ECTS credits ECTS credits: 3
ECTS Hours Rules/Memories Student's work ECTS: 45 Hours of tutorials: Expository Class: Interactive Classroom: 30 Total: 75
Use languages Spanish, Galician
Type: Ordinary subject Master’s Degree RD 1393/2007 - 822/2021
Center Faculty of Biology
Call: Second Semester
Teaching: With teaching
Enrolment: Enrollable | 1st year (Yes)
The objectives are intended to learn theoretical and practical bases of those techniques that are widely used in neurobiology to explore cytoarchitecture, neural circuits, neurochemistry and gene expression.
(a) Learn the skills needed to carry out histological processing of brain and spinal cord for light and electron microscopy, including general staining, histochemistry, neuronal tracers, immunohistochemistry, and in situ hybridization. The functioning and applications of the different types of the different types of microtomes and light and electron microscopes, as well as of the laser confocal microscope, will also be learned.
(b) Achieve a level of knowledge of the different techniques as to be able to apply them and to discuss the protocol of a specific technique.
(b) Assess and solve theoretical and real scientific problems by using the knowledge acquired during the course.
PRACTICES PROGRAM
Practice 1. Fixation and embedding of nervous tissue.
Classroom: Chemical fixation: Types of fixatives, fixation methods, proper selection of fixatives. Embedding: Types and properties of embedding substances for light and electron microscopy.
Laboratory: Fixation and embedding of nervous tissue for light and electron microscopy using different fixatives and embedding substances according to the method to be carried out.
Practice 2. Microtomy.
Classroom. How to get sections from nervous tissue. Types of microtomes.
Laboratory: Obtaining sections from embedded and non-embedded tissue depending on the procedure to follow.
Practice 3. General staining methods and observation of nervous tissue sections.
Classroom. Common staining methods to visualize the nervous tissue, types of dyes and their applications. Histochemical staining.
Laboratory. Nissl staining method. Golgi Method. Histochemical staining of nitric oxide synthase positive neurons and glia.
Practice 4. Light and electron microscopy immunohistochemistry.
Classroom. Introduction to the immunohistochemical methods. Indirect immunohistochemistry for light and fluorescent microscopy, and pre-embedding and post-embedding immunohistochemistry for electron microscopy.
Laboratory: Detection of glial and neuronal markers at light, fluorescent microscopes, as well as by electron microscopy by using the pre-embedding method.
Practice 5. Identification of axonal pathways by using tract tracing techniques.
Classroom: Types of neuronal tracers, methods for tracer application, “in vitro” and “in vivo” experiments.
Laboratory: Labeling of the brain by using HRP or BDA as tracers, and of the spinal cord by using FDA or TRDA as tracers.
Practice 6. “In situ” hybridization.
Classroom. Types of probes. Preparation and labeling of probes. “In toto” and on tissue sections in situ hybridization.
Laboratory: “In situ” hybridization to detect the expression of a gene “in toto”.
Practice 7. Analysis of results and discussion of real experimental approaches.
Classroom: Observation, discussing, photographing, and reporting the obtained results.
Laboratory: Designing a protocol to solve a particular scientific problem.
Armengol, J.A., Miñano, F.J. 1995. Bases Experimentales para el Estudio del Sistema Nervioso. Vol. 1. Universidad de Sevilla. Sevilla.
Bancroft, J.D., Gamble, M. 2007. Theory and Practice of Histological Techniques (6ª ed). Churchill Livingstone. London.
Bolam, J.P. 1992. Experimental Neuroanatomy: A Practical Approach. Oxford University Press. Oxford.
Carter, M., Shieh, J.C. 2009. Guide to Research Techniques in Neuroscience. Academic Press. Amsterdam.
Celis, J., Carter, N., Simons, K., Small, J., Hunter, T., Shotton, D. 2006. Cell Biology: A Laboratory Handbook. (3ª ed). Academic Press. London.
Cuello, A.C. 1993. Immunohistochemistry II. John Wiley & Sons. New York.
Gerfen, Ch.R., Rogawski, M.A., Sibley, D.R., Skolnick, P., Wray, S. 2007. Short Protocols in Neuroscience: Cellular and Molecular Methods. John Wiley & Sons, Inc. New Jersey.
Hayat, M.A. 2000. Principles and Techniques of Electron Microscopy: Biological Applications (4ª ed). Cambridge University Press. Cambridge.
Kiernan, J.A. 2008. Histological and Histochemical Methods: Theory and Practice. (4ª ed). Scion Publishing Ltd. Oxford.
Morel, G., Caballero, T.G., Cavalier, A. Gallego, R. 2000. Hibridación in situ en Microscopía Óptica. Universidad de Santiago de Compostela.
Spacek J. 1989. Dynamics of the Golgi method: a time-lapse study of the early stages of impregnation in single sections. Journal of Neurocytology, 18: 27-38.
Záborsky, L., Wouterlood, F.G., Lanciego, J.L. 2006. Neuroanatomical Tract-Tracing 3. Molecules, Neurons, and Systems. Springer Science + Business Media. New York.
In this course the student will:
- Prepare fixative solutions and embed samples for both light and electron microscopy.
- Use different types of microtomes and know their applications.
- Perform immunohistochemical and in situ hybridization experiments.
- Perform experiments with neuronal tracers.
- Use different types of light and electron microscopes.
- Write a report on the work carried out in the laboratory and on the results of the experiments developed.
- Analyze and discuss the methods described in scientific publications in relation to those carried out during the course.
At the end of the course the student will be able to:
- Design experimental protocols to study the nervous system.
- Process histological material for light and electron microscopy.
- Select the most appropriate method depending on the scientific problem to be addressed.
- Handle the different devices and equipment used in neuroanatomy.
- Analyze and summarize the experimental outcomes.
- Resolve technical problems and modify or adapt technical protocols to particular situations or specific material.
- Use of theoretical knowledge to solve real case studies.
- Properly present the obtained results in scientific context.
Interactive lessons: seminars or practices
Tutorials in small groups or individualized
- Obligatory attendance of the course.
- Student participation in discussions and in the daily work during the course (10%).
- Assessment of the capacity to design experimental protocols by a written examination (30%).
- Evaluation of the final informs (30%).
- Analysis and critical discussion of course-related techniques used in specific scientific publications (an article per student; 30%).
- The course will be passed with no less than 50 %.
Interactive lessons: seminars or practices: 30 hours
Preparation of a final inform and other homework: 24 hours
Tutorials in small groups or individualized: 1 hour
Evaluation activities: 20 hours
In this course, techniques widely used in neurobiology to study normal cytoarchitecture, neurochemistry, neural circuits, and gene expression are addressed. A basic knowledge of microscopes and common laboratory material handling: micropipettes, pHmeters, etc, is required. In any case, the development of experiments during the course will provide sufficient dexterity to behave easily in the laboratory.