Week 7 Lecture 1: Techniques for Neural circuits

Week 7 Lecture 1: Techniques for Neural circuits

1. Fluorescent dyes

   • Opaque dyes usually give low contrast
   • Fluorescent dyes are excited by short wavelengths and emit longer wavelength light against low background

2. Antibodies

   • Made up by B Cells in response to a foreign antigen in body
   • Most common type in blood are IgG immunoglobin - bind strongly to antigen and attract other white blood cells to attack

3. Direct immunohistochemical detection

   • Antibodies against Chloine acetyltransferase are raised in an animal, purified and labelled with dye
   • Disadvantage - attaching dye to antibody can mess up its affinity and making antibodies can be difficult and expensive

4. Indirect immunohistochemistry

   • first antibody against ChAT is made normal way
   • Antibody is raised in a different species against rabbit IgG
   • Secondary antibody is tagged with a fluorescent dye. One batch of secondary antibody can then be used in dozens of experiemnts to detect any primary antibodiy raised in rabbit against any antigen

5. Multiple labelling immunohistochemistry

   • Combination of antibodies revels in one piece of tissue,

6. In situ hybridisation

   • Used to detect specific mRNA within a cell - signal from which the related protein is translated
   • Probes can be RNA or DNA sequences, with variety of labels

7. Retrograde tracing

   • Inject a special dye into muscle which is taken up by neurons that project to muscle

8. Anterograde tracing

   • inject a special dye into sensory nervous system. e.g. into dorsal root ganglian which is taken up by sensory neurons and transported to their axon terminals

9. Intracellular recordings

   • Give accurate measurements of membrane potential including resting potential, synaptic potentials and action potentials
   • Current can be injected through microelectrode to measure cell membrane resistance, capacitance, action potentials and excitability
   • Limitations
     • Can only make recordings from cell bodies
     • Require major dissection to access cell bodies
     • Unstable - easily dislodged by movement

10. Extracellular recording

    • Extracellular recordings is often carried out of nerve trunks - with axons that are too fine for intracellular recording
    • Recordings are typically made between an electrode close to nerve and a indifferent electrode in the bath
    • Key to recordings is that the small currents around the cells are not short-circuited by surrounding fluid
    • Achieved by insulating the recording point from surrounding fluid either using a barrier or using tight suction electrode
    • Measure the gross currents flowing around the outside of a nerve cell or its axons
    • Limitations
      • Only record action potentials - they do not record membrane potential or synaptic potentials
      • Shape of recordings does not directly reflect what is happening across the cell membrane
      • Difficult to combine with dye: fills so structure of recorded neuron cannot usually be determined