Nerve Conduction And Synapses

Question 0

Miniature epps

Answer 0

Observed in absence of nerve stimulation
Magnitude always a multiple of 0.4mV = vesicular release by exocytosis quantal release, occurred randomly and were discrete depolarisations –> not in fact noise representing continual ACh release from boutons
del Castillo and Katz 1954 aided interpretation –>
Used Mg2+ to show that as concentration increased size of epp decreased in stepwise rather than continuous relationship, lead to their ‘vesicle hypothesis’ in 1956
Boyd and Martin altered external Ca and recorded changes in the depolarisation of the membrane in steps of 0.4mV

Question 1

K+ dependence of membrane potential

Answer 1

Hodgkin and Horowicz 1959 on frog muscle fibres
Much closer to constant field equation than Nernst
More depolarised than prediction of lower [K+] of 10mM –> greater influence of other ions as membrane must be permeable to things other than K+

Question 2

Electrogencity if Na+k+ pump

Answer 2

Snail neurone
Inject sodium chloride (charge neutral –> no polarisation) but generates hyper generation as pump is activated
Ouabain inhibitor of pump causes depolarisation
–> pump at rest –> pump is electrogenic and makes contribution to resting potential

Question 3

Voltage Clamp

Answer 3

Hodgkin and Huxley
Squid giant axon
Electronic, manual mechanism to control cell voltage - feedback loop
Direct measurement of ionic flux whilst holding the membrane at a particular value
Showed Na and K permeability of a nerve axon were gated by a change in membrane potential

Question 4

Patch clamping

Answer 4

Neher and Sigworth 1980
Relatively wide tip to form very tight electrical seal. Isolate patch of membrane to record individual icon channels
Two currents (Na and K) are shown to be separated by blocking one of the fluxes (tetradoxin --> Na inhibits action potential, tetraethylammonium --> K prevents repolarisation )

Question 5

Channels packed into nodes of ranvier

Answer 5

Immunofluorescence
Purify protein –> antibody formation –> label antibodies and expose to tissue
Binds to target (Na+ channels) for their location

Question 6

Acetylcholine at NMJ confirming Loewi

Answer 6

Henry Dale
Took 2 dog hearts (myogenic)
> bathed in solution
> one had nerves removed
> release chemical in one with neurones into solution, then placed solution on non-nerve heart
–> caused slowing of heart beat –> chemical in solution must be the cause

Question 7

Determine where ACh is stored

Answer 7

Homogenisation / centrifugation studies carried out in cerebral cortex show that ACh and ChAT occur in a large particle similar in size to mitochondria

Question 8

Resting membrane potential

Answer 8

Cathode ray oscilloscope
When an intracellular micro electrode is passed through the plasma membrane, there is an immediate change in the potential difference
This shows that the charges are localised at the membrane due to attraction between the positive and negative ions, the rest of the cell is neutral

Question 9

Na-K ATPase ion ratio

Answer 9

In 1957 Post and jolly
Cooled erythrocytes to a temperature at which Na-KATPase could no longer function and only diffusion occurred
When temperature was increased, showed every 3 sodium out, 2 potassium pumped in
Hyperpolarised by 0.5 and 10mV

Question 10

All or nothing

Answer 10

E D Adrian et al 1920s
Size of action potential is constant and independent of initial stimulus - once threshold reached, stimulus has no further effect
Seen by direct observation of consistency of action potential size on motor and sensory neurones

Question 11

Discovery of ACh

Answer 11

Otto Loewi 1921
Perfusion fluid from a heart that was stimulated by the vagus nerve could reduce amplification of the normal beat in the absence of vagal stimulation
Demonstrated AP causes release of chemical substances that can pass on the effects of that neurones stimulation
Named the substance vagusstoff later ACh

Question 12

Recycling of ACh

Answer 12

Potter 1970
Monitored ACh synthesis from radioactively labelled choline that the store of ACh in a motor nerve terminal is degraded and re-synthesised three times in an hour

Question 13

ACh vesicles

Answer 13

Stored in 50nm vesicles which appear clear in EM
1960 centrifugation of brain homogenates
Differential centrifugation showed that the ACh is located in particles of similar size to mitochondria- vesicles
Fraction containing ACh was then separated with the application of a sucrose density gradient

Question 14

ACh release is by exocytosis

Answer 14

Heuser and Reese in 1973
Continuous stimulation of pre-synoptic neuron –> vesicles become depleted and pre-synaptic membranes become enlarged
Indicates vesicle membrane was becoming incorporated into pre-synoptic membrane
Conclusive evidence by Heuser and colleagues in 1979, with freeze-fracturing in EM showing vesicles in exocytosis

Question 15

Each quantum/vesicle contains 5000-10000 ACh molecules

Answer 15

Kuffler and Yoshikami
Ionophoresis of ACh onto postsynaptic membrane in snake muscle
Determined number of molecules to mimic mmep

Question 16

Vesicles are stored in active zones

Answer 16

Vesicles are stored within active zones within the presynaptic membrane and cannot move laterally within the pre-synaptic neuron terminal
Shown by photobleaching
A fluorescent dye is used to label vesicles and a small cluster is bleached
Bleached stop remains static during ACh release from NMJ
Held in place by synapsins

Question 17

ACh receptors

Answer 17

Radioactively labelled a-bungarotoxin binds to ACh receptors
When exposed with photographic emulsion, the radioactive label ell cause precipitation of silver grains
Causes image to turn black where bungarotoxin has bound
Receptors gathered directly opposite the synaptic cleft