11- In-vitro techniques Flashcards
What are three ways we have explored how the nervous system works?
- Basic observations
- Moderate interventions - hypothesis testing
- correlating behaviour and evironment (eg pavlov)
- Detailed interventions
- Classical in vivo and in vitro experiments
- in vivo and in vitro experiments allow for ________ ________ mechanismac
Allows for hypothesis testing mechanism
What are four typical mechanistic questions in neuroscience?
- Functions of ion channels
- Emergent properties of ion channel assemblies
- Emergent properties of neuronal groups
- Actions of receptors on all of these
What does emergent mean in the sense of mechanistic testing?
Emergent means that properties of a system arise that you would not have predicted from the properties of individual elements of the system
eg consciousness is an emergent propery of neurons
What is Patch clamp great for looking at and what is EEG great for observing?
- Patch-clamp
- Good for single channel or whole-cell
- current time resolution
- NOT IDEAL for network analysis
- Scalp EEG (electroencephalogram)
- great for measuring synchronous events in networks of millions of neurons, but not for the analysis of quantal synaptic events in individual neurons
How do you match question with research technique?
- First - what is your question
- Good idea to determine if it mediates a behavioral effect
- Start in vivo
- inject into brain ventricles, then regions where it is expressed or where fibres containing it are found
- Observe behaviour changes if any
After performing in vitro techniques, what would be the next steps of research
- maybe generate very specific hypothesis
- unlikely that a highly-specific hypothesis will arise from behavioural experiments alone
- maybe if existing data already support it
- unlikely that a highly-specific hypothesis will arise from behavioural experiments alone
- More likely, generate very general hypothesis
- Test hypothesis experimentally
Example of a general hypothesis
Peptide X will change the activity of neurons in (brain area of interest Y)
Ways of testing a general hypothesis (in cellular neuroscience)?
- Measure drug effect on action potential frequency measured extracellularly
- Measure action potential activity in intracellular recording
- Fill individual neurons, or an entire slice with a Ca++ sensitive dye, using imaging to measure activity changes
What is required for extracellular field potential recordings?
Extracellular field potential recordings requires organized structure (current flows must be aligned)
What are orthodromic responses?
- Stimulate presynaptic fibres
- Observe postsynaptic responses
What are antidromic responses?
- Stimulate postsynaptic axons
- observe cell body responses
What are advantages of evoked field potential recordings
- Record responses of many neurons at once
- technically simple, stable responses
What are disadvantages of Evoked firled potential recordings?
- Requires organized structure (current flows must be aligned)
- Overall measure of activity, mechanistic understanding requires supporting experiments
What is a single-unit extracellular recording?
Follow activity of one neuron in vivo or in vitro
Electrode remains very near cell, but not inside it
What are advantages of single-unit extracellular recording?
- Characterize firing activity of single neurons
- can observe differential responses in different cell types
- Can permit prolonged recordings
- Can permit lots of recordings one after another
What are disadvantages of Single-unit extracellular recording?
- Only works on active neurons (silent cells might be more important for a given response)
- Assumes neuron remains the same (hard to tell if cell is the same when there are lots of active cells nearby)
- Hard to identify neuron you recorded afterwards
- Does not record subthreshold behaviour
What are four intracellular recording methods?
- sharp microelectrode recordings
- whole-cell patch clamp
- loose patch
- perforated patch
What is bridge current clamp (sharp intracellular micropipette recording)
- “Wheatstone bridge”
- balances resistances of pipette and bridge to neutralize effect of pipette resistance
- now replaced by operational amplifiers
- balances resistances of pipette and bridge to neutralize effect of pipette resistance
- Circuit injects current proportional to product of DC current passed and pipette resistance
- Adjust circuit so initial response to current step reflects only cell membrane properties
The image shows a sharp micropipette intracellular recording measuring rheobase, what is the reason?
Generate current ramp in current clamp. Determine current needed to reach AP threshold
The arrowheads in the image show _______ from sharp micropipette recording
The arrowheads indicate antidromic stimuli
Note: constanct latency, sharp rise of AP from baseline (red arrow)
Recall: antidromic: stimulate postsynaptic axons and observe cell body response
What does the vertical arrow indicate in the Sharp intracellular recording of antidromic potential?
The vertical arrow indicates collision cancellation
Where AP would have occurred if the spontaneous AP had not happened
How does the access resistance differ between sharp and patch electrodes?
- In addition to the much higher seal resistance, the access resistance is far less with the larger patch pipette tip
- Tip resistance determines how much current can flow through the pipette
- the more current can flow = the larger the signals
Sharp vs patch pipette tips - diameter difference
- Patch pipette tip has a larger diameter shank than sharp pipette
- Much greater conduction volume, therefore lower resistance
What are the four patch clamp modes and what do they have in common?
- All start with gigaohm seal, membrane patch is “glued” to the inside of pipette
- “Cell-Attached mode”
- “Whole Cell Mode”
- “Inside-out patch”
- “Outside-out patch”
How are N-type Ca++ currents explored in conventional whole-cell patch recordings?
- Recombinant N-type VDCC and Recombinant u-opioid receptors in somatic cell line (tsA 201)
- Cs+ and TEA in the pipette and bath solutions, 0 K+, and Ba++ as the charge carrier
- Strong depolarizing prepulse (PP - grey traces) relieves “reluctant” state of the channel, caused by constitutive activity of the u-opiod receptors
What is the alternative for conventional whole-cell and the dialysis of the cytoplasm that can cause important signaling elements to be washed out?
Make your own ion channels
Perforated patch recording
- Use pore-forming antibiotics (nystatin, amphotericin B, gramicidin) inside pipette
- Form gigaohm seal, let abx perforate membrane
- Once access through membrane is adequate, record as with whole-cell
- Higher access resistance than conventional whole-cell
- Can rupture patch at end to fill cell with dye
How is a cell-attached recording done?
- Form seal, observe channel activity
- If studying ligand-gated ion channel, can include agonist in pipette
