Lecture 9: Quantal Transmission

Question 1

Katz and Miledi (1967) showed that Neurotransmitter Release is?
2.

Answer 1

1. DEPENDENT on DEPOLARISATION
2. INdependent of Na+ or K+ Channels

Question 2

Relationship between Ca+2 INFLUX and transmitter RELEASE?

Answer 2

“AMPLITUDE” of POST-SYNAPTIC POTENTIAL CORRELATES … with… “AMOUNT” of PRE-SYNAPTIC INWARD Ca+2 “CURRENT”

Question 3

Relationship between Ca2+ influx and
transmitter release DIAGRAM

Answer 3

SLIDE 5

Question 4

What TWO relationships SYNAPSE have?

Answer 4

The synapse has CLOSE RELATIONSHIP WITH:
1. Ca+2 channels
2. Synaptic Active Zone

Question 5

How/where does Ca trigger release? (6)

Answer 5

1. Preparation of Choice —> NEUROMUSCULAR JUNCTION

–> 2. Relatively SIMPLE
–> 3. easily ACCESSIBLE
–> 4. “LARGE”
–> 5. ONE MUSCLE CELL is generally INNERVATED by only ONE PRESYNAPTIC AXON (though multiple branches)
–> 6.neurotransmitter directly opens 1 ION CHANNEL TYPE

Question 6

NEUROMUSCULAR JUNCTION DIAGRAM

Answer 6

SLIDE

NEED TO KNOW THE DRAWING/DIAGRAM
HOW DOES IT WORK?

Question 7

Quantal nature of release - WHAT IS MEPPS?
Who discovered? 3

Answer 7

1. Fatt and Katz 1954 Discovery of MEPPs in frogs
2. Miniature EndPlate Potentials ± 0.5 mV in frog

3.MEPPS DEPENDENT on “acetylcholine (Ach)” RELEASE

Question 8

Quantal nature of release RECORDINGS

Answer 8

SPONT.

EVOKED.

FOUND ON SLIDE 9

Question 9

why are MEPPs always same size?

Answer 9

1. MEPPs (Miniature End-Plate Potentials) ARE ALWAYS THE SAME SIZE
2. This is because each synaptic vesicle releases a FIXED AMOUNT OF NEUROTRANSMITTER (e.g., acetylcholine).
3. The uniform size of MEPPs is due to the CONSISTENT NUMBER OF NT MOLECULES IN EACH VESICLE
4. The release process and receptor activation are highly regulated and consistent.

Question 10

why are MEPPs always same size?

One channel opening per MEPP? = 2

Answer 10

NO (Del Castillo and Katz 1954)

Actually ± 2000 channels per MEPP = 5000
molecules of ACh

Question 11

In experiments where calcium (Ca²⁺) LEVELS ARE LOW researchers observe specific patterns in neurotransmitter release at the neuromuscular junction

Key Observations in Low Ca²⁺:

Answer 11

1. ‘FAILURES’
   Sometimes, there is no neurotransmitter release, leading to “failures” where no Miniature End-Plate Potential (MEPP) is detected.
2. ‘MEPPs’:
   When neurotransmitter release does occur, it results in MEPPs, WHICH ARE OF CONSISTENT SIZE
3. ‘Events that are Integer Multiples of MEPPs’:
   “INTEGER MULTIPLES ONLY!”
   Occasionally, the responses are observed as integer multiples of the MEPP size (e.g., 2x MEPP, 3x MEPP). Only these multiples are seen, not fractional or irregular amounts.

And the fact that specific amount of NT causes MEPP

Question 12

Why experiment in Low Ca+2 conc? = 4

Answer 12

1. Calcium’s Role:
   Calcium helps trigger the release of neurotransmitters by causing synaptic vesicles to fuse with the membrane.
2. Low Calcium Conditions:
   When calcium is low, vesicle fusion and neurotransmitter release happen less often.
3. Quantization of Release:
   Even though release is less frequent, it still happens in fixed amounts (quantum), with each vesicle releasing the same amount of neurotransmitter.
4. Integer Multiples:
   When release does occur, the size of the response is always a whole number multiple of the smallest response (MEPP), showing that neurotransmitter release is still quantal but happens less often.

Question 13

why are MEPPs always same size?

Conclusion? found from Experiments in low Ca2+
– Failures, MEPPs and multiples of MEPPs.
– And the fact that specific amount of NT causes MEPP

Answer 13

1. Conclusion: NEUROTRANSMITTER RELEASED IN FIXED AMOUNT: ‘QUANTA’
2. Each quantum produces fixed post-synaptic potential
   = quantal synaptic potential
3. A normal EPP is multi quanta, MEPP is single
   quantum.

Question 14

Specific Amount of Neurotransmitter Causes MEPP:

Answer 14

The size of each MEPP is determined by the amount of neurotransmitter released from a single vesicle.

Each vesicle contains a fixed amount of neurotransmitter.

Question 15

Quantal Synaptic Potential:

Answer 15

1. Each quantum of neurotransmitter produces a fixed post-synaptic potential, which is the MEPP.
2. The MEPP is a single quantum response.

Question 16

EPP vs. MEPP:

Answer 16

1. ‘EPP (End-Plate Potential):’
   MULTI-QUANTA
   Represents the total response when multiple vesicles release neurotransmitter, leading to a larger potential.
2. ‘MEPP (Miniature End-Plate Potential):’
   SINGLE QUANTUM
   Represents the response from the release of a single vesicle, showing the effect of one quantum of neurotransmitter.

Question 17

What is the basis for quanta?

Answer 17

1. Electron Microscopy (EM) Evidence
   - De Robertis and Bennett, 1954: Early electron microscopy (EM) studies revealed the presence of synaptic vesicles at the neuromuscular junction.

2.EM showing ‘active zones’ - vesicle clustered areas

3. 1 vesicle = 1 quantum
   - smallest unit of neurotransmitter release (a quantum) is associated with the release from one vesicle.
4. Confirmation by “freeze fracture” (Heuser and Reese 1970s).
   - Capture of release moment of vesicles
   - confirmed that each vesicle releases a discrete, quantal amount of neurotransmitter, supporting the idea that neurotransmitter release is quantal.

Question 18

TEM of free-fractured cell membrane

Answer 18

1. occurs between the two (bi) phospholipid layers
   - can see exposed proteins sticking out of the two layers
   - individual phospholipids are TOO SMALL TO SEE
2. electron microscopy technique
3. used to examine the internal structure of cell membranes.
4. It involves rapidly “freezing” the sample, “fracturing” it, and then “imaging” the fractured surface.
5. visualizing the arrangement of vesicles and active zones at the synaptic terminal.

Question 19

Understanding Quantal Transmission:

Answer 19

1. 1 packet of neurotransmitter = 1 quantum
2. each quantum gives approximately the SAME POST-SYNAPTIC RESPONSE = “QUANTAL AMPLITUDE”
3. MEPPs are SPONTANEOUS, PROBABILISTIC OCCURRENCE (like tossing a coin)

Question 20

Key variables in quantal transmission

WHAT IS ‘p’?

What is ‘pn’?

Answer 20

p = the average release probability.

pn = Probability of releasing ‘n’ quanta of
neurotransmitter (mean probability of failure = 1-p)

Question 21

Key variables in quantal transmission

WHAT IS q?

Answer 21

q = Amplitude (size) of a quantal response

AMPLITUDE = SIZE

Question 22

Key variables in quantal transmission

WHAT IS n?

Answer 22

n = number of quanta available for release

Question 23

Key variables in quantal transmission

WHAT IS m?

Answer 23

m = mean quantal content (Mean EPP (evoked)/Mean
MEPP amplitude)

= the average number of quanta of transmitter released per stimulus

Question 24

Key variables in quantal transmission - Determining these parameters is

Answer 24

“doing a quantal analysis” of synaptic transmission

Question 25

How to determine key variables?

5 MAIN STEPS

Answer 25

1. RECORD SPONTANEOUS AND STIMULATED endplate potentials (EPP) from NMJ.
2. Low Ca2+ high Mg2+ (High Ca2+ lots of quanta released)
3. Some stimulation evokes no release = “failures”
4. PLOT HISTOGRAM of number of responses/amplitude
   - X axis is amplitude of end=plate potentials (mV)
   - Y axis is number of observations
5. FIT A STATISTICAL MODEL as best as possible to the data to determine amplitude peaks

Question 26

determining key variables = OUTCOMES

HOW TO DETERMINE ‘p’?

Answer 26

p can be determined from statistical Model

Question 27

determining key variables = OUTCOMES

HOW TO DETERMINE ‘q’?

5 main steps

Answer 27

q (quantal size or amplitude)

1. ‘usually MEAN of SPONTANEOUS mini’s’
2. ‘usually AMPLITUDE OF 1ST PEAK in a response histogram’
3. ‘Peaks I, II, III etc = # of quanta released’
   Peaks I, II, III, etc.: These peaks in the histogram of postsynaptic responses correspond to different multiples of the quantal size (q). For example:
   Peak I: Represents the response to 1 quantum (1 vesicle).
   Peak II: Represents the response to 2 quanta (2 vesicles).
   Peak III: Represents the response to 3 quanta (3 vesicles), and so on.
4. ‘Spread of q due to some variation in neurotransmitter
   content per vesicle’
   - due to differences in neurotransmitter content per vesicle.
   - not all vesicles contain exactly the same amount of neurotransmitter, which can cause some variation in the size of MEPPs.
5. ‘Distance between peaks’
   -in the response histogram helps in determining the value of q in quantal transmission.
   - The interval between peaks reflects the size of a single quantum

Question 28

determining key variables = OUTCOMES

HOW TO DETERMINE ‘n’?

Answer 28

1. Determining N is MORE DIFFICULT
2. REQUIRES QUANTITATIVE ANATOMICAL STUDY
3. How to DEFINE ‘N’ is also UNCLEAR - usually defined as a CLUSTER OF VESICLES, CLEFT, POSTSYNAPTIC SPECIALISATION

Question 29

Key Issues in Quantal Analysis

because there is

Answer 29

Issues in DETERMINING QUANTAL SIZE

Issues in VARIATION in MEPP size

Question 30

ISSUES Determining Quantal Size (q): 4

Answer 30

1. ‘At NMJ’: EASIER TO MEASURE ACCURATELY
   - LARGE SYNPASE
   - REPSONSE CLEAR
2. ‘In CNS’:
   - HARDER to measure due to:

…3. ‘Signal Decay’: POSTsynaptic signals WEAKEN as they TRAVEL along DENDRITES.

…4. ‘Small Size and Multiple Inputs’:
- CNS synapses are SMALL and RECEIVE INPUTS FROM MANY NEURONS, making it TRICKY TO ISOLATE AND MEASURE INDIVIDUAL QUANTA

Question 31

3 ISSUES in Variation in MEPP Size:

Answer 31

1. NUMBER OF NEUROTRANSMITTER MOLECULES:
   - Different vesicles may have varying amounts of neurotransmitter, affecting MEPP size.
2. REUPTAKE AND BREAKDOWN:
   - How neurotransmitters are reabsorbed or broken down can impact MEPP size.
3. NUMBER OF RECEPTORS:
   - More receptors on the postsynaptic membrane can increase the MEPP size for a given amount of neurotransmitter

Question 32

Example of Quantal Analysis

Context, Findings, Explanation, Result

Answer 32

1. Context:
   Studying Clcn3−/− mice (mice lacking the chloride channel CLC-3).
2. Findings:
   Reduced Miniature Inhibitory Postsynaptic Currents (mIPSCs):
   
   3. ‘Amplitude:’ The size of individual inhibitory postsynaptic currents is SMALLER
   4. ‘Frequency:’ The rate at which these currents occur is LOWER
3. Explanation:
   ‘Chloride Channel (CLC-3)’: This channel is important for maintaining proper chloride ion balance.
   
   6. “Impact of Absence”: Without CLC-3, the release of neurotransmitters is impaired.
4. Result:
   “Reduced Quantal Content”: Fewer neurotransmitter vesicles are released, or the vesicles contain less neurotransmitter, leading to smaller and less frequent inhibitory signals.

Summary
In Clcn3−/− mice, the absence of the CLC-3 chloride channel leads to smaller and less frequent inhibitory postsynaptic currents due to reduced neurotransmitter release, showing decreased quantal content.