PCOL3022 Lecture 9: Cellular Techniques in Neuropharmacology

What is Electrophysiology?

- Study of electrical properties of cells or tissues

- Cells have membranes with channels that allow ion passage
- Electrophysiology measures the movement of these ions

What does Electrophysiology tell us?

- How drugs modify the way ion channels activate/open/inactivate/desensitise
- How drugs change neuronal excitability, synaptic transmission, and synaptic plasticity
- Where drugs are acting
- How drugs interact with mutated proteins

How do we measure Electrophysiology?

- Dependent on the size of the current and of the cell

- E.g. Giant squid axons are large enough to just stick a wire into

What are Extracellular Recordings?

- Non-invasive

- When action potentials occur, current flows in and out of the cell --> changes the cell voltage
- An extracellular electrode can measure these changes in voltages

- Small electrodes: Can measure activity of a single neuron (single-unit)
- Larger electrodes: Can record several neurons (multi-unit)

- Recording is of REVERSE of action potential (as we're recording the extracellular environment)

- Main use is in vivo (in anaesthetised or awake, behaving animals)

Extracellular Recordings Disadvantages

- Hard to control the administration of drugs
- No control over cell of interest
- Cannot determine where drug acts

Micro-electrode/Intracellular Electrodes

- Very fine glass electrode inserted into the cell. Metal wire in a liquid-filled glass electrode is connected to an amplifier

- Can measure voltage or current across the membrane and compare it to a reference electrode

- Mainly used in oocytes (large frog-egg cells that can accommodate 2 electrodes) and rarely in neurons

Advantages of Microelectrode/Intracellular Electrodes

- Oocytes express proteins well
- Technically simple
- Useful for drug screening

Disadvantages of Microelectrode/Intracellular Electrodes

- Frog oocytes, not human neurons
- Difficult to exchange drugs
- Hard to get good electrical control as there is only one electrode
- Recording is noisy
- Cell is compromised (has hole from electrode)

What is Patch-clamping?

- Can either be outside-out or whole-cell

- Can control or clamp the voltage of most mammalian neurons
= Amplifier maintains membrane potential and measures current required to maintain it
= Can measure voltage-dependent ion channel responses
= E.g. When GIRKs open and K+ exits the cell, more positive charge is required to maintain voltage

Patch Clamping Advantages

- Low noise, high resolution
- Can measure/study a single or small number of proteins
- Can control the intracellular milieu of cells

- Versatile: Can measure several proteins in same cell, many different applications (single cells, cultured cells with mutated proteins, brain slices, in vivo)
- Accesses inside of cell (can manipulate signalling systems one cell at a time)

Patch Clamping Disadvantages

- Technically complex
- Expensive (equipment > $100,000)
- Not suitable for all cells (e.g. some adult rat neurons)
- One cell at a time (low throughput)
- Loss during cell preparation (from inside and outside of the cell)

What is Outside-out Patch-Clamping?

- Microelectrode forms tight seal with membrane and a patch of membrane containing a single ion channel is removed

What is Whole-cell Patch-clamping?

- Once microelectrode is sealed to membrane, patch is ruptured to give electrode access to whole cell (multiple channels)

- Can study ion channels in isolated cells, cell cultures, and cells from brain slices or in vivo

- Study how drugs alter synaptic transmission (overall effect + more detailed approach)

- Size of post-synaptic current depends on: (1) amount of Glu released, (2) How many post-synaptic AMPA receptors there are, (3) Location of AMPA receptors, (4) Phosphorylation state of receptors, (5) Voltage of post-synaptic cell, (6) Ion inside + outside of the cell

- Can study both acute drug action and long-term potentiation/learning

What are Current Clamps?

- Apply known constant or time-varying current
- Measures changes in membrane potential caused by applied current
- Move "natural" response to opening ion channel
- Mimics current produced by synaptic input

What are Voltage Clamps?

- Apply known membrane voltage and measures trans-membrane current required to maintain voltage
- Controlling voltage means we can limit changing variables

Pulled Patches

- Very low noise and high resolution (as there is no hole in the cell membrane)

What are Fluorescence-based assays?

- Optical non-invasive measure of drug action
- Fluorescent dye responds to signaling pathways of interest
- Rapidly increased use due to advancement of fluorescent materials

- Fluorophores can respond to:
= membrane voltage
= ions
= cAMP
= kinase activity
= apoptosis

What do Fluorescent-based assays tell us?

- Whether drugs target the protein of interest
- Dose-response for a drug acting at the protein of interest
- How drug interacts with the mutated protein
- How drug alters longer-term processes

How do Fluorescent-based assays work?

- Cells (+/- expressed proteins) are cultured in plates
- Fluorescent material is added
- Use a plate reader or microscope to excite fluorophores and measure emitted fluorescence
- Measure at baseline and in response to drug application or other stimuli
- Latest tech can apply multiple drugs and measure over an extended period

Fluorescent-based assays Advantages

- Population response
- Very easy to do
- Non-invasive
- Real-time readout
- Long-term recordings
- Can measure multiple readouts at once

Fluorescent-based assays Disadvantages

- No access to inside of the cell (requires membrane permeable drugs to access intracellular environment)
- No control over membrane potential
- Fluorescent material may interfere with measurements
- Cannot wash off drugs
- Slow temporal resolution than electrophysiology
- Can be hard to know exactly what process is being measured

What is Immunohistochemistry?

- Combination of histological, immunological, and biochemical techniques for identifying specific tissue components through a specific antigen/antibody reaction tagged with a visible label
- Allows localisation of antigen within a cell or tissue

What can Immunohistochemistry tell us?

- Which receptors, enzymes, transporters, lipids, etc are in the brain region of interest
- What receptors might be a good target for treating a disease
- Whether a drug changes the expression of something
- Whether a drug changes the sub-cellular location of receptors, etc
- Whether a drug changes the levels of a cellular event such as apoptosis, actin depolymerisation

Immunohistochemistry -- 1st step

1. Tissue preparation
- Brain slices, cells on cover plates
- Tissue usually fixed with PFA (paraformaldehyde) to prevent tissue breakdown and keep tissue locked in place (PFA crosslinks proteins)
- NB: some tissue prep can cover up desired antigen

Immunohistochemistry -- 2nd step

2. Minimise non-specific labeling
- Only want antibody to react with specific antigen
- Block non-specific sites with bovine serums, etc
- Can use detergent to create holes in membranes and allow antibodies to target intracellular antib

Immunohistochemistry -- 3rd step

3. Primary antibody
- Wash on and leave (30-120 mins)
- Wash off

Immunohistochemistry -- 4th step

4. Secondary antibody
- Interacts with primary antibody
- Can be linked to a fluorophore or to an enzyme that produces a coloured product (immuno-electron microscopy)

- Fluorescent secondaries:
= Requires fluorescent or confocal microscope
= High structural resolution possible
= Advanced 3D image reconstruction and signal
= Quantification
= Multiple labelling
= Live cells

Immunohistochemistry Advantages and Disadvantages

- Can have false negatives or false positives
- Response level is variable, dependent on experimental conditions
- Requires appropriate control (i.e. in tissue lacking antigen, or with no primary antigen)
- Tissue must come from human/animal
- Often sliced very small (10 um)