Transduction: detection of sound

Question 1

What is the evidence for the importance of tip links?

Answer 1

1. They only occur along the axis of sensitivity
2. Transduction disappears when treat the hair bundles with BAPTA (severs tip links - Assad et al 1991). If break tip links with BAPTA, then leave for 24-48 hours -
   transduction comes back! (tip links reform - Zhao et al 1996)

Question 2

What is the difference between endolymph & perilymph?

Answer 2

Layer of tissue across top of hair cells (joining them together) = very electrically tight layer of tissue

Hair bundle electrically isolated from basal pole of hair cell, apart from through the transduction route

Endolymph: 140mM potassium (high), ~35µM calcium
Perilymph: high sodium, low potasium ~1.3µM calcium

Question 3

What is the membrane potential of a hair cell?

What is the driving force for potassium?

Answer 3

~ -55mV resting potential
~80mV in endolymph

Driving force for potassium = electrical gradient: difference between inside cell compartment and endolymph ~ 135mV

Electrical gradient as concentration of potassium ~140mM inside cell and in endolymph

For calcium, electrochemical gradient (although low in endolymph, calcium is extremely low inside the cell)

Question 4

What happens when a vibration pushes the hair bundle in a positive direction?

Answer 4

Towards the tallest stereocilium

• Mechanically-gated transducer channel opens
• Positive charge (potassium + small amount of calcium) flows into the cell and depolarises cell
• Voltage-gated calcium channels in basolateral membrane activated
• In an IHC, calcium influx causes more transmitter release onto afferent nerve (in OHC, activation of voltage-gates acts on prestin)

Question 5

What happens when the hair bundle is pushed in the negative direction?

Answer 5

Towards shortest stereocilium

• Tension on tip link released, allows open channels to close
• Shuts off current
• Hyperpolarises very slightly
• Any open calcium channels will close
• Reduction in afferent firing

Question 6

What happens to transduction when the hair bundle is in the resting position? Evidence for this?

Answer 6

A small amount of current / neurotransmitter release

When bundle is pushed in negative direction, can see a small amount of current switched off, therefore in resting position should have small number of open channels as can record a current that goes away when pushed negatively

Number of channels open at rest varies between IHCs & OHCs

Question 7

How do you prepare an experimental preparation of the hair cells?

Answer 7

• Remove the bone that encases the cochlea (sometimes have to remove organ of Corti/surrounding tissues to get in dish)
• Remove tectorial membrane (once in the dish - if you want access to the hair bundles)

Aim: remove sensory epithelia from animal without damaging transduction

• Using immature animal is key to avoid damaging tip links

Question 8

How can you make recordings of transducer currents? (experimental method)

Answer 8

Whole-cell patch clamping

• Hair cells sit on strip of tissue (doesn’t usually work if isolate hair cells - too much mechanical disruption)
• Burrow in with patch pipette and voltage clamp membrane (rupture membrane between pipette & cell, gain electrical access)
• Simple feedback mechanism: amplifier detects and records changes

Question 9

What are the advantages/disadvantages of rupturing the cell membrane with the patch pipette?

Answer 9

✔︎ Can introduce chemical compound into the cell
✔︎ Can record membrane currents through any kind of ion channel (receptor operated, voltage operated or mechanically operated)

But, may wash out key signalling components of cell

Question 10

What are some key differences between studying transduction in hair cells in vivo vs experimentally?

Answer 10

In experiment: damage electrically sealed compartment, replace endolymph with experimental solution: high sodium, low potassium (and no Mg as Mg can block things)
If used endolymph, would be bathing basolateral pole in high potassium, membrane potential destroyed/leaky membrane (sodium and potassium similar size, both travel through channel)

Compromises:

1. Much smaller driving force (as no endolymph) - set holding potential to -80mV (if go too negative cell won’t survive for long)
2. Differences in ions flowing (sodium rather than potassium - high potassium would destroy membrane potential)
3. Room temperature (usually a disadvantage as processes are slow at room temp, in this case, advantage as slow enough to record elements of transduction)

Question 11

When recording transduction in non mammilian vertebrates - which preparations to use? Why?

Answer 11

Turtle auditory hair cells (Fettiplace, Ricci)
Bullfrog vestibular hair cells (auditory stimuli tend to be much faster than vestibular)

✔︎ Easy preparation to obtain
✔︎ Fairly resilient in an experimental situation
✔︎ Cold-blooded (so working at right temperature for the animal!)
✔︎ Have low frequency hair cells <1kHz or even vestibular: slowness can be advantage as difficult to study something that responds at 20kHz

Question 12

What did Fettiplace et al (2001) show with turtle auditory hair cells?

Answer 12

Step change stimulus (rather than sinusoid) using physical push with glass probe

Produced family of currents: bigger push = bigger current, eventually max out current

1. Current turns on then very rapidly turns off (adapts) - even though stimulus is maintained (especially with small pushes i.e. more physiological pushes)
2. Adaptation ~80% in turtles (channels open
   then close again - higher value in mammals)
   Bundle resets sensitivity during continuous sound stimulation so you can then push it again (if it doesn’t reset then push was larger than physiological levels)

Question 13

What did Fettiplace R. (2006) show?

Answer 13

Two experiments

1. Bundle in resting position,then simple pushes to get family of currents
   Peak of current (y) plotted against push (Δx - um), shows current switched on at ~0.2 and maxes out ~0.4/0.6 - would expect greater sensitivity in Vivo (not sure why)
2. From rest, push bundle 0.4um - get big current which then adapts, then family of pushes from that position: get very similar currents but displaced (I.e. At 0.6um, same peak current as 0.2um in first experiment)
   Shows that current can respond in very similar fashion despite ‘preloading’ bundle

Question 14

What are the properties of the rat cochlea?

Answer 14

P11-P12 = onset of hearing (not fully developed, but can first respond to sound)

Total range 500Hz - 65KHz
Low frequency = apex - tend to work in 3-10kHz range as already fast for recordings

Can take out whole structure more easily as not fully developed (like turtle/bullfrog - it is more robust than human - but it is a mammal)

Question 15

What did Kennedy et al (2003) show?

Answer 15

First data on mammalian hair cells

OHC, glass probe push stimulus & patch pipette buried into cell (patch clamp with compensatory current)

By convention, show what is happening through channel - not what the amplifier is injecting!!

Shows mammalian hair cells operate at different speed to turtle/bullfrog

Can’t really measure current turning on as so fast - slope reflects how far pushing with pride and very fast switch off ~0.1ms

ADAPTATION TIME CONSTANT ~0.1ms

Question 16

Does the transducer current change during development?

Answer 16

Transduction develops very early

Kennedy et al (2003) found very little change over development (P5 P13 P18)
- almost no change in size of current or adaptation rate

Other things much change the process from deaf to hearing

Question 17

How can the in Vivo environment be mimicked experimentally?

Answer 17

Kennedy et al (2003)

1. Hold cell at -80mV (normal driving force), push bundle then record what happens
2. Very quickly dial amplifier to -130mV (mimicking endocochlear potential), push bundle, record current and dial back up
   (With bigger driving force, bigger current through the mechanically gated channel)

Current gets faster as well as bigger - thought to be because more calcium comes in with the bigger current

Question 18

What evidence shows that calcium is adaptation dependent?

Answer 18

Kennedy et al (2003)

Cells don’t stay healthy if low calcium around basolateral pole, therefore 1.3mM used (35uM in endolymph too low for basolateral pole, so 1.3mM is used around hair bundle)

Push hair bundle (stepwise stimulus) - family of currents

At 1.5mM, 1nA inward current with adaptation
At conc closer to endolymph (hard to achieve) - larger current and slower adaptation

• Decreasing external Ca²⁺ slows adaptation: therefore, adaptation process itself is likely to require calcium in order for it to happen

Question 19

What are the effects of DECREASING external calcium on the hair cell?

Answer 19

Slows adaptation rate
Increases current size
Increases amount of current in at rest

(Kennedy et al 2003)

Question 20

What are the properties of the transducer current in mammals?

Answer 20

Fast onset of current (too fast to measure in mammals)

Fast rate of adaptation: (10x faster than non mammal)

Properties don’t seem to change with development - reasonably well demarked atleast by P5

Lowering extracellular calcium (around the BUNDLE):

* increases current size
* decreases adaptation rate

However, in these experiments:

* different ions flowing
* no endocochlear potential 
* room temperature

Question 21

How can we extrapolate from experiments on hair cells to in Vivo performance? What factors are different?

Answer 21

• Calcium concentration
• Driving force
• Differences between K⁺ & Na⁺
• Temperature

Typically see 1.6 nA current with adaptation time constant: 230µs

In vivo: may convert to 6nA current, time constant 29µs - fast enough to follow the stimulus on a cycle by cycle basis

(here, factoring in temp and endocochlear potential that occurs in vivo) - too fast to measure, so have to estimate