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Synaptic transmission
- Synapses– functional contacts between neurons
- Two general classes– chemical and electrical synapses
- Chemical– neurons talk to each other by release of neurotransmitters
- Electrical– direct, passive flow of current between neurons
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Thus far we’ve discussed how neurons generate action potentials that propagate down axons with high fidelity over cm’s to to meters of space and the ion channels in the membrane that underly voltage dependent excitability.
But is through synapses that neurons actually talk with one another and it is also through synapses that the nervous system effects behavior function enabling us to interact with the world around us– in other words there are synapses between pairs of neurons that form the basis of inter-neuronal communication as well as synapses on muscle fibers that neurons use to get our muscles to contract.
Now there are two general classes of synapses, chemical... and electrical...
todo: motor neuron - muscle fiber model
Electrical and chemical synapses have different mechanisms for transmission
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Electrical synapses
- Less common than chemical synapses
- The cell membranes of two cells are linked together via gap junctions
- Current flows directly from one neuron to another via gap junctions– form large pores between cells made up of connexin proteins
- The signal is very fast– the only limit is diffusion
- Signals can go in both directions
- Are used to synchronize electrical activity among populations of neurons
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These electrical synapse or gap junction synapses are thought to be more common among inhibitory interneurons in the brain—
quadrillion synapses, 10^15 in our nervous system
Gap junctions allow current to flow from one cell to the next
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connexins— extracellular loops and disulfide bridges
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3.5nm separating the apposed lipid bilayers connected through connexon hemichannels
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20-40nm separation at a chemical synaptic cleft
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passive ionic current flow, small substance like ATP and second messengers
Current in the presynaptic cell is not felt directly by post-synaptic cell for a chemical synapse.
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Electrical synapses
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In Crayfish an action potential in one neuron spreads quickly to the next in fraction of a millisecond.
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Electrical synapses
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In hippocampal neurons gap junctions can make neurons fire in synchrony
Electrical Synapses: putative functions
- Synchronization of the electrical activity of large populations of neurons
- the large populations of neurosecretory neurons that synthesize and release biologically active peptide neurotransmitters and hormones are extensively connected by electrical synapses
- brainstem neurons involved in breathing
- Synchronization may be required for neuronal development, including the development of chemical synapses
- Synchronization may be important in functions that require instantaneous responses, such as reflexes and pacemakers
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quadrillion synapses, 10^15 in our nervous system
important in diseases of pathological oscillations/synchrony like childhood epilepsy, etc
Electrical synapses and synchronization characterisitc of cells that stimulate pulses of pituitary hormones (e.g oxytocin/vasopressin secretion).
medulla and pons, medulla: nucleus ambiguous, pre-botzinger complex, solitary nucleus
inferior olivary nucleus: source of climbing fiber input to cerebellar cortex. ultastructure adn ephys (Llinas 1974) found electrical coupling between pairs of neurons in cat inferior olive. Same thing demonstrated later in guinea pig, rat, mouse. Also dye coupling. 2-8Hz synchronous oscillasions. 1
thalamic reticular nucleus (thin interneuron layer) of dorsal thalamus. Spatially localized coupling (cells 40 um apart). 1
hippocampus. between pyramidal neurons and also interneurons. 1
in neocortex only rarely found between pyramidal neurons, often between interneurons. 'Late spiking' L1 interneurons make electrical synapse with other neurons of the same class 83% of time but with other interneuron types only 2% of time. Maybe necessary for gamma frequency rhthyms.
retina has widespread electrical coupling. Extensive between amacrine cells, scoptopic vision impaired in Cx36 KO mice from loss in rods and cones and between amacrine cells and bipolar cells.
Cx36 in both olfactory epithelium and olfactory bulb. between granule cells. between mitral cells in same glomerulus.
Early in development, first postnatal week in rat electrical coupling extensive between motor neurons in spinal cord. Declines during first postnatal week but still present in adult.
gap junction proteins: connexins (chordates), innexins (invertebrates). Similar topologies but dissimilar gene/amino acid sequences. Also pannexins in
connexins : 20 isoforms in humans and mice. 40 connecxin orthologues across species. Cx36 36kDa protein, hexamer possibly only forming hemichannels homotypically, specific to neurons. 1
50% of mammalian connexins widely expressed in CNS. Some strong in astrocytes (Cx26,30,43) or oligodendrocytes (Cx29,32,47) 1
gap junctions first found and studied in invertebrates. Innexins for gap junctions in drosophila, c elegans molluscs, annelids, playhelminthes. Mammalian pannexin genes are similar to innexins and Px1 and Px2 mRNA is present in pyramidal neurons and interneurons of the hippocampus.
gap junctions may be sensitive to Ca2+ influx, at least at high concentrations. But are very sensitive to small intracellular (but not extracellular) pH changes and intracellular pH changes occur doing neuronal activity.
Carbenoxolone (from licorice root) not very specific for Cx36.
Quinine selectively blocks Cx36,50,45. Mefloquine is a derivative that is 100x more potent.
Cx36 KO mouse has no obvious behavioral phenotype other than retinal deficits1 .
c elegans: 959 total cells in adult hermaphrodite. 302 are neurons, 58 are glia. Every cell in worm expresss innexins, most of the 20+ isoforms are expressed in nervous system and every neuron is believed to form gap junctions. 7000 synapses. 6393, 890 electrical junctions. 1410 NMJ.
Chemical synapses
- The majority of connections use chemical synapses
- They form at the synaptic cleft
- Presynaptic cells have synaptic vesicles that have neurotransmitters in them
- Post-synaptic cells have neurotransmitter receptors on the plasma membrane
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Synapse structure as seen by electron microscopy
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- synapse, Gray type 1 is asymmetrical synapse. Usually excitatory synapse. Spherical vesicles.
- synapse, Gray type 2 is symmetrical synapse. Usually inhibitory synapse. Elongated vesicles.
Synaptic transmission
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- Action potential in the presynaptic neuron opens voltage-gated Ca²⁺ channels
- Ca²⁺ influx raises [Ca²⁺]i in the nerve terminal
- Elevated [Ca²⁺]i triggers the fusion of synaptic vesicles to the plasma membrane of the presynaptic neuron and exocytosis
- Neurotransmitter is released into the synaptic cleft where it diffuses about
- Neurotransmitter binds to specific neurotransmitter receptors in the postsynaptic neuron causing ion channels in that cell to open or close
- The neurotransmitter is inactivated and/or removed from the synaptic cleft (active transport into presynaptic neuron or glial cells or both)
- The vesicular membrane is recovered by endocytosis and recycled
neurotransmitter receptors :
- direction action through ligand gated channels
- indirect action through G protein coupled receptors
The steps of synaptic transmission
- Neurotransmitter synthesized and/or packaged into vesicles
- Action potential enters the presynaptic terminal
- Voltage-gated calcium channels open because of depolarization
- Calcium influx occurs rapidly. Ca²⁺ concentration difference is 1000x across the cell membrane
- Vesicles fuse with membrane because of calcium flux
- Neurotransmitter release into synaptic cleft
- Neuroransmitter binds to receptors on postsynaptic cell
- Postsynaptic ion channels open or close
- Postsynaptic current flux occurs across post-synaptic cell membrane
- Neurotransmitter removed from synaptic cleft by enzymatic degradation or glial cell uptake
- Vesicle membrane recycled via endocytosis
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The discovery of the neurotransmitter acetylcholine
- Otto Loewi– wanted to figure out how stimulation of vagus nerve caused the heart to slow down
- Vagus nerve (cranial nerve X) has both sensory and motor axons. Regulates heartbeat
- Loewi transfered a solution generated from one heart to slow down another heart even without stimulation
- Demonstrated a diffusible substance was released upon stimulation
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The vagus nerve is responsible for such varied tasks as heart rate, gastrointestinal peristalsis, sweating, and quite a few muscle movements in the mouth, including speech (via the recurrent laryngeal nerve). It also has some afferent fibers that innervate the inner (canal) portion of the outer ear (via the auricular branch, also known as Alderman's nerve) and part of the meninges.
The vagus nerve (/ˈveɪɡəs/ vay-gəs), historically cited as the pneumogastric nerve, is the tenth cranial nerve or CN X, and interfaces with parasympathetic control of the heart and digestive tract. The vagus nerves are paired; however, they are normally referred to in the singular.
The vagus nerve supplies motor parasympathetic fibers to all the organs except the suprarenal (adrenal) glands, from the neck down to the second segment of the transverse colon. The vagus also controls a few skeletal muscles, notable ones being:
- Cricothyroid muscle
- Levator veli palatini muscle
- Salpingopharyngeus muscle
- Palatoglossus muscle
- Palatopharyngeus muscle
- Superior, middle and inferior pharyngeal constrictors
- Muscles of the larynx (speech).
This means that the vagus nerve is responsible for such varied tasks as heart rate, gastrointestinal peristalsis, sweating, and quite a few muscle movements in the mouth, including speech (via the recurrent laryngeal nerve).
It also has some afferent fibers that innervate the inner (canal) portion of the outer ear (via the auricular branch, also known as Alderman's nerve) and part of the meninges. This explains why a person may cough when tickled on the ear, such as when trying to remove ear wax with a cotton swab.[citation needed]
Afferent vagus nerve fibers innervating the pharynx and back of the throat are responsible for the gag reflex.
The discovery of acetylcholine
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Otto Loewi, 1921
Free acetylcholine acts on muscarinic receptors which hyperpolarize the cells of the SA node and slow the conduction of the action potential through the AV node. This slows heart rate. Acetylcholine also decreases Ca2+ influx which lowers the heart's force of contraction.
This figure no longer is in 6e.
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The discovery of acetylcholine
Otto Loewi (Austrian)– on the discovery of vagus nerve substance:
"In the night of Easter Saturday, 1921, I awoke, turned on the light, and jotted down a few notes on a tiny slip of paper. Then I fell asleep again. It occurred to me at six o'clock in the morning that during the night I had written down something most important, but I was unable to decipher the scrawl. That Sunday was the most desperate day in my whole scientific life. During the next night, however, I awoke again, at three o'clock, and I remembered what it was. This time I did not take any risk; I got up immediately, went to the laboratory, made the experiment on the frog's heart, described above, and at five o' clock the chemical transmission of nervous impulse was conclusively proved."
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Acetylcholine (ACh) shown to be the vagus factor
- Sir Henry Dale purified ACh (1914) and showed that it is vagus nerve substance
- Can apply ACh to muscle and evoke an end plate potential (EPP)
- Henry Dale and Otto Loewi shared Nobel prize (1936):
"for their discoveries relating to chemical transmission of nerve impulses"
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curare used as a paralyzing poison by South American indigenous peoples for hunting that causes respiratory asphixiation (diaphragm muscle paralysis) in prey
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alkaloid arrow poisons that are competitive and reversible inhibitors of nicotinic acetylcholine receptor (nAChR)
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ACh action has same pharmacology as vagus nerve substance in that it is sensitive to curare (a plant poison that kills by preventing muscle contraction). Competes with curare for receptor binding
Formal criteria that define a neurotransmitter
- Must be present in the presynaptic neuron
- Must be released in response to a depolarization and be Ca²⁺ dependent
- Must have specific receptors localized on the post-synaptic cell
- Note– It does not have to function uniquely as a neurotransmitter (it may have other functions). e.g. glutamate, glycine, ATP
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There are a few criteria that define a neurotransmitter...
Criteria that define a neurotransmitter
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Criteria depicted here
It depends on how you count, but maybe 30 - 100 different molecule types, with 10 of them doing 99% of the work. More than 100 different neurotransmitters have been identified.
There are two main broad categories of neurotransmitters: "Small molecule" neurotransmitters (glutamate, GABA, acetylcholine, biogenic amines (dopamine, serotonin, noradrenaline, and histamine)) and neuropeptides (opioid peptides, substance P). ATP/purines and unsaturated fatty acids like endocannabinoids (anandamide, 2-AG) also can act as neurotransmitters.
This box figure also no longer in 6e.
Synaptic transmission is quantal
- Synaptic transmission is quantal (composed of discrete units)
- The initial evidence was obtained from studying the release of acetylcholine at neuromuscular junctions
- The synapses between spinal motor neurons and skeletal muscle are simple, large, and peripherally located. Easy to study
- These motor synapses form structures at the neuromuscular junction called end plates. This is where the action happens
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How have we come to learn about the properties of chemical synaptic transmission?
End plate potential
A presynaptic action potential releases a lot of ACh, opening channels in the muscle cell. The resulting depolarization in the muscle cell at the neuromuscular junction is called an end plate potential (EPP).
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Muscle fibers are excitable cells. They are multinucleated myocytes. They too generate action potentials.
End plate potentials evoked by motor neuron stimulation almost are almost always above threshold and result in an action potential along the muscle fiber.
It is the synaptic potential at the neuromuscular junction.
motor unit is a motor neuron’s axon terminals and all the skeletal muscle fibers it innervates (10 for extraocular muscles, 1000 for thigh muscles). Motor pool is a bunch of motor units of same fiber type.
Miniature end plate potentials (MEPPs)
- Spontaneous changes in potential even in the absence of an action potential
- Same shape as EPPs but smaller (1 mV vs 50+ mV)
- Sensitive to agents that block ACh receptors
- Removing Ca²⁺ from media reduces EPPs to MEPPs
- Thus EPPs are a bunch of MEPPs added up
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Spontaneous MEPPs and subthreshold EPPs evoked in low [Ca2+] have similar amplitudes
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0.5mV depolarizations.
- in the absence of stimulation there is spontaneous postsynaptic membrane transients called minature EPPs. Small amplitude.
- Bath in low calcium and stimulate you get small subthreshold EPPs that are about the same size as the MEPPs.
- Examination of the muscle membrane potential at high gain reveals small, spontaneous depolarizations. These are miniature end plate potentials (MEPPs)
This work was on frog neuromuscular junc in 1950s but subsequent investigations have demosntrated these synaptic properties for all chemical synapses studied to date.
Quantal neurotransmission
- Lowering [Ca²⁺] reduces the amount of total transmitter (no. of vesicles) released by an AP
- Here [Ca²⁺] is so low that often presynaptic APs fail to release any ACh. But sometimes APs will release 1 to 6 quanta
- The distribution of stimulated EPPs in low [Ca²⁺] has multiple modes (several local maxima). Multiples of the smallest EPP amplitude (e.g. 0.4 mV)
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If you measure the amplitudes of these small low calcium EPPs and plot their distribution, e.g. this histogram here you can see a certain statistical distribution that indicates these amplitudes fall into discrete steps or quanta showing that the smallest amplitude ones that are about the same size as the spontaneous MEPPs must be result of neurotransmitter release from single synaptic vesicles.
Poisson statistics used to analyse independent occurence of unitary events. Red curve shows what the distribution would expected to be if neurotransmitter release is quantal, made up of discrete message packets (vesicles) made of multiples of MEPP amplitudes (e.g. 0.4 mV)
Quantal neurotransmission
- The MEPP is the quantal event of neurotransmission. It represents the postsynaptic response to the release of a single vesicle of neurotransmitter
- The EPP is the result of the synchronized release of many vesicles. It is the sum of many MEPPs
- Bernard Katz, Nobel prize (1970)
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One MEPP = one synaptic vesicle
- Synaptic vesicles are full of neurotransmitter
- In motor neuron one vesicle contains approximately 10,000 molecules of neurotransmitter
- About the same amount needed to invoke an MEPP
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Synaptic vesicles recycle
- All that vesicle fusion– why doesn’t the membrane keep growing and growing?
- Synaptic vesicle membranes get recycled quickly
- Are endocytosed in clathrin coated vesicles which fuse to endosome and bud off again
- Can use a pulse chase experiment to show this
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Local recycling of synaptic vesicles in presynaptic terminals
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(Experiments by Heuser and Reese, 1973). HRP enzyme forms dense reaction product, can be visualized easily in electron microscopy.
Clathrin has a unique three arm structure that forms little geodesic dome coverings around membrane segments and dynamin forms a ring that pinches or 'buds' off the vesicle.
Local recycling of synaptic vesicles in presynaptic terminals
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Calcium is required for synaptic vesicle fusion
- Voltage clamping shows that there is an inward Ca²⁺ flux in presynaptic cells that is voltage dependent
- Ca²⁺ can be visualized entering cell after depolarization
- Injection of Ca²⁺ into the presynaptic neuron can drive a post-synaptic potential
- Chelating Ca²⁺ in the presynaptic cell can inhibit post-synaptic potential
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The role of calcium
- If extracellular Ca²⁺ is removed or Ca²⁺ entry is blocked, there will be no release
- Voltage-gated Ca²⁺ channels in the presynaptic membrane provide Ca²⁺ to trigger the release of neurotransmitter
Voltage-clamp presynaptic neuron and
block Na⁺/K⁺ currents with TTX/TEA
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(Augustine and Eckert 1984)
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The role of calcium
- Intracellular injection of Ca²⁺ into the presynaptic terminal will stimulate release
- Intracellular injection of Ca²⁺ chelator will inhibit release
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- microinjection of Ca²⁺ into squid giant axon presynaptic terminal (Miledi, 1973)
- microinjection of Ca²⁺ chelator BAPTA into squid giant axon presynaptic terminal (Adler et al, 1991)
Fluorescent dye that binds calcium (Smith et al 1993)
squid giant axon from contacts the contractile muscular mantle responsible for water expulsion and squid jet propulsion
Many proteins are involved in synaptic vesicle cycling
- Many specific proteins have been isolated from presynaptic terminals
- Some of these proteins are required for different steps of vesicle cycling: budding, docking, priming, fusion
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Model after Takamori et al 2006
Don't memorize this.
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Presynaptic proteins implicated in synaptic vesicle cycling
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NSF: ATPase NSF important for fusion of vesicle with membranes of the golgi apparatus. NEM sensitive fusion protein.
snaps: soluble NSF-attachment proteins
snares: SNAP receptors
Model after Takamori et al 2006
Molecular mechanisms of synaptic vesicle exocytosis
- SNARES ('SNAP' receptors) tether the vesicle to plasma membrane
- SNAP-25 is a plasma membrane SNARE that regulates the assembly of two other SNAREs
- Syntaxin is a plasma membrane SNARE
- Synaptobrevin is a vesicle SNARE
- Synaptotagmin is a vesicle Ca²⁺ sensor and helps trigger vesicle fusion
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Many proteins specific to presynaptic terminals have been isolated. These proteins are required for different steps of vesicle cycling: budding, docking, priming, fusion.
Just know there are is a calcium sensitive protein called synaptotagmin and that there are proteins like SNAREs that help dock and pinch membranes together
- NSF
- NEM-sensitive fusion protein (orig found to be important for fusion of vesicles with membranes of Golgi apparatus)
- ATPase
- SNAPs
- soluble NSF attachment proteins
- SNARES
- 'SNAP receptors'
Model based on crystal structure work for SNAP25 from Sutton 1998, Madej 2014, Zhou Nature 2015
Molecular mechanisms of synaptic vesicle exocytosis
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Model based on crystal structure work for SNAP25 from Sutton 1998, Madej 2014, Zhou Nature 2015
Vesicle proteins are the targets of many toxins
- Tetanus toxin– cleaves synaptobrevin
- Botulinum toxins– cleave syntaxin and snap25 (causes botulism)
- alpha-latrotoxin– black widow causes a massive exocytosis of vesicles. Somehow bypasses Ca²⁺ requirement, likely affecting synaptotagmin
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from https://en.wikipedia.org/wiki/Botulinum_toxin:
Cleavage of the SNARE proteins inhibits release of acetylcholine.[45] Hence, botulinum toxins A, B, and E specifically cleave SNAREs, preventing "neurosecretory vesicles" from docking/fusing with the interior surface of the plasma membrane of the nerve synapse, and so block release of neurotransmitter. In inhibiting acetylcholine release, nerve impulses are blocked, causing the flaccid (sagging) paralysis of muscles characteristic of botulism[45]
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Synaptic vesicle toxins
Tetanus toxin and various types of botulinum toxin act by preventing exocytosis.
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Synaptic transmission summary video
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