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PMCOL 371 > 11 - In-vitro Techniques > Flashcards

11 - In-vitro Techniques Flashcards

1
Q

What are three ways we have explored how the nervous system works?

A
  • Basic observations
  • Moderate interventions - hypothesis testing
    • correlating behaviour and evironment (eg pavlov)
  • Detailed interventions
    • Classical in vivo and in vitro experiments
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2
Q

in vivo and in vitro experiments allow for ____ ____ mechanismac

A

Allows for hypothesis testing mechanism

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3
Q

What are four typical mechanistic questions in neuroscience?

A
  1. Functions of ion channels
  2. Emergent properties of ion channel assemblies
  3. Emergent properties of neuronal groups
  4. Actions of receptors on all of these
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4
Q

What does emergent mean in the sense of mechanistic testing?

A

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

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5
Q

What is Patch clamp great for looking at and what is EEG great for observing?

A
  • 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
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6
Q

How do you match question with research technique?

A
  • 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
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7
Q

After performing in vitro techniques, what would be the next steps of research

A
  • maybe generate very specific hypothesis
    • unlikely that a highly-specific hypothesis will arise from behavioural experiments alone
      • maybe if existing data already support it
  • More likely, generate very general hypothesis
  • Test hypothesis experimentally
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8
Q

Example of a general hypothesis

A

Peptide X will change the activity of neurons in (brain area of interest Y)

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9
Q

Ways of testing a general hypothesis (in cellular neuroscience)?

A
  • 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
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10
Q

What is required for extracellular field potential recordings?

A

Extracellular field potential recordings requires organized structure (current flows must be aligned)

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11
Q

What are orthodromic responses?

A
  • Stimulate presynaptic fibres
  • Observe postsynaptic responses
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12
Q

What are antidromic responses?

A
  • Stimulate postsynaptic axons
  • observe cell body responses
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13
Q

What are advantages of evoked field potential recordings

A
  • Record responses of many neurons at once
  • technically simple, stable responses
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14
Q

What are disadvantages of Evoked firled potential recordings?

A
  • Requires organized structure (current flows must be aligned)
  • Overall measure of activity, mechanistic understanding requires supporting experiments
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15
Q

What is a single-unit extracellular recording?

A

Follow activity of one neuron in vivo or in vitro

Electrode remains very near cell, but not inside it

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16
Q

What are advantages of single-unit extracellular recording?

A
  • 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
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17
Q

What are disadvantages of Single-unit extracellular recording?

A
  • 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
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18
Q

What are four intracellular recording methods?

A
  • sharp microelectrode recordings
  • whole-cell patch clamp
  • loose patch
  • perforated patch
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19
Q

What is sharp microelectrode recording?

A
  • Use fine-tipped glass micropipette
  • Filled with strong salt solution (eg KCl, K-Acetate)
  • Requires preparation and micromanipulator to be very stable mechanically
  • More complex than extracellular
  • Requires DC-coupled amplifier (measures off-set as well as transient responses)
20
Q

What information does sharp microelectrode recording provide?

A

Provides detailed information on properties and behaviour of a single neuron

21
Q

What are advantages of sharp microelectrode recording?

A
  • Measure subthreshold changes in membrane potential
  • Can influence activity of neuron by passing current into cell (bridge circuit)
  • Can be used to voltage-clamp (switch clamp)
  • Can measure changes in membrane conductance, synaptic responses
  • Can measure reversal potential for ionic currents
22
Q

What are diadvantages of sharp microelectrode recording?

A
  • Technically demanding
    - Micropipette tips <200nm diameter (high resistance)
    - electrode time constant is slow (tau=RC) making rapid switching difficult
    - Pipette pokes through the membrane, so possible for current to leak past outside of pipette as it passes through membrane (= low seal resistance)
  • Information acquired only from one cell at a time
23
Q

What is bridge current clamp (sharp intracellular micropipette recording)

A
  • “Wheatstone bridge”
    - balances resistances of pipette and bridge to neutralize effect of pipette resistance
    - now replaced by operational amplifiers
  • 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
24
Q

The image shows a sharp micropipette intracellular recording measuring rheobase, what is the reason? (pg.24)

A

Generate current ramp in current clamp. Determine current needed to reach AP threshold

25
Q

The arrowheads in the image show ____ from sharp micropipette recording (pg. 26)

A

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

26
Q

What does the vertical arrow indicate in the Sharp intracellular recording of antidromic potential?(pg. 26)

A

The vertical arrow indicates collision cancellation

Where AP would have occurred if the spontaneous AP had not happened

27
Q

What is a switched single electrode voltage clamp?

A
  • One pipette shares voltage sensing and current passing functions
  • High frequency switching is much faster than membrane time constant
  • Essentially the cell does not notice the current passing “goes away” for a few hundred microseconds
28
Q

What are advantages of switched single electrode voltage clamp?

A
  • Single pipette inside cell (was developed for sharp micropipettes, aslo works with patch pipettes)
  • Voltage sensing means the clamp compensates for actual measured potentials (clamps pipette tip)
  • Can be very fast with the right amplifier, pipette
  • “true” current clamp recording, bridge circuit amplifier
29
Q

What are disadvantages of switched single electrode voltage clamp?

A
  • With sharp pipette, high resistance means limits on current passing
  • Speed of clamping membrane slower than 2-electrode clamp
30
Q

What is a whole-cell patch clamp recording

A
  • Use relatively large tip glass micropipette
  • Filled with iso-osmotic salt solution (eg K-gluconate)
  • Requires similar mechanical stability to other intracellular recording modes
  • Technically even more complex, patch-clamp amplifier (voltage-clamp, current clamp, DC mode)
  • Provides considerably more options than sharp pipette recordings
31
Q

What are advantages of Whole-cell patch clamp recording

A
  • All the strengths of sharp pipette recordings
  • Novel modes of voltage clamp
  • Cytoplasmic contents readily replaced w/ pipette sol’n (altered ionic composition)
  • Greatly increased fidelity of recording
    - Much higher seal resistance = less current leaks past pipette
    - Larger diameter tip means pipette resistance much lower
32
Q

What are disadvantages of Whole-cell patch clamp recordings?

A
  • Dialysis of intracellular contents means some important cytoplasmic components can be lost
  • Need to replace energy substrate in cell (ATP, GTP) lost by dialysis
  • Some other technical considerations (liquid junction potential, pipette thickness)
  • Assume potential at back of pipette is potential at cell membrane (clamp potential accurate for small spherical cells only)
33
Q

How does the access resistance differ between sharp and patch electrodes?

A
  • 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
34
Q

Sharp vs patch pipette tips - diameter difference

A
  • Patch pipette tip has a larger diameter shank than sharp pipette
  • Much greater conduction volume, therefore lower resistance
35
Q

What are the four patch clamp modes and what do they have in common?

A
  • 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”
36
Q

What is whole cell mode?

A

Patch ruptured, tight seal, big hole

37
Q

What is inside out?

A

Single channels, outside mimics cytoplasm

38
Q

What is outside out patch?

A

Single channels, pipette solution mimics cytoplasm

39
Q

How are N-type Ca++ currents explored in conventional whole-cell patch recordings?

A
  • 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
40
Q

Whole cell and imaging

  • The large lumen permits ____
  • Simultaneous ____and ____ measurments are straightforward
A

Whole cell and imaging

  • The large lumen permits easy dialysis of pipette contents into the cell
  • Simultaneous electrophysiological and Ca++ measurments are straightforward
  • Makes filling cells with dyes simple
    - can also fill with dye for later identification
41
Q

Convential whole-cell causes significant ____ of cytoplams

A

Convential whole-cell causes significant dialysis of cytoplams

  • can wash out elements important for signaling
42
Q

What is the alternative for conventional whole-cell and the dialysis of the cytoplasm that can cause important signaling elements to be washed out?

A

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
43
Q

What is used for single-channel recordings?

A
  • On-cell (“cell-attached”) recording the simplest
    - Form seal, observe channel activity
    - If studying ligand-gated ion channel, can include agonist in pipette
44
Q

How is a cell-attached recording done?

A
  • Form seal, observe channel activity
  • If studying ligand-gated ion channel, can include agonist in pipette
45
Q

When is a macropatch used?

A

For channels or transporters with very small conductance

46
Q

A loose-patch is similar to what kind of recording?

A

Extracellular recording

PMCOL 371 (18 decks)

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