Synapses

Purves et al. Chapter 5

Major point

Cell theory (cells being separated) implies that cells must communicate with each other through extracellular connections and most communication is through chemical messages
- "synapse" - = "clasp" (sherrington)

Fig. 5.1AB p. 78 "electrotonic synapse"
Figure of chemical synapse is like Fig. 5.3 p. 80 (only simpler)
Vesicles, spine, receptors (the ionotropic type, i.e. channels) are shown

Alumna

Johnnie Moore took this course in 2002. She published this paper: Moore et al., Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca 2+-evoked release of catecholamine, Am J. Physiol Cell Physiol 291, 270-281, 2006
Topics to discuss:
confocal
synaptotagmin
NGF
calcium
PC

"Electrical synapses"

Fig 5.1A B p. 78

Gap junction is an exception to the above generalization in that cells are coupled electrically with cytoplasmic continuities (small ones).
Gap junctions are used in crayfish escape,

"Landmark" paper EJFurshpan and DDPotter, Mechanism of nerve-impulse transmission at a crayfish synapse, Nature 180, 342, 1957 and J. Physiol. 145, 289, 1959. "Landmarks" was a department in The Journal of NIH Research, includes interviews. (ref=vol 8, Oct 1996, 51-57)

They are also used to connect myocardial cells electrically at intercalated disk.
Conductance is high - 120 pS.

Fig 5.1C p. 78
Gap junction is patch of hexamers forming big channel in register with adjacent cell.
Proteins, called connexins, are very diverse.
They are often named with a number that represents the molecular weight.

Recent paper CDLandisman & BWConners, Long-term modulation of electrical synapses in the mammalian thalamus, Science 310, 1809-1813, 2005. There are gap junctions with connexin36 (Cx36) in thalamic reticular nucleus (TRN). There are chemical synapses using metabotropic glutamate receptors (mGluRs) from neocortex. Activating this corticothalamic path causes long-term decrease in electrical synapse strength. This is like long term depression, a model for learning at the level of the chemical synapse.(Each item of terminology that might be unfamiliar to you now will be covered later.)

Pore is big enough to give cytoplasmic continuity for medium sized molecules (dyes).
In addition to electrical coupling, there can be communication by molecules.
In EM, membranes appear very close but not fused
Extracellular tracers (heavy metal Lanthanum) proves there is extracellular space.

History

In 1906 Sir Charles S. Sherrington (England) published Integrative Action of the Nervous System and later (1932) won the Nobel prize for "functions of neurons." He coined the term "synapse."
In studies of the spinal reflex, he determined that the spinal motor neuron was the "final common pathway" (for integrative action of the nervous system). Spinal reflexes were studied in spinal animals in which the spinal cord was transected to prevent descending cortical influence. There are no inhibitory neuromuscular junctions in vertebrates, so whether the spinal motor neuron fires, based on summed inhibitory and excitatory influences, is the last chance to integrate neural influences.

Fig. 5.4 p. 81
1926 Otto Loewi (Austria) experiment he dreamed, stimulate vagus (10th cranial nerve, parasympathetic), take substance and show that it slows a heart in another dish, vagus substance = acetylcholine (ACh) a monamine transmitter.
1930's Sir Henry H. Dale (England) acetylcholine
share 1936 Nobel "chemical transmission of nerve impulses"

Fig. 5.22AB p. 103
Sir John C. Eccles 1963 Nobel (with Hodgkin & Huxley) EPSP & IPSP
Excitatory or Inhibitory PostSynaptic Potentials (Eccles, using spinal motor neurons)
Excitatory and Inhibitory integrate in cell, and axon hillock "decides" whether to fire.

Fig. 5.21A p. 102
glutamate is the excitatory transmitter
EPSP - depolarize (unless clamped positive to reversal potential)
increase sodium and potassium conductance
inferred because reversal potential is near zero (in voltage clamp)

Fig 5.21BC p. 102
GABA (gamma amino butyric acid) is the inhibitory transmitter
IPSP - hyperpolarize (unless clamped negative to reversal potential)
increase potassium and chloride conductance
inferred because reversal potential negative to resting potential (in voltage clamp)
and by changing Cl- gradient by using ion specific electrodes to inject Cl-

1970 Nobel Sir Bernard Katz (England) Ulf von Euler (Sweden) Julius Axelrod (US) "humoral transmitters...nerve terminals....storage release inactivation"

Fig. 5.7A p. 86
classic experiment by Katz showing that the transmission at the neuromuscular junction is "quantal." Quantum is one vesicle. EPP (end plate potential) is reduced to meep's (miniature end plate potentials, 0.4 mV) by lowering extracellular Ca2+ ion, and nerve stimulation elicits responses the size of 0, 1, 2, or 3 meep's according to the Poisson distribution. "end plate" potential is big and effective in generating muscle action potential. Usually 200 vesicles give 40 mV potential.

Fig. 5.8 A p. 87
Here is a classic pictures, work by Hueser and Reese, of vesicle release at the neuromuscular junction, a freeze fracture electron micrograph

also a transmission electon micrograph (Heuser) where the vesicle release is called an omega figure because it is shaped like the Greek letter.

Just to put into perspective the degree to which much of this information is background,
TRANSPARENCY shows the version of this picture the Biology Department teaches to freshmen.
Specifics shown in this figure:
Ca2+ enters presynaptic terminal upon arrival of the action potential.
The receptor shown is a channel passing Na+ (this situation can vary).
Neurotransmitter is broken down (true for acetylcholine, but this situation also varies).

Fig. like Fig 5.3 p. 80 (only simpler)
Chemical synapses
Presynaptic membrane, cleft, Postsynaptic membrane (intracellular density seen in EM [electron microscopy]), vesicle
Specifics in this figure
Note that the post-synaptic membrane is up on a spine.
Membrane is recycled, and endocytotic pits and vesicles are coated (with clathrin); coated pit from my work another (not related to synapses).

Fig 5.13B p. 91
There is a protein called dynamin that helps pinch off vesicles. It is the product of the temperature sensitive Drosophila paralytic mutant called shibire. At restrictive temperature, there is a block in endocytosis of vesicles (another view).

vesicles and T-shaped ribbons in Drosophila

Here is a transmission electron micrograph of a synapse

Vertebrate - inputs to cell or dendrite (spine)
Invertebrate - cell is usually away from action surround "neuropil(e)." Here is a picture from my work on Drosophila, retina (compound eye is off top, cartridges of synaptic connections are at bottom, cell bodies of post-synaptic neurons are between.

Vesicle release

General: vesicles are interesting, transmitter is very concentrated, there are pumps to move transmitter "up hill" (against gradient) into vesicle, sometimes part of synthesis is in vesicle.

Fig 5-13, p. 91
very modern, interesting and detailed
also, interesting Box 5B, pp. 93-95, on diseases and toxins that affect neurotransmitter release
there are vesicle membrane proteins, target (presynaptic) membrane proteins, and cytoplasmic proteins
Ca2+ in through Q or N type voltage gated channel
(N stands for "neither," as opposed to T=transient or L=long lasting, the N channel is blocked by omega toxin from Conus [snail genus])

Vesicle proteins:

Synaptobrevin / VAMP (vesicle-associated membrane protein) = v-SNARE (SNAP receptor)
Botulinum and Tetanus toxin (clostridial toxins) are proteases which cleave synaptobrevin
Botulism (Clostridium botulinum) anaerobic, improper canning (need to heat to kill spores) - block release
When I ws 10, in the Cold War, we discussed, at the dining room table, how 1 teaspoon in the reservoir would kill the city. Now. 45 years later, people take it (injected) to get rid of face wrinkles.
Tetany is term for sustained muscle contraction based on twitches adding up.
Tetanus toxin cleaves synaptobrevin in inhibitory interneurons.
The disease is contracted in deep (because it is an anaerobic bacterium) dirty puncture wounds.
You would die with muscles contracted, called "lock-jaw."
There is a vaccine and boosters every 10 years are suggested.

Synaptotagmin - binds calcium
synapsins get phosphorylated (by CaM Kinase II and PKA) interact with actin
rhabphilin receptor

Target membrane proteins:

Syntaxin = t-SNARE = unc-18 (uncoordinated C. elegans roundworm mutant)
Neurexin - black widow spider venom (alpha Latrotoxin) causes too much release
Neuroexins bind to synaptotagmin

Cytoplasmic:

NSF - N-ethylmaleimide sensitive factor (ATPase activity when complex dispersed)
SNAP - soluable NSF associated protein
Rab3 (like ras, small GTP binding protein) (lots of rab's, specific for transport)
rabphillin

Summary:

Fig. 5.14 AB p. 92
SNAREs and SNAP (docking)
In addition to SNAREs and SNAP, Ca binding synaptotagmin is for fusion

Fig. Box 5B pp. 93-95
BoTX and TeTX sites.

Test questions from 2005 -2012 that relate to this outline

Why would pheochromocytoma cells be a reasonable model for studying neurotransmitter release?

can culture adrenal medulla cancer cells which release catecholamines

"Ramon y Cajal's 'cell theory' implies that something like a chemical synapse MUST exist (even though they has not been demonstrated yet)" Explain.

the alternate, Golgi's reticular theory, has continuous processes, while separate cells require communication between cells

Describe the structural or molecular specializations that form an "electrical synapse."

in gap junction, connexons are formed from hexamers of connexins in register from one cell to the next

"There are no inhibitory neuromuscular junctions in the vertebrate." What does that information tell us about "the final common pathway in the integrative action of the nervous system?"

it must be in the spinal motor neuron since there is no integration after that

What kind of technique would be needed to determine the reversal potentials for the IPSP and the EPSP?

voltage clamp

Activation of the nerve elicited (what? - be specific) at the end plate when Katz decreased the extracellular calcium ion concentration.

miniature end plate potentials, 0, 1, 2, or 3 of them

Describe either the freeze-fracture or the transmission electron microscope image of a vesicle in the process of release.

holes (pits), omega figures

Why would there be transporter molecules in the vesicle membrane?

to maintain a steep gradient against leakage

On the spinal motor neuron, there are synapses onto the dendrites and cell body. A picture was shown of the first synaptic area behind the Drosophila compound eye, and the point was made that invertebrate neuropils we would see later in the semester are similarly organized. How does this synaptic organization differ from that of the vertebrate?

the cell body of the postsynaptic neuron is on the outside of the ganglion, aloof from synapses

How could Shibire mutants have temperature sensitive paralysis when the mutation does not block vesicle release?

it blocks recycling of vesicle membrane

Why would a doctor give a patient a clostridial protease that cleaves synaptobrevin?

cosmetic dermatologists would discourage wrinkles in the face by blocking neuromuscular junctions

What is the target membrane that the vesicle-SNARE latches onto?

presynaptic

What nerve did Otto Loewi from Austria use in the first demonstration that a neurotransmitter substance was used in signaling?

vagus

A patch with hexamers in register between two adjacent cells describes what kind of intercellular communication structure?

gap junction

"The spinal motor neuron is the final common pathway in the integrative action of the nervous system." Why is the spinal motor neuron (as opposed to the muscle cell) the final place where integration of signals can take place?

there is excitation, no inhibition, on the muscle cell

Starting at a normal resting potential (not voltage-clamped), what happens to the membrane potential of the postsynaptic cell if a GABA-releasing presynaptic cell is activated?

it hyperpolarizes

A micropipette that was used to selectively inject chloride ions into neurons told us (what) about postsynaptic potentials?

IPSPs are based in part on an increase in chloride conductance

In Sir Bernard Katzís Nobel Prize-winning research, he turned end plate potentials into 0, 1, 2, or 3 miniature end plate potentials. Answer either (1) How? Or (2) Why?

(1) by decreasing calcium ions (2) to show that vesicles were the unit of synaptic transmission

Name a protein involved in retrieval of vesicle membrane.

clathrin dynamin

After calcium ions come in through channels in the presynaptic membrane, it binds to a calcium-binding protein to mediate vesicle release. Answer either (1) What is its name? or (2) Where is it (which specific compartment involved in vesicle release)?

(1) synaptotagmin (2) bound to vesicle membrane

Why do they refer to "omega figures" in the ultrastructure of synapses?

vesicles in the process of release are shaped like Greek letter omega

What kind of synapse involves opening of a chloride channel?

inhibitory

Suppose you voltage clamp a postsynaptic neuron to a level negative to the reversal potential. What would you record (postsynaptically) if you activated a cell making a GABAergic
synapse?

in this case it would depolarize

Name a protein involved in recycling of synaptic vesicle membrane.

clathrin, dynamin

What is the quantum of transmission that Katz found to elicit a miniature end plate potential of 0.4 mV?

vesicle

Compared with the conductance of a voltage-gated ion channel, what is the conductance of a one gap junction channel?

huge

What happens to the conductance of the postsynaptic menbrane during the EPSP?

it increases

What is a connexin used for?

gap junction

How does Ca2+ get into the cell to affect vesicle release?

through a calcium channel

What do the clostridial toxins from anaerobic bacteria do?

cleave Synaptobrevin / VAMP (vesicle-associated membrane protein) = v-SNARE, inhibit vesicle release

Katz was interested in the end plate potential. Where is the end plate?

the "post-synaptic membrane" of the muscle cell

Two SNAREs hook onto eachother, v-SNARE and t-SNARE. What are v- and t-?

vesicle and "target" (presynaptic membrane)

What happens to the end plate potential when extracellular Ca2+ is decreased?

gets small

Why do tetanus and botulinum toxins have opposite effects on motor activity?

both inhibit vesicle release, tetanus is in inhibitory interneurons

What would not happen at the restrictive temperature in a temperature sensitive mutation affecting the protein dynamin?

coated pits would not pinch off to vesicles

Why did Sherrington consider the spinal motor neuron rather than the muscle cell itself to be the "final common pathway" of "the integrative action of the nervous system?"

because it receives + and - inputs while the muscle only gets + input

What happens to the conductance at the postsynaptic ionotropic receptors for K+ and Cl- for the IPSP?

they go up

Not just any old Ca2+ channel would work on the presynaptic membrane. It has to be voltage gated. Why?

to detect the arrival of the action potential

What are connexin proteins, such as Cx36, used for?

gap junctions

With extremely low extracellular Ca2+, why would the potential recorded at the end plate be several different sizes for several different nerve stimulations?

because you might get o, 1, 2, ... miniature end plate potentials

What does parasympathetic output via the vagus do to the heart rate?

slows it

The toxin from Clostridium botulinum (BoTX) cleaves an important protein. Precisely where is this protein localized?

on the vesicle

Why are vesicles in the process of release sometimes called omega figures?

a TEM of it is shaped like the Greek letter

Electrical "synapses" use what membrane specialization in common with heart muscle cells?

gap junctions with connexons made up of connexin protein

What happens to the conductance of the postsynaptic membrane when GABA (gamma amino butyric acid) elicits an IPSP (inhibitory postsynaptic potential)?

increases (for potassium and chloride)

"Activating the corticothalamic path causes long-term decrease in electrical synapse strength." These electrical synapses are made of what protein?

connexin

Why did the Nobel prize winning Sir Charles Sherrington refer to the vertebrate spinal motor neuron as "the final common pathway in the integrative action of the nervous system?"

there can be no integration (of + & - inputs) further out since each muscle cell has only (one) + connection

Graded depolarizations and hyperpolarizations on the dendrites and cell body pool. As a result, an action potential is initiated (where)?

Axon hillock

Determining reversal potentials was instrumental in establishing the conductances mediating the EPSP and IPSP (excitatory and inhibitory postsynaptic potentials). What was done, in addition to Voltage clamping and recording from the postsynaptic membrane, to determine the reversal potentials?

stimulate the presynaptic neuron

With very low Ca2+, upon stimulating the motor neuron's axon, the end plate potential (EPP) was usually 0.4, 0.8, 1.2, etc mV. What other size was observed?

zero

Recycling of vesicle membrane begins with a clathrin coated pit. A mutation of what protein prevents the pit to pinch off to a vesicle?

dynamin (the shibire gene product)

There are synaptic connections to the dendrites and cell bodies of vertebrates. How is the situation strikingly different for invertebrates?

cell bodies are on outside of neuropil, away from where connections are

If heat resistant bacterial endospores survive improper canning, by what mechanism would eating those tomatoes affect transmitter action?

bacterial botulism toxin would cleave synaptobrevin (v-SNARE)

Give one of the several reasons why alumni Michelle Li, also Johnnie Moore, used PC12 cells in their published research.

they release catecholmines

Compare the conductance of a gap junction channel with that of a potassium channel.

way higher

What's with the "36" when they call the gap junction protein in the thalamus "Cx36?"

molecular weight

How did Nobel Prize winner Loewi prove that a substance released by the vagus slows the heart?

solution bathing heart slowed by vagus stimulation slows another heart

What property of the neuromuscular junction rationalizes Sir Sherrington referring to the spinal motor neuron as "the final common pathway" of "the integrative action of the nervous system?

since there is only excitation at the vertebrate n.m.j, the motor neuron is the last place excitation and inhibition can integrate

If you observed a coated vesicle near a synapse, say whether it is in the process of exocytosing and justify your answer.

no. endocytosing, those are the pits with clathrin

Relate vesicle release with either (1) improperly canned goods, (2) dangers of terrorism against civilians, or (3) facial cosmetic treatment.

BoTox decreases release, form heat resistant endospores turning into anaerobic bacteria that make a potent toxin that can be injected to decrease face wrinkles

Why, in Sir Bernard Katz's Nobel Prize-winning experiment, did they refer to "end plate potentials" instead of "postsynaptic potentials?"

neuromuscular junction was used to model a synapse

"Silencing of synaptotagmin in PC12 cells inhibits Ca2+-evoked catecholamine release." Why does calcium elicit release if synaptotagmin is not inhibited?
 
Calcium, coming in through calcium channels, mediates vesicle release

Consider the mepp (miniature end plate potential) and answer either (1) What cellular mechanism delimits it to be way smaller than the full end plate potential? Or (2) Suppose your preparation were giving you just a few mepps on the average ­p; what would you do to the preparation to restore the full end plate potential?
 
Fewer vesicles are released with low extracellular calcium, add calcium
 
There are synaptic connections to the dendrites and cell bodies of vertebrates. How is the situation strikingly different for invertebrates?
 
Synapses are in a neuropil(e) and cell bodies are on the outside of ganglia
 
At first they were surprised that a mutation in dynamin (shibire) would exhibit paralysis when moved to the restrictive temperature. Rationalize why.
 
If vesicle membrane fails to recycle, eventually vesicles would not be released
 
"Botulism and tetanus toxins cleave synaptobrevin." Precisely what membrane is synaptobrevin on?
 
vesicle
 

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