Publicaciones por año
Publicaciones del Departamento de Histología y Embriología
Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis
Sci Rep 2022 12(1):4439
Silvia Olivera-Bravo 1 , Carmen Bolatto 2 3 , Gabriel Otero Damianovich 2 , Matías Stancov 2 , Sofía Cerri 2 3 , Paola Rodríguez 2 , Daniela Boragno 2 , Karina Hernández Mir 3 , María Noel Cuitiño 2 , Fernanda Larrambembere 2 , Eugenia Isasi 2 3 , Diego Alem 4 , Lucía Canclini 4 , Marta Marco 2 5 , Danilo Davyt 6 , Pablo Díaz-Amarilla 2
1 Cell and Mol Neurobiol Lab, NCIC Department, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. solivera@iibce.edu.uy. 2 Cell and Mol Neurobiol Lab, NCIC Department, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. 3 Histology and Embryology Department, Faculty of Medicine, Universidad de La República (UdelaR), Montevideo, Uruguay. 4 Genetic Department, IIBCE, Montevideo, Uruguay. 5 Tumoral Biol Area, Clin Biochem Department, Faculty of Chemistry, UdelaR, Montevideo, Uruguay. 6 Pharm Chem Lab, Organic Chemistry Department, Faculty of Chemistry, UdelaR, Montevideo, Uruguay.
DOI: 10.1038/s41598-022-06470-7
PMID: 35292673
Pubmed: https://pubmed.ncbi.nlm.nih.gov/35292673
Texto completo: https://doi.org/10.1038/s41598-022-06470-7
Abstract:
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.
SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice
J Biol Chem 2022 298(3):101711
Mariana Bresque 1 , Karina Cal 2 , Valentina Pérez-Torrado 3 , Laura Colman 4 , Jorge Rodríguez-Duarte 5 , Cecilia Vilaseca 6 , Leonardo Santos 7 , María Pía Garat 7 , Santiago Ruiz 7 , Frances Evans 8 , Rosina Dapueto 1 , Paola Contreras 9 , Aldo Calliari 2 , Carlos Escande 10
1 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay; Laboratory of Vascular Biology and Drug Development, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay. 2 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay; Departamento de Biociencias, Facultad de Veterinaria, Universidad de la República (UdelaR), Montevideo, Uruguay. 3 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay; Departamento de Inmunobiología, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay; Área Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay. 4 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay; Departamento de Química Orgánica, Facultad de Química, Universidad de la República (UdelaR), Montevideo, Uruguay. 5 Laboratory of Vascular Biology and Drug Development, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay. 6 Departamento de Fisiología, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay. 7 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay. 8 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay; Laboratory of Neuroinflammation and Gene Therapy, Institut Pasteur Montevideo, Montevideo, Uruguay. 9 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay; Departamento de Fisiología, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay. 10 Laboratory of Metabolic Diseases and Aging, INDICYO Program, Institut Pasteur Montevideo, Montevideo, Uruguay. Electronic address: escande@pasteur.edu.uy.
DOI: 10.1016/j.jbc.2022.101711
PMID: 35150745
Pubmed: https://pubmed.ncbi.nlm.nih.gov/35150745
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(22)00151-X
Abstract:
Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.
Pathophysiological and molecular considerations of viral and bacterial infections during maternal-fetal and -neonatal interactions of SARS-CoV-2, Zika, and Mycoplasma infectious diseases
Biochim Biophys Acta Mol Basis Dis 2022 1868(1):166285
Gonzalo Ferreira 1 , Fernanda Blasina 2 , Marianela Rodríguez Rey 2 , Gabriel Anesetti 3 , Rosana Sapiro 3 , Luisina Chavarría 4 , Romina Cardozo 4 , Grazzia Rey 5 , Luis Sobrevia 6 , Garth L Nicolson 7
1 Laboratory of Ion Channels, Biological Membranes and Cell Signaling, Dept. of Biophysics, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay. Electronic address: ferreira@fmed.edu.uy. 2 Dept. of Neonatology, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay. 3 Dept. of Histology and Development, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay. 4 Laboratory of Ion Channels, Biological Membranes and Cell Signaling, Dept. of Biophysics, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay. 5 Dept. of Clinical Ginecology and Obstetrics B, Facultad de Medicina, Universidad de la Republica, Uruguay. 6 Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics, Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile; Department of Physiology, Faculty of Pharmacy, Universidad de Sevilla, Seville E-41012, Spain; Medical School, Faculty of Medicine, Sao Paulo State University (UNESP), Brazil; University of Queensland Centre for Clinical Research (UQCCR), Faculty of Medicine and Biomedical Sciences, University of Queensland, Herston, QLD 4029, Australia; Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, 9713GZ Groningen, The Netherlands. 7 Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, CA, USA.
DOI: 10.1016/j.bbadis.2021.166285
PMID: 34624499
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34624499
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0925-4439(21)00218-0
Abstract:
During pregnancy, a series of physiological changes are determined at the molecular, cellular and macroscopic level that make the mother and fetus more susceptible to certain viral and bacterial infections, especially the infections in this and the companion review. Particular situations increase susceptibility to infection in neonates. The enhanced susceptibility to certain infections increases the risk of developing particular diseases that can progress to become morbidly severe. For example, during the current pandemic caused by the SARS-CoV-2 virus, epidemiological studies have established that pregnant women with COVID-19 disease are more likely to be hospitalized. However, the risk for intensive care unit admission and mechanical ventilation is not increased compared with nonpregnant women. Although much remains unknown with this particular infection, the elevated risk of progression during pregnancy towards more severe manifestations of COVID-19 disease is not associated with an increased risk of death. In addition, the epidemiological data available in neonates suggest that their risk of acquiring COVID-19 is low compared with infants (<12 months of age). However, they might be at higher risk for progression to severe COVID-19 disease compared with older children. The data on clinical presentation and disease severity among neonates are limited and based on case reports and small case series. It is well documented the importance of the Zika virus infection as the main cause of several congenital anomalies and birth defects such as microcephaly, and also adverse pregnancy outcomes. Mycoplasma infections also increase adverse pregnancy outcomes. This review will focus on the molecular, pathophysiological and biophysical characteristics of the mother/placental-fetal/neonatal interactions and the possible mechanisms of these pathogens (SARS-CoV-2, ZIKV, and Mycoplasmas) for promoting disease at this level.
Pericyte Mapping in Cerebral Slices with the Far-red Fluorophore TO-PRO-3
Bio Protoc 2021 11(22):e4222
Sandra P Mai-Morente 1 , Juan P Irigoyen 1 , Victoria M Carriquiry 1 , Virginia M Marset 1 , Mariana Di Doménico 2 , Eugenia Isasi 3 , Verónica Abudara 1
1 Departamento de Fisiología, Facultad de Medicina, Universidad de la República, General Flores 2125, Montevideo, CP 11 800, Uruguay. 2 Departamento de Biofísica, Facultad de Medicina, Universidad de la República, General Flores 2125, Montevideo, CP 11 800, Uruguay. 3 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, General Flores 2125, Montevideo, CP 11 800, Uruguay.
DOI: 10.21769/BioProtoc.4222
PMID: 34909443
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34909443
Texto completo: https://bio-protocol.org/e4222
Abstract:
This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.
Increased mitochondrial activity upon CatSper channel activation is required for mouse sperm capacitation
Redox Biol 2021 48:102176
Juan J Ferreira 1 , Adriana Cassina 2 , Pilar Irigoyen 3 , Mariana Ford 3 , Santiago Pietroroia 3 , Nikita Peramsetty 1 , Rafael Radi 2 , Celia M Santi 4 , Rossana Sapiro 5
1 Department of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University in St. Louis, School of Medicine, St. Louis, MO, United States; Department of Neuroscience, Washington University in St. Louis, School of Medicine, St. Louis, MO, United States. 2 Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO) Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 3 Departamento de Histología y Embriología and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina UDELAR, Montevideo, Uruguay. 4 Department of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University in St. Louis, School of Medicine, St. Louis, MO, United States; Department of Neuroscience, Washington University in St. Louis, School of Medicine, St. Louis, MO, United States. Electronic address: Santic@wustl.edu. 5 Departamento de Histología y Embriología and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina UDELAR, Montevideo, Uruguay. Electronic address: rsapiro@fmed.edu.uy.
DOI: 10.1016/j.redox.2021.102176
PMID: 34753004
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34753004
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S2213-2317(21)00336-0
Abstract:
To fertilize an oocyte, sperm must undergo several biochemical and functional changes known as capacitation. A key event in capacitation is calcium influx through the cation channel of sperm (CatSper). However, the molecular mechanisms of capacitation downstream of this calcium influx are not completely understood. Capacitation is also associated with an increase in mitochondrial oxygen consumption, and several lines of evidence indicate that regulated calcium entry into mitochondria increases the efficiency of oxidative respiration. Thus, we hypothesized that calcium influx through CatSper during capacitation increases mitochondrial calcium concentration and mitochondrial efficiency and thereby contributes to sperm hyperactivation and fertilization capacity. To test this hypothesis, we used high-resolution respirometry to measure mouse sperm mitochondrial activity. We also measured mitochondrial membrane potential, ATP/ADP exchange during capacitation, and mitochondrial calcium concentration in sperm from wild-type and CatSper knockout mice. We show that the increase in mitochondrial activity in capacitated wild-type sperm parallels the increase in mitochondrial calcium concentration. This effect is blunted in sperm from CatSper knockout mice. Importantly, these mechanisms are needed for optimal hyperactivation and fertilization in wild-type mice, as confirmed by using mitochondrial inhibitors. Thus, we describe a novel mechanism of sperm capacitation. This work contributes to our understanding of the role of mitochondria in sperm physiology and opens the possibility of new molecular targets for fertility treatments and male contraception.
Immunohistochemical description of isotocin neurons and the anatomo-functional comparative analysis between isotocin and vasotocin systems in the weakly electric fish, Gymnotus omaroum
Gen Comp Endocrinol 2021 313:113886
Paula Pouso 1 , Rossana Perrone 2 , Ana Silva 3
1 Depto. Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Unidad Bases Neurales de la Conducta, Depto Neurofisiologia Celular y Molecular, IIBCE, Montevideo, Uruguay. Electronic address: ppouso@fmed.edu.uy. 2 Unidad Bases Neurales de la Conducta, Depto Neurofisiologia Celular y Molecular, IIBCE, Montevideo, Uruguay; Instituto de Fundamentos y Métodos, Facultad de Psicología, Universidad de la República. 3 Unidad Bases Neurales de la Conducta, Depto Neurofisiologia Celular y Molecular, IIBCE, Montevideo, Uruguay; Laboratorio de Neurociencias, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1016/j.ygcen.2021.113886
PMID: 34411583
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34411583
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0016-6480(21)00179-9
Abstract:
The vasopressin-vasotocin (AVP-AVT) and oxytocin-mesotocin-isotocin (OT-MT-IT) families of nonapeptides are of great importance in shaping context-dependent modulations of a conserved and yet highly plastic network of brain areas involved in social behavior: the social behavior network. The nonapeptide systems of teleost fish are highly conserved and share a common general organization. In this study, we first describe the presence of IT cells and projections in the brain of an electric fish, Gymnotus omarorum. Second, we confirm that IT neuron types and distribution in the preoptic area (POA) follow the same general pattern previously described in other teleost species. Third, we show that although IT and AVT neurons occur intermingled within the POA of G. omarorum and can be classified into the same subgroups, they present subtle but remarkable differences in size, number, and location. Finally, we show that unlike AVT, IT has no effect on basal electric signaling, reinforcing the specificity in the actions that each one of these nonapeptides has on social behavior and communication.
Functional expression of P2Y2 receptors in mouse ovarian surface epithelium (OSE)
Mol Reprod Dev 2021 88(11):758-770
Ana Patricia Juárez-Mercado 1 , Rebeca Chávez-Genaro 2 , Tatiana Fiordelisio 3 , Adriana González-Gallardo 1 , Mauricio Díaz-Muñoz 1 , Francisco G Vázquez-Cuevas 1
1 Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro, México. 2 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 3 Laboratorio de Neuroendocrinología Comparada, Departamento de Ecología y Recursos Naturales, Biología, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad Universitaria, CDMX, México.
DOI: 10.1002/mrd.23545
PMID: 34694051
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34694051
Texto completo: https://doi.org/10.1002/mrd.23545
Abstract:
Ovarian surface epithelium (OSE) is a cell monolayer surrounding the ovary; it is involved in the regulation of the ovulatory process and the genesis of ovarian carcinoma. However, intercellular messengers regulating signaling events, like proliferation in the OSE, have not been completely described. Purines have emerged as novel intercellular messengers in the ovary, in which expression of purinergic receptors has been reported in different cell types. In the present work, we described the functional expression of P2Y2 receptor (P2Y2R), a purinergic receptor widely associated with cell proliferation, in the OSE. The expression of P2Y2R by immunofluorescence and RT-PCR, and its functionality by Ca2+ recording was demonstrated in primary cultured OSE. Functional expression of P2Y2R was also exhibited in situ, by recording of intracellular Ca2+ release and detection of ERK phosphorylation after injection of a selective agonist into the ovarian bursa. Furthermore, P2Y2R activation with UTPγS, in situ, induced cell proliferation at 24 h, whereas continuous stimulation of P2Y2R during a complete estrous cycle significantly modified the size distribution of the follicular population. This is the first evidence of the functional expression of purinergic P2Y2R in the OSE and opens new perspectives on the roles played by purines in ovarian physiology.
A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance
Biomed Res Int 2021 2021:2641068
Frances Evans 1 , Julio A Hernández 2 , Federico Cabo 3 , Silvia Chifflet 3
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay. 2 Sección Biofísica, Facultad de Ciencias, Universidad de la República, Iguá s/n esq. Mataojo, 11400 Montevideo, Uruguay. 3 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay.
DOI: 10.1155/2021/2641068
PMID: 34722759
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34722759
Texto completo: https://doi.org/10.1155/2021/2641068
Abstract:
Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.
Neonatal androgenization in rats affects oocyte maturation
Reprod Sci 2021 28(10):2799-2806
Gabriel Anesetti 1 , Rebeca Chávez-Genaro 2
1 Laboratorio de Biología de la Reproducción, Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. ganesett@fmed.edu.uy. 2 Laboratorio de Biología de la Reproducción, Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
DOI: 10.1007/s43032-021-00559-6
PMID: 33825168
Pubmed: https://pubmed.ncbi.nlm.nih.gov/33825168
Texto completo: https://doi.org/10.1007/s43032-021-00559-6
Abstract:
Androgens are relevant in order to achieve a normal growth and maturation of the follicle and oocyte, since both excess and absence of androgens may affect the correct ovarian function. The current study analyzes the impact of neonatal androgenization in the first ovulation and oocyte maturation in response to exogenous gonadotrophin stimulation. Neonatal rats were daily treated with testosterone, dihydrotestosterone, or vehicle during follicle assembly period (days 1 to 5). At juvenile period, rats were stimulated sequentially with PMSG and hCG. Ovulation, ovarian histology, hormonal milieu, morphological characteristics of meiotic spindle, and in vitro fertilization rate in oocytes were analyzed. Our data shows that oocytes from androgenized rats displayed a major proportion of aberrant spindles and altered meiotic advance that control animals. These alterations were accompanied with an increase in both fertilization rate and aberrant embryos after 48 h of culture. Our findings showed a direct impact of neonatal androgens on oocyte development; their effects may be recognized at adulthood, supporting the idea of a programming effect exerted by neonatal androgens. These results could be relevant to explain the low fertility rate seen in polycystic ovary syndrome patients after in vitro fertilization procedures.
Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions
J Vis Exp 2021 (174)
Carmen Bolatto # 1 , Silvia Olivera-Bravo # 2 , Sofía Cerri 3
1 Developmental Biology Laboratory, Histology and Embryology Department, Faculty of Medicine, Universidad de la República; cbolatto@fmed.edu.uy. 2 Cell and Molecular Neurobiology Laboratory, Clemente Estable Biology Research Institute (IIBCE), Ministerio de Educación y Cultura. 3 Developmental Biology Laboratory, Histology and Embryology Department, Faculty of Medicine, Universidad de la República; Cell and Molecular Neurobiology Laboratory, Clemente Estable Biology Research Institute (IIBCE), Ministerio de Educación y Cultura. # Contributed equally.
DOI: 10.3791/62620
PMID: 34459813
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34459813
Texto completo: https://doi.org//10.3791/62620
Abstract:
The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.