Methods for studying the Nervous system

Methods for studying the Nervous system

1. Histochemistry and Cytochemistry

   • Hematoxylin and Eosin
     • Hematoxylin
       • Basic dye
       • Components with megative charge
       • Purple
     • Eosin
       • Acidic dye
       • Components with positive charge
       • Pink
   • Golgi stain
     • Silver salts
     • Affinity for nervous cells
     • Black
   • Nissl stain
     • Uses various stains
       • Cresyl violet
       • Thionine
       • Toludine blue
       • Methylene blue
       • Aniline blue
     • High affinity for DNA and RNA
     • Used for neurons ´cytoplasm
   • Enzyme Histochemistry
     • Acetylcholinesterase stain
       • Present in
         • basement membrane between nerves
         • Neuromuscular juntions
       • Product
         • Rubeanic acid
     • The product of an enzymatic reaction is visualized
   • Binding of a dye

2. Immunohistochemistry

   • Binding of a coloured-lablled antibody
   • It is limited to 1 or 2 dyes
   • It is more permanent
   • Low contrast

3. Immunofluorescence

   • Binding of a fluorescent-lablled antibody
   • Different lablled fluophores can be used
   • It can fade over time
   • High contrast

4. Autoradiography

   • Incorporation of radioactive atoms
     • H-thymidine
     • H-uridine
   • Proteins and Nucleic acids are tagged
   • Photographic emulsion over a tissue

5. In-situ hybridization

   • Localization of mRNA or DNA through hybridization
   • Use of a complementary strand (nucleotid probe)

6. Electron microscopy

   • Provides morphological data of ultrastructure of cells
   • Types
     • TEM
       • Interaction of a beam of electrons with a specimen
       • The electron beam passes through the specimen
     • SEM
       • The electron beam does not pass through the specimen
       • The surface of the specimen is scanned

7. Anatomic tracing

   • Injection of tracers into brain regions
   • Tracking their transport along axons and dendrites

8. Unbiased counting method

   • Used for quantify cells or synapses
   • It eliminates systematic errors
     • section thickness
     • overcounting
     • human bias

9. Optogenetics

   • Control of genetically modified neurons through ilumination
   • Modified channels
     • Chanelrodopsins
       • Conduct cations and depolarize neurons
       • Blue ilumination
     • Halorhodopsins
       • Conduct cloride ions into the cytoplasm
       • Yellow ilumination
     • Optorhodopsins
       • Activation of intracellular loops
         • Signaling that activates proteins
       • Green light