Foundations I Fall, 2016 Synaptic Transmission I Neuromuscular - - PowerPoint PPT Presentation

Synaptic Transmission I

Foundations I Fall, 2016

Neuromuscular Junction

Neuromuscular Junction

junctional folds

Neuromuscular Junction

Del Castillo and Katz (1954)

Transmitter Release

0.9 mM Ca++ and 14 mM Mg++

m

Early experiments revealed that mEPPs could not result from the effects of a single molecule of ACh since iontophoresis or bath experiments with small concentrations (many molecules) did not give a discernible response. Thus, the mEPPs must result from the action of thousands of ACh molecules. A statistical treatment was necessary since this is a phenomenon (mEPP size) that varied in amplitude and frequency. Del Castillo and Katz assumed that at the neuromuscular junction there was a large population of n units of undetermined nature that respond to a nerve impulse. So what controls the size of the mEPPs? How does the transmitter get released? Is it expelled in a continuous, graded fashion or is it constrained to some sorts of units (quanta), like energy? To make the mEPPs as small as possible, [Ca2+]e was greatly lowered, Mg2+ was raised and a blocker of ACh receptors was added.

Fatt and Katz and Del Castillo and Katz (1951, 1952, 1954)

Transmitter Release

If the probability of a single unit responding is p, and if each unit has an independent and equal p, then the mean number of units responding to each stimulus is given by:

m = np

Del Castillo and Katz (1954)

Transmitter Release

mean quantal content

total n of available units p of a unit responding

nx = N n! (n − x)!x! pxq( n− x)

Under these conditions, the relative occurrence of multiple events (i.e., mEPPs of sizes corresponding to integral multiples of the minimum mEPP size) is given by the Binomial distribution:

where N = the number of trials, nx is the number of events consisting of x units (quanta) and q = 1-p., i.e., the probability of the unit not responding.

P

x = n x

N P

x =

n! (n − x)!x! p

xq (n− x)

What is the probability of getting a response of size (number) x? But there is a problem calculating this...

Transmitter Release

substituting for n from last slide

x

... the values of n and p are unknown! P

x = m x

x! e−m

If one assumes that p is low (<0.1 or so), and equal for all n and that n is very large, the binomial distribution approaches the Poisson distribution described as: m can be easily estimated in 2 ways:

• 1. m= mean amplitude of synaptic potential/mean amplitude of minimal synaptic

potential

n0 = Ne

−m

• 2. If one only counts the failures (x=0, mEPP amplitude = 0), the Poisson

distribution degenerates to

Del Castillo and Katz (1950s)

Transmitter Release

e−m = N n0

divide by N and take ln

m = ln N n0

integrate

Thus, neurotransmitters are released in small packets called vesicles

poisson distribution mepp amplitude distribution

1 vesicle= 1 quantum= Nobel Prize (Fatt, 1970)

The fit between the experimental data and the Poisson distribution was excellent.

How many quanta are released at one time? But this is at the neuromuscular junction, not a synapse... ... and this was under stringent conditions that strongly interfered with transmitter release presynaptically... ... not to mention pharmacological blockade of post-junctional ACh receptors ... So how closely does this describe “normal” synaptic transmission?

Korn, Triller and Faber, 1982

record intracellularly from postsynaptic neuron stimulate intracellularly with HRP-filled microelectrode in presynaptic neuron label presynaptic neuron after experiment and count number of boutons

interneuron Mauthner AD spike and collateral IPSP

Korn et al., 1982

Mauthner Cell

n is very small and p is huge!

Korn et al., 1982

Korn et al., 1982

Transmitter release at this synapse is always monoquantal i.e., the presynaptic boutons operate in an all or none binary fashion!!!

Parabola: y=Ax-Bx2 Y = PSC variance and x = PSC mean A and B are adjusted to fit parabola

then

Mean quantal size (Q) = A/(1+CV2) Probability of release Pr= x(B/A)(1+CV2) Number of release sites N= 1/B (curvature of parabola)

Koós et al., 2004

25 25 ms 100 pA mV

Postsynaptic Spiny neuron Presynaptic FS interneuron Postsynaptic Spiny neuron Presynaptic Spiny neuron

0.2 mV 1.5 mV 20 ms

A1 A2 B1 B2

• 47 mV
• 48 mV

FS -->Spiny IPSP Spiny --> Spiny IPSP

Tepper et al., 2004

25 pA 100 ms

Traces 21-40 Traces 11-20 Traces 1-10

• 136 pA
• 71 pA
• 18 pA

Auger et al., 1998

cerebellar interneuronal IPSCs in vitro from verified single release site

Auger et al., 1998

up to 30% of release events at this cerebellar synapse are multiquantal

Sir Charles Sherrington (1857-1952)

“In view therefore, of the probable importance physiologically of this mode of nexus between neurone and neurone it is convenient to have a term for it. The term introduced has been synapse.” - C.S. Sherrington, 1906

Central Synaptic Transmission

synaptic vesicle

• 1. A presynaptic impulse invades the terminal bouton and depolarizes it
• 2. The depolarization opens voltage sensitive Ca++ channels leading to a transient influx of Ca++
• 3. [Ca]i triggers a sequence of biochemical events resulting in the fusion of a synaptic vesicle

membrane with the terminal membrane.

Steps in Synaptic Transmission

Steps in Synaptic Transmission

• 4. The vesicle contents are extruded into the synaptic cleft in a process called exocytosis.

Exocytosis

• 5. [Ca]i is immediately deactivated by uptake into mitochondria and presynaptic vesicles.
• 6. Released transmitter diffuses across synaptic cleft (very fast, no more than a few tens of

microseconds).

• 7. Transmitter combines with postsynaptic receptors.
• 8. Receptor linked with ion channels and/or intracellular second messengers is activated

and alters the permeability to certain ions and/or or cause an intracellular biochemical event

Steps in Synaptic Transmission

• 4a. The vesicle membrane is incorporated into the presynaptic terminal membrane within ~

50 µsec of fusion. Excess membrane and vesicle components are recycled by endocytosis at sites outside the active zone into clatherin-coated vesicles that eventually lose the coating and are re-used

Synaptic Transmission II

Most synaptic potentials are due to an increase in conductance to an ion or ions

slope=I/V R=V/I R= 1/Slope

Voltage Clamp

Synaptic Transmission II

The increase in conductance causes a synaptic current to flow across the membrane Isyn=nγ(Em-Esyn)

where γ is the single channel conductance and n is the number of channels open. Recall that (Em-Esyn) is called the driving force

The synaptic current is dependent on the driving force on the ions which depends on the potential difference between the membrane potential and the reversal potential. The point at which the driving force is zero is called the reversal potential. This is just another way of saying that the reversal potential is that membrane potential at which there is no net current flow.

What is the difference between a reversal potential and an equilibrium potential?

Synaptic Transmission II

Kaila et al., 1989

Bicarbonate depolarizes the GABA equilibrium potential by up to 10 mV depending on pH.

A

a brief but necessary aside

Cl- free + HCO 3 HCO3 free

• Text

Cl- free + HCO 3

What does Isyn=nγ(Em-Esyn) imply about the relation between membrane potential and synaptic potentials?

Synaptic Transmission II

Shifting the membrane potential (usually by injecting current through the recording electrode) must alter the driving force, thus altering the synaptic current thereby altering the amplitude of the synaptic response.

IPSP amplitude (mV) Em (mV)

reversal potential

stim

Synaptic Transmission II

mV mV mV mV mV

Lacey et al., 1987

Voltage Clamp

Isyn=nγ(Em-Esyn)

Synaptic Transmission II

What is the reversal potential for the action of baclofen?

Because at that point there is no current flow due to the synapse, i.e., the control and the baclofen curves intersect. This can

• nly happen when Em=Esyn

reversal potential

What is this point? Em

What is this point?

Em after baclofen

What does baclofen do to the membrane potential of the cell?

baclofen hyperpolarizes the cell by about 20 mV

A standard method to test for the synaptic nature of a membrane potential change (and to measure the reversal potential) is to inject current and alter the membrane potential and see the effect on the amplitude of the response.

Synaptic Transmission II

Synaptic Transmission II

Hyperpolarizing the membrane makes an EPSP get larger and and IPSP get smaller Depolarizing the membrane makes an EPSP get smaller and an IPSP get larger Em

Synaptic potentials that make the postsynaptic neuron more likely to fire an action potential are termed excitatory postsynaptic potentials (EPSP) and those that reduce the probability of the postsynaptic neuron firing are termed inhibitory postsynaptic potentials (IPSP). Note that this means that not all depolarizing synaptic potentials (DPSPs) are EPSPs.

Synaptic Transmission II

Everything depends on the relation between the reversal potential of the synaptic potential and the spike threshold, not on whether or not the synaptic potential is depolarizing or

• hyperpolarizing. For example, if the spike threshold were -55 mV, and the resting membrane

potential were -75 mV, then a synapse that opened up a Cl- conductance which had reversal potential of -65 mV would elicit a depolarization, but that depolarization would be an IPSP, not an

• EPSP. No matter how much such a conductance were increased, the membrane potential would

never rise above threshold, and the neuron would be effectively voltage-clamped at -65 mV.

EPSP

C

• 2. The potential change is much longer than the conductance

change

Synaptic Transmission II

Two other important points:

• 1. Very few ions need to move in order to create large changes in

Em

Eccles 1962

Voltage Dependence of Central EPSPs

Most EPSPs are due to an increase in GNa+ and GK+ which produces an inward current

Why is Erev ~= -10 - 0 mV?

Are there different channels for K+ and Na+?

Central IPSPs

IPSPs are due to an increase in GCl- (e.g. GABAA receptor)

• r GK+ (e.g., GABAB, dopamine D2, adrenergic α2, and 5HT1

receptors) causing an outward current.

ErevIPSP = ECl- or EK+

5 mV 4 ms

STN Stimulation

• 62 mV

• 47.3
• 54.6
• 71.0
• 77.4
• 80.7
• 84.3
• 87.7
• 91.8

20 ms 5 mV

Control

B B B B B B B B B B

1 2 3 4 5 6 7 8

• 120 -100 -80
• 60
• 40
• 20

Control

Membrane Potential (mV) Response Amplitude (mV)

• 63.2
• 73.1
• 81.9
• 89.5
• 98.0
• 103.0

After 50 µM Bicuculline

J J J J J J J Bic

What is this? monosynaptic EPSP +

• verlapping IPSP

Equilibrium potential is too hyperpolarized for an EPSP

Gulledge and Stuart, 2003

Gulledge and Stuart, 2003

What is the difference between soma and dendrite that accounts for this?

Gulledge and Stuart, 2003

Gulledge and Stuart, 2003

Gulledge and Stuart, 2003

Gulledge and Stuart, 2003

Timing is everything. That, and the fact that synaptic conductances are of much shorter duration than synaptic potentials.

Synaptic potentials that make the postsynaptic neuron more likely to fire an action potential are EPSPs and those that reduce the probability of the postsynaptic neuron firing are IPSPs. Not all DPSPs are EPSPs Not all hyperpolarizing synaptic potentials are IPSPs

Patterns of neurotransmitter release are correlated with release probability

paired pulse ratio (PPR) PPR < 1 = paired pulse inhibition PPR > 1 = paired pulse facilitation Changes in PPR are associated with changes in transmitter release, i.e., are evidence of a presynaptic locus of effect

High p synapses show paired pulse depression and long-term depression

Both are due to changes in p

Low p synapses show paired pulse facilitation and long-term potentiation

Short Term Plasticity

synaptic depression synaptic facilitation

g a p

gap junction

spot desmosome