Synaptic Transmission:

What happens when the action potential reaches the synaptic bouton?

I. Overview

Two major types of synapses: Electrical and Chemical

II. Electrical Synapses

A. Structure - Gap junctions between cells.

1. Structural features

a. Presynaptic cell - which is anatomically indistinguishable from the

b. Postsynpatic cell.

2. Gap junctions are sites where two cells juxtapose one another and are physically attached by connexons.

a. Connexons are aqueous channels composed of six connexin subunits. Connexin is the name of the integral membrane protein.

b. A connexon from one cell aligns with a connexon from the other cell to form an aqueous pore.

3. Site of electrical continuity w/ current flowing passively through the connexons.

B. Function – very rapid transmission of electrical signals with essentially no delay between the pre- and post-synaptic membrane.

III. Chemical Synapses

A. Structural features

1. Presynaptic cell – histologically distinguished by the presence of synaptic vesicles full of neurotransmitter

2. Postsynaptic cell – lacking synaptic vesicles, but having neurotransmitter receptors (which can be detected immunohistochemically)

3. Synaptic cleft – space separating pre- and postsynaptic cells.

B. Studying the Chemical Synapse (Katz and Fatt, 1950's)

1. Model System – The Neuromuscular Junction

a. Advantages

i. Relatively accessible due to peripheral location

ii. Muscle is

o amenable to intracellular recording, and

o excitable

b. Disadvantage – electrode can be dislodged during muscle contraction in response to motor neuron stimulus

o Solution – lower extracellular calcium or block NTR's with curare

2. Experimental set-up

a. Stimulating electrode stimulates motor neuron

b. Recording electrode in muscle

3. Experiments and observations

a. Stimulation of motor neuron (in the presence of calcium); record from muscle

i. Suprathreshold endplate potential (EPP) elicited (~50 mV)

ii. Muscle conducts action potential (and contracts)

b. Observation of miniature endplate potentials (MEPPs) in the absence of stimulation of the motor neuron.

i. ~0.5 mV in amplitude

ii. Similar in shape to EPP (hence name)

c. Stimulation of motor neuron in the presence of low calcium concentrations

i. EPPs generated ~0.5 mV in amplitude at lower limit

ii. EPPs were integer multiples of MEPPs in amplitude

4. Conclusion/Conjecture - MEPPs represented quantum of released NT

C. Cytology and Functional Studies of Chemical Synapse

1. EM revealed

a. Synaptic vesicles in the presynaptic terminal

b. aligned within 40 nm of Ca²⁺ channels in the active zone.

2. EM combined with pharmacology (4-AP) and physiology revealed that there was about a 1:1 relationship between quanta released and the number of vesicles fusing.

D. Vesicle Recycling

1. These findings raised a problem: How does the neuron prevent the synaptic bouton from ballooning as a consequence of membrane addition as a consequence of vesicle fusion?

2. Experiments

a. Hypothesis: Membrane is recovered (returned to vesicle population) through endocytosis.

b. Approach

i. Load synaptic cleft with HRP

ii. Stimulate motor neuron.

iii. Fix tissue at intervals.

iv. Examine by EM.

c. Results

i. At early time points HRP was in coated vesicles.

ii. At intermediate time points, HRP was in the endosome.

iii. At late time points, HRP was in synaptic vesicles.

d. Conclusion (taken with other results) – vesicle cycle takes about 1 min from packaging to re-packaging.

IV. Role of Calcium in Transmitter Secretion

A. Fatt & Katz experiments hinted that calcium was important in regulating the amount of NT secreted.

B. Axon terminal contains voltage-gated calcium channels

1. AP of abnormal shape can be observed in the axon terminal even in the presence of TTX.

2. Voltage-clamp experiments revealed an inward current

a. blocked by cadmium, a calcium-channel blocker

b. post-synaptic neuron failed to respond in the presence of cadmium

3. Calcium imaging experiments confirmed a rise in intracellular calcium concentrations in the synaptic terminal.

4. Microinjection of calcium (in the absence of electrical stimulation) into the presynaptic neuron

a. leads to a post-synaptic response.

b. demonstrating elevation in intracellular calcium is sufficient to induce NT release.

5. Injection of calcium buffers (chelators) into the presynaptic neuron

a. prevents post-synaptic response.

b. demonstrating that the elevation of calcium is necessary (and that membrane depolarization is not sufficient).

V. Molecular Mechanism of Transmitter Secretion – Possible Sites of Action for Calcium

Stage of Secretion	Calcium-sensitive protein and its proposed role
Reserve (after budding but before docking)	Unphosphorylated synapsin tethers vesicles to the cytoskeleton.
Docking (when the vesicles come close the presynaptic membrane, but before they're primed)	Synapsin, phosphorylated by Ca^2+/CaM-dependent protein kinase, releases vesicles from the cytoskeleton and enables them to get close to the membrane.
Fusion	Synaptotagmin binds calcium, which enables it to insert into membranes and bind the SNAREs (See below). This somehow leads to vesicle fusion.

A. Other synapse-associated proteins

1. SNAREs = SNAP receptors which braid themselves into a protein complex that primes the vesicles for fusion.

a. synaptobrevin – associated with the vesicle

b. syntaxin and SNAP-25 – associated with the presynaptic membrane

2. SNAP = soluble NSF-attachment proteins

3. NSF = NEM-sensitive fusion protein (NEM = N-ethylmaleimide)

B. Effect of some toxins

o Nicole was right. Both botulinum toxins and tetanus toxins sever SNARE proteins and thereby prevent the vesicles from fusing. However, their cellular sites of action are different.

1. Botulinum toxin – prevents release of NT from motor neurons

2. Tetanus toxin – prevents release of NT from inhibitory neurons in the spinal cord, leading to hyperactivity of motor neurons.