analysis have revealed that the zebrafish has three different receptors for galanin; GALR1a, GALR1b, and GALR2, while it lacks GALR3 [10]. Recent phylogeny and synteny analysis of the gene family have shown that zebrafish GALR2 is subdivided into GALR2a and GALR2b [7]. A recent study has shown that galr1a expression is mainly restricted to the brain and intestines, while galr1b mRNA was present in all tissues [9]. However, nothing has been reported about GALR2a and GALR2b in the zebrafish CNS.