Neuro - Synaptic Transmission

Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.
edu
Synaptic Transmission
(These notes supplement the information I will present in my lecture, as well as that found in Chapter 8: Synaptic Transmission between Neurons in the textbook by J. Nolte.) From an evolutionary perspective, we humans can attribute our vastly superior level of intelligence, range of behaviors and flexibility, and hence our domination of the animal kingdom to the high level of synaptic connectivity in the human brain. The chemical synapse is the hub of neuronal connectivity in the CNS. -This class will address the following topics in what we currently know about synaptic transmission. 1) What is a neuronal synapse, and how are pre- and postsynaptic membranes and proteins organized to facilitate rapid and efficient communication betweens neurons? 2) What happens when an action potential invades a synapse, and what role does Ca+ entry, synaptic vesicles, and SNARE proteins play in regulating transmitter release? 3) Neurotransmitters and receptors in the CNS: differences in the kinetics and types of ligand gated ion channel and G-protein coupled receptor-mediated responses. 4) Termination of transmission: Transporters, degradative enzymes, and glia. 5) Diseases involving a disruption in synaptic transmission.
Vesicles and synaptic transmission

The idea that chemicals get released from nerve endings comes from studies of the neuromuscular junction (NMJ) in the 1940s.The best studies chemical synapse is the NMJ. This is the site where peripheral neurons innervate muscle. The NMJ is also typical of a chemical synapse in the CNS in that the nerve terminal at the end of the axon has a specialized pouch like morphology where the axon is thicker. Also, on the postsynaptic side (a muscle cell in the case of the NMJ), ligand gated receptors for acetylcholine (nicotinic receptors) clustered for efficient signal transmission. This part of the muscle is called the endplate [endplate because a saucerlike appearance where axon elaborates terminals],
- Katz work on frog motor neuron stimulation of muscle contractions lead to the discovery of endplate potentials (EPPs) or transient depolarization in membrane potential in the muscle. They also found that the plant toxin curare blocked the muscle responses. D-curarine is a competitive antagonist at nicotinic receptors, and is used as anesthesia in some surgeries to relax skeletal muscle. -Katz et al., also noticed smaller deflections in traces of electrical activity from muscle that looked like EPPs even without stimulating the axon. These deflections were called spontaneous and because scaled down in size (< 1mV whereas EPPs are ~ 40-50 mV), called miniature EPPs MEPP or minis. The EPP size was much less than needed for threshold for postsynaptic action potential or contraction. But like the EPPs the mini EPPs were sensitive to curare, but were observed even calcium free media.
Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu -Studies in low Ca++ showed that the evoked EPPs are about the same size as spontaneous mEPPs. Interestingly, saw a frequency distribution that showed the size of the EPP varied in an integral fashion (i.e., responses were 1, 2, 3 times the unit size). The unit-sized responses were given the name quanta, and one quanta was proposed to evoke a response <1 mV Electron microscopic studies of the NMJ showed membrane bound organelles in the nerve terminal. Biochemists purified these vesicles and found they were filled with acetylcholine (Ach). These are now called synaptic vesicles. The vertebrate-specific toxin from black widow spider -latrotoxin dumps all the synaptic vesicles (even in Ca++ free media). Years of study by EMs of pre-synaptic terminals, and neurotransmission yielded the following general principles: 1) nerve terminal contains multiple synaptic vesicles, and multiple types of synaptic vesicles, 2) some vesicles are close to synapse, whereas as some removed from synapse, and 3) the synaptic vesicle might be the quanta or indivisible packet or sac of neurotransmitter released.
Packaging of neurotransmitters into vesicles

In addition to acetylcholine, neurotransmitters like glutamate, GABA, dopamine, norepinephrine and serotonin, as well as neuropeptides like NPY, vasopressin, oxytocin, melanocortin or the opiates are also packaged in synaptic vesicles, and are found in axonal boutons. Not all vesicles are alike. There are at least two varieties: small clear vesicles and large dense core vesicles (LDCVs). The LDCVs are not necessarily near the synapse active zone, whereas at least a fraction of small clear vesicles appear to be docked at the synapse proper. GABA, acetylcholine and glutamate are in clear vesicles whereas neuropeptides tend to be in LDCVs. Monoamines can be in either type depending on cell type. Nerve terminals can contain both clear core and LDCVs. When this happens the terminal is said to release co-transmitters. The concentration of Ach in a synaptic vesicle at the NMJ is about 100 mM or ~ 10, 000 molecules of Ach per vesicle based on size. How does so much transmitter get squeezed into a vesicle? For small molecule transmitters and monoamines, this is the job of proteins called transporters that pump neurotransmitter into vesicle against a concentration gradient using energy in the form of electrochemical gradient of co-transported ions. But peptide transmitters like ACTH, melanocortin, and the enkephalins, are synthesized and packaged into DCVs in the cell body, these DCVs are in turn transported along microtubular cytoskeleton to the terminal by motor proteins like kinesin that uses ATP (sometimes up to 400 mm/day). In contrast, small molecule transmitters are synthesized from precursors, and pumped into vesicle in the axon terminal. The enzymes for converting precursors to neurotransmitters are transported down the axon by microtubules. The transport of these enzymes and precursors is (~100 X) slower than for the DCVs.
Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu
Dynamics of synaptic vesicle release

Evoked release of neurotransmitter requires a rise in presynaptic Ca2+. This Ca2+ rise comes in from extracellular sources via voltage sensitive Ca2+ channels that are situated in the plasma membrane, some are located in the active zone where docked vesicles are released. Indeed, Ca++ channels in presynaptic terminal are a major determinant in evoked release of a bolus of synaptic vesicles by an action potential (as opposed to spontaneous release of a much smaller number of synaptic vesicles in the absence of an action potential invading the bouton). Presynaptic Ca2+ channels are mainly of the P/Q and N types, they can be modulated by G proteins resulting from GPCR stimulation as well as various kinases and phosphatases. The opening of Ca2+ channels in presynaptic terminal following AP invasion of bouton is a very fast event, i.e., the channels open quickly (0.1 ms) during peak action potential. As voltage decreases during descending phase of AP, there is a greater increase in Ca2+ influx via the channels. The local increase in Ca2+ in these domains around the channel might be 1000 fold. So, the Ca++ concentration might increase from ~ 200 nM to >/ 200 microM. Vesicles are located near Ca2+ channels are released sooner. The role of Ca++ in release of synaptic vesicles was figured out by injecting Ca2+, or Ca2+ chelator like EDTA. The Ca++ acts over short distances, 10s of nanometers, and within v. little time (~ .2 msec). DCVs get released with stronger depolarizations due to more global increase in Ca++ throughout the terminal (or due to opening of channels further removed from active zone that have a higher threshold for opening, e.g., P and Q type channels).
There are two well known disorders associated with presynaptic Ca2+ channels LEMS and FHM. Lambert-Eaton Myesthenic Syndrome (LEMS) is associated with certain cancers and arises from production of autoantibodies against P/Q type voltage gated Ca2+ channels. LEMS results in reduced evoked Ach release and failure in neuromuscular transmission. Seen as reduced evoked end plate potentials, muscle weakness and fatigue (but the amplitude of the spontaneous MEPP is normal). LEMS can be treated with immunosuppressant drugs and plasma exchange. Another is Familial hemiplegic migraine (FHM), a monogenic type of inherited migraine caused by mutations in the gene for the pore-forming subunit of P/Q-type Ca++ channels. Studies suggest the mutations increase open probability of the channel and increased probability of glutamate release, so lead to increased strength of excitatory transmission due to enhanced action-potential evoked Ca2+ influx. It is estimated that at the active zone of the typical glutamatergic synapse in the CNS that there are ~ 2 to 20 vesicles in a fusion ready state, i.e., they are also called docked or pre-docked at the presynaptic membrane. There is good evidence for an organizing structure for the docking of synaptic vesicles at ribbon synapses. Ribbon synapses are an interesting variation on the typical central excitatory synapse, seen mostly in sensory neurons. Vesicles tethered to the ribbon proved a pool for sustained release that is ~5X greater than docked pool available for fast release. 3
Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu
The molecular machinery of vesicle fusion

Release of neurotransmitter from synaptic vesicles into the cleft requires fusion of docked vesicles with the plasma membrane. This fusion is a Ca2+ dependent process involving molecular machinery that is pre-assembled. Somehow Ca2+ influx triggers membrane fusion. The machinery is made of SNARE proteins which mediate membrane fusion in a wide variety of cells, not only neurons. SNARE proteins form complexes that bridge the donor and acceptor membrane. They do this by forming molecular zippers involving a 4-stranded coil-coil domains. The formation of these coil/coils in the zippers is thought to release enough energy to drive the 2 membranes together. Some SNARE proteins found at synapses are the targets of clostridial toxins like botulinin toxin and tetanus toxin. Synaptotagmin, a synaptic protein that binds Ca++ as well as SNARES and phospholipids is thought to facilitate membrane fusion. Synapaptotagmin binds PIP2, a phospholipid enriched in the plasma membrane via a C2 domain. The C2 domain is proposed to bind PIP2 in a Ca++ dependent fashion. The Ca2+ sensor has low affinity, so only a fraction of pool is released. Different isoforms of synaptotagmin are also found on LDCVs as well as synaptic vesicles. Exocytosis of synaptic vesicles is followed by rapid endocytosis of membrane and membrane proteins. Synaptic vesicle release is probabilistic, not a certainty. Many more vesicles are docked than are released. The reserve pool is typically 5-10x the size of the released pool. The Ca++ responsiveness of vesicles can vary, number of releasable vesicles can vary, Ca++ channel dynamics and magnitude of Ca++ influx can vary, as can the shape of action potential. Synaptic transmission is also highly regulated by G proteins, kinases and phosphatases.
Ligand-Gated and G-protein coupled Postsynaptic Receptors

The response of a neuron is a function of the type and number of receptors expressed. About half the synapses in the brain are excitatory, 30-40% inhibitory, and the rest involve small molecular or peptide transmitter. The binding of neurotransmitter to ligand gated ion channel (LGIC) receptors open ion channels in postsynaptic membrane. Excitatory transmitters, like glutamate, aspartate and acetylcholine open channels that permit the influx of positive ions like Na+ or Ca++, whereas inhibitory transmitters like GABA or glycine gate negatively charged Cl- ions. The ion fluxes change the membrane potential of the postsynaptic cell. In contrast, G-protein coupled receptors (GPCRs) accomplish this effect on postsynaptic membrane hyperpolarization or depolarization via 2nd messenger or G-protein mediated modulatory effects on ion channels. The structure of the pore forming unit of LGICs is similar to that of a variety of classes of voltage gated ion channels. LGIC contain multiple sites for regulation by second messengers.
Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu LGICs mediate rapid poststynatpic effects, with changes in membrane potential detected within milliseconds of an action potential invading the presynatpic terminal. The postsynaptic responses mediated by LGICs might only last only for 10s of millisecs, too. Ligands unbind quickly due to clearance mechanisms (transporters, degradative enzymes). LCIC numbers influence size of the post synaptic responses, along with other factors (i.e., channel open time, desensitization time). LGIC are also typically clustered at the synapses via interactions with synaptic proteins that are somehow tethered to synapse. AMPA and NMDA receptors are the main excitatory postsynaptic LGICs in the CNS. They play distinct roles at excitatory synapse, with both contributing to the postsynaptic potential. That is, the excitatory postsynaptic potential is usually multi-component. AMPA receptors open in a ligand dependent fashion whereas NMDA receptors also require the membrane to be depolarized before opening. At resting membrane potentials, the pore of the NMDA receptor is blocked by Mg2+ ions. Depolarization (usually through AMPA receptor stimulation) is necessary to remove the Mg2+ block. Another interesting feature of NMDA receptors is that glycine or D-serine is required as a co-agonist. Glia are a source of D-serine, so glia play at least two roles in modulating excitatory transmission: release of D-serine and uptake of glutamate via glutamate transporters. The drug APV blocks NMDA receptors. While most of the current let in is via AMPA receptors, NMDA receptors let in Ca2+ which activates a variety of enzymes linked to synaptic potentiation. Additionally, NMDA receptors show a slower onset, and a longer lasting response. GPCRs have a 7 transmembrane structure related to that of bacterial rhodopsin. Natural and endogenous ligands that bind GPCR range from stimuli that activate sensory systems to esoteric peptides (such as orexins, kisspeptin), as well as the typical cast of synaptic transmitters in the brain including glutamate, GABA, DA, NE, 5HT. GPCRs stimulate heterotrimeric G-proteins, and activate a range of effectors. In contrast to LGICs, GPCRs tend to localized perisynaptically. They have higher affinity for ligands, and their activation depends on rate of removal of neurotransmitter as well as amount of neurotransmitter released. Although GPCR/G protein complexes might be preformed, the onset of the signal is slow compared to that of LGICs. GPCR signaling is terminated in various ways including GTP hydrolysis, receptor endocytosis and sometimes receptor degradation. Effect of GPCR on channels (via liberated Gproteins) might be detected for 100s of msec or longer (via second messengers and 3rd tier of 2nd messenger activated signaling cascades). GPCRs directly modulate presynaptic Ca++ channels, postsynaptic K+ channels, as well as 2nd messenger systems via heterotrimeric G proteins. The CB1 cannabinoid receptor provides an interesting example of this type of presynaptic regulation as it is activated by a retrograde signaling.
Shutting off synaptic transmission

Terminating neurotransmission is accomplished in part by diffusion and reduced release or receptor desensitization. However, the bulk of this task is performed by neurotransmitter transporters and degradative enzymes. Most transporters are found on presynaptic terminals. However, transporters for glutamate are located on astrocytes as well as on presynaptic 5
Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu terminals. Glutamate transporters in glia are also important in recycling glutamate in the form of glutamine to the excitatory nerve terminal. The catecholaminergic transporters are targets for drugs of abuse, used in treating ADHD or depression, and/or to promote weight loss. Various neurological or mental disorders are associated with degradative enzymes for neurotransmitters include acetylcholine esterase (a drug target in myasthenia gravis), monoamine oxidase (violence, treatment of mental disorders), catechol o methyl transferase (DA).
Synaptic Integration
Dendritic integration: usually EPSPs are subthreshold, need many to generate an action potential at the axon hillock. Summation will depend on frequency of EPSPs (#APs/unit time) as well as distance from soma (but there are mechanisms in dendrite to correct for inputs distal to the soma). During summation postsynaptic membrane stores change in capacitance that next action potential adds to. Synaptic plasticity, e.g., synaptic potentiation or depression, are adaptive responses that are thought to underly learning and memory, or information storage in general. Plasticity can be influenced by both presynaptic and postsynaptic events. For example of presynaptic: terminals with high probability of release, but small pool of vesicles will be prone to depression. Synaptic depression can also be facilitated by postsynaptic mechanisms such as rapid receptor desensitization or removal from the synapse. In addition, whether a synapse can be potentiated or depressed is a function of previous history. So if pool of vesicles is exhausted due previous depolarization and to slow rececycling, the subsequent response is likely to be weaker, too. On the other hand, some stimuli which open NMDA receptors lead to insertion of AMPA receptors, and a strengthening of subsequent post-synaptic responses is detected.
STUDY GUIDE FOR SYNAPTIC TRANSMISSION - BERGSON BRAIN & BEHAVIOR Please be knowledgeable about the following: 1. What defines a synapse? How is a quanta defined? 2. What is the difference between dense core synaptic vesicles and small clear synaptic vesicles? 3. What is the difference between the neuromuscular junction (NMJ) and other synapses? 4. What is required for rapid neurotransmitter release after a presynaptic action potential? Does neurotransmitter release occur after each action potential?. 5. Which neurotransmitter is responsible for synaptic inhibition in the forebrain? 6. How does clostridial neurotoxin block neurotransmitter release? 7. How is it possible that small decreases in calcium can produce large changes in neurotransmitter release? 8. What are the mechanisms by which neurotransmitters are removed from the synaptic cleft? 9. What is meant by coincidence detection? To which neurotransmitter does this process apply and why? 10. How does the activation of NMDA receptors produce excitotoxicity and neuronal death? 11. What are the two major classes of postsynaptic receptors? What are the differences between these two classes? 12. What is the difference between point-to-point vs. diffuse synaptic transmission? 13. What is the pathophysiology seen in myastenia gravis? How does this manifest into clinical symptoms of the illness?
Chemical Signaling by CNS Neurons Clare Bergson, Ph.D. cbergson@mail.mcg.edu, 1-1926, CB3616
5/31/2005
Synaptic Transmission
Phase I: January 4, 2010 Lecturer: Clare Bergson cbergson@mail.mcg.edu
5/31/2005
Ramon y Cajal
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connection b/w two neurons in CNS or a neuron with a muscle cell (motor)
synaptic cleft is the area b/w the dendrite and axon where neurotransmitters get released
1) transmitter gets synthesized and stored 2) action potential arrives 3) depolarization = Ca2+ channels open
S Y N A P T I C
T R A N S M I S S I O N
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Background Tripartite System Presynaptic terminal Transmitter synthesis Transmitter release Postsynaptic response Receptor diversity and function Glia Regulation of transmission Transmitter removal Adaptive synaptic responses
does some regulation of reabsorption of glutamate
Chemical transmission at neuronal synapses
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Chemical Synapse
axon terminal
Motor neuron
Muscle
NMJ
presynaptic neuron
The synapse is the basis for point to point communication between nerve cells (as well as nerve and muscles)
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Synaptic Release is Quantal
quantal = energy of neurotransmission
Fatt and Katz (1952): Spontaneous mEPPs
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responses came in response to certain aplitudes.... called quanta every x amnt = response the number of transmitter in each vesicle relates to amnt of reaction
Physiological evidence for release of synaptic quanta*
** ** in low Ca++-containing media
*indivisible packet of neurotransmitter

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Morphological evidence for synaptic quanta
muscle endplate
Synaptic Vesicles Membrane-bound organelles 40-60 nm in size
Robertson (1956)1st EM of synaptic vesicles at the NMJ
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Are vesicles the basis of quanta? Ceccarelli, Hurlbut..(1990)
Frog NMJ
vesicles typically contain a large quantity (~10,000) of neurotransmitter (e.g. ACh)
-latrotoxin a drug
= no muscle response in cells
-latrotoxin dumps all the synaptic vesicles (even in Ca++ free media)
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shows: difference in end plate potentials
Myasthenia gravis: apparent effects on quantal size

Elmquist et al., 1964
the end plate potential is smaller than normal
antibodies against the receptors for ACh or nicotinic receptors
neostigmine = an AChE inhibitor
Actual data Results predicted from model (or after treatment with neostigmine, an AchE inhibitor)
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Presynaptic ultrastructure
nerve terminals contain numerous synaptic vesicles- not randomly distributed some vesicles are docked at an active zone, others are held in reserve only vesicles that are already docked can release neurotransmitter quickly (the readily-releasable pool)
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Two types of synaptic vesicles
D E N S E
different neurotransmitter vesicles gabbet vesicles are flat and big???? dense = neuropeptides (entethalon, beta-endorphins, angiotensin)
C L E A R
with clear a lot are close to being docked... and another pop further away "RESERVE"
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glutamate, aspartate, GABA, glycine are the ones she "mentions a lot" her words, not mine
Small clear core vesicles
Either
LDCV
large dense core vesicles
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prozac prevents reuptake of serotonin
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Neurotransmitter packaging
neurotransmitter concentrated in synaptic vesicles by active transport: vesicular transporters utilize pH or membrane potential vesicles contain a very high concentration of neurotransmitter Peptides are usually packaged into LDCVs in the cell body
ATP dependent
early on
so for peptides, they get packaged after golgi and before they get sent, but others are packaged as tehy are going through the axon
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small molecules are sent through axon THEN put into vesicles.... peptides are packaged before being sent and an enzyme will quickly alter it to make it a neurotransmitter
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Co-Transmitters
vesicles closer to synapse are released (Ca2+ dependent)
doesn't always get released synaptically if there is high frequency stimulation, more gets released in higher quantities
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Calcium channels are mutisubunits that open with membrane polarization adn are highly regulated. concentrated in axonal pluton (?)/ highly regulated by second messengers calcium channel is a pore that lets in calcium. highly regulated and thre are diseases that mess with this stuff
Calcium channel structure ( 1 subunit)
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Ca++ Channelopathies and Neurological Diseases

Lambert-Eaton myasthenic syndrome (LEMS)
against Ca channels
Auto-antibodies vs. P/Q-type Ca++ channels
Familial hemiplegic migraine (FHM)

Mutations in the gene for the pore-forming subunit of P/Q-type Ca++ channels lead to enhanced evoked Ca++ influx.
exercise helps with these..... but iwth myasthenia gravis exercise worsens symptoms
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Calcium influx triggers release (fusion) of docked vesicles

release requires a very high, local [Ca++]
Rapid <0.5 msec, 100-1000x increase
Release is brief
Channels close Ca++ diffuses away
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Ca2+ channels are situated close to the docked vesicles
Calcium domains
Docked
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Docked vesicles
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some sensory reponses (e.g. audio) are arranged along filaments. as as sensory neurons become stimulated, there is no limit to how much can be released can be prolonged, less desensitization
Docked vesicles at ribbon synapses
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Chemical Signaling by CNS Neurons Clare Bergson, Ph.D. botox cbergson@mail.mcg.edu, 1-1926, CB3616
tSNARE = target vSNARE = vesicle
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snares can be cleaved by botchuline and tetnis toxins and they inactivte synaptic fxns
What drives vesicle fusion?

SNARE proteins on vesicles and on target plasma membrane Molecular machinery involved in membrane fusion events
on vesicles
Synaptobrevin (VAMP)=vSNARE Syntaxin=tSNAREon target SNAP25=tSNARE
SNAREs are targets of clostridial toxins
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BOTOX treatment
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Before
After
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Synaptotagmin: a candidate Ca++ sensor

binds Ca2+ with low affinity located near Ca2+ channels interacts with phospholipids and SNARE proteins found exclusively on synaptic vesicles genetic ablation abolishes evoked but not spontaneous release
MEPP = mini end plate potential
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How does calcium trigger vesicle fusion?
Docked vesicles located near Ca++ channel vSynaptotagmin binds Ca++ and translocates to plasma membrane
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The reliability of release can be modulated

the probability of release can be decreased or increased by second messengers (G-proteins, cAMP) the probability of release can be decreased or increased by activity presynaptic inhibition modulation can occur at several points
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Synaptic vesicles are recycled
~30 sec
Incomplete fusion
Complete fusion
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Presynaptic summary:
an action potential invades a presynaptic terminal, where some vesicles are already docked and waiting... calcium channels open, flood nearby vesicles with a high concentration of Ca2+ Ca2+ ions bind a low affinity sensor, greatly increasing the probability of vesicle fusion/release...
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presynaptic summary (continued):

if vesicles fuse, these release a high concentration of neurotransmitter calcium channels close very quickly as the action potential repolarizes... Ca2+ quickly diffuses away from vesicles that are still docked, the probability of vesicle fusion returns to a low value... neurotransmitter concentration in the synaptic cleft falls quickly
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Neurotransmitter release is probabilistic

a docked vesicle may or may not fuse/release after an action potential calcium merely increases the probability that release will occur (transiently) synaptic reliability is determined in part by the number of releasable vesicles
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B. Synaptic depressionpool with high probability of release, but small pool, or slow vesicle recycling
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Short-term Plasticity (altered reliability) =f[previous history, stimulation pattern]
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Postsynaptic receptors
Major classes of postsynaptic receptors:
Ligand-gated ion channels (LGICs) G-protein-coupled receptors (GPCRs)
many ligands bind both classes multiple receptors can be present at a given synapse
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Point-to-point vs. diffuse synaptic transmission

Point-to-point: rapid information transfer, directly alter firing (usually LGICs) Diffuse: slower modulatory actions, may or may not alter firing (usually GPCRs)
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LGIC
GPCR
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LGICs: general properties

structurally related to voltage-gated ion channels; multiple subunits divided into families of related channels selective for monovalent cations (Na+, K+) or anions (Cl-, HCO3-) a few are also permeable to Ca2+ ligands usually small molecules
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LGIC structure
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LGICs: affinity
LOW affininity for neurotransmitter (Kd in the micromolar range) ligands unbind fairly quickly must be close to the release site to see a high concentration of ligand (are clustered at postsynaptic density)
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NR1
LGICs are clustered
GluR1
syn
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LGICs: kinetics
ligand binding (2 molecules) increases probability of channel opening LGIC open probability is raised for 2-20 ms following release, starting <1ms following release bind, open, close, open...close, unbind can close persistently with ligand bound (desensitize) if ligand persists
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LGICs: numbers
at CNS synapses, 10-100 LGICs open in response to a single vesicle receptor number may change rapidly quantal PSPs are small (~0.1mV) signal proportional to channels bound (no amplification)
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Specific LGICs
glutamate receptors (GluRs) AMPA NMDA GABAA receptors (GABA-Rs) glycine receptors (GlyRs) nicotinic Ach, 5HT3, ATP.
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NMDA receptors
a type of glutamate receptor pore blocked by magnesium at hyperpolarized potentials can only open during depolarization (coincidence detection) requires glycine or D-serine as co-agonist pass calcium (fairly unique) important for some types of plasticity
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NMDA Receptor Co-agonists

serine racemase
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LGICs: Clinical Significance

Glutamate receptors mediate excitotoxicity (stroke, epilepsy) GABAA receptors are sites of action of benzodiazepines, barbiturates, anesthetics, ethanol nAch blockers for anesthesia
D-curarine
targets of auto-immune disease
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GPCRs: general properties

all related 7TM domain proteins ligands diverse (small molecules, peptides; odorants, light) exert actions by coupling to heterotrimeric G-proteins activated G-proteins activate a large number of downstream effectors
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GPCR Structure
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GPCRs: affinity
HIGH affinity for ligands (Kd in nanomolar range) remain bound by ligand for some time so need not be located at a synapse binding depends on distance from a synapse, and number of vesicles released
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Receptor affinity
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GPCRs: kinetics
time is required for multiple proteinprotein interactions onset is slow (10-100 ms) peak is late (100 ms-seconds) action terminated by GTP hydrolysis bound receptors can activate multiple G-proteins, multiple effectors, large capacity for amplification/divergence
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Receptor kinetics
G A B A A ( L G IC )
G A B A B (G P C R )
200
400
600
T im e ( m s )
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GPCRs: effectors
voltage-gated calcium channels K+ channels (open or close) adenylate cyclase (cAMP) phospholipase (IP3; DAG) many others, incl. transcription factors many effects of GPCRs do not change membrane potential
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Specific GPCRs
glutamate (metabotropic GluRs; mGluRs) GABAB muscarinic acetylcholine dopamine, opiate, 5HT, adrenergic...
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CB1 receptors and retrograde endocannabinoid signaling
Copyright 2009 American Physiological Society
Kano, M. et al. Physiol. Rev. 89: 309-380 2009;
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GPCRs: clinical significance

targets (direct or indirect) of a huge list of clinically useful drugs (too numerous to mention)
E.g., L-dopa for Parkinsons Disease
GPCRs important for modulatory actions
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LGICs
low affinity located at synapse fast/brief no amplification 1 signal (Vm) small molecules point-to-point
GPCRs
high affinity peri/extrasynaptic slow/prolonged amplification divergent signals small/large molecules diffuse
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Termination of transmission Neurotransmitter Removal

removal of neurotransmitter from the synaptic cleft is accomplished in part by diffusion (amino acids) rapid sodium-dependent re-uptake keeps the resting concentration of NT low (transporters) enzymatic degradation is important for acetylcholine, ATP, and monoamines
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Catecholamine Transporters as Drug Targets
ADHD
ADHD
Depression Short term weight loss
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Astrocytes are enriched in Glutamate Transporters
Theodosis, D. T. et al. Physiol. Rev. 88: 983-1008 2008

Copyright 2008 American Physiological Society
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ALS and glutamate transport
(Glt-1)
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Synaptic integration
Unitary EPSPs are usually subthreshold Neurons integrate many E and I inputs from entire structure Spatial and temporal summation AP initiation in initial segment
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Postsynaptic vs. Action Potentials
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Distal events are filtered by dendrites
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Dendritic Integration of Synaptic Events
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Neuro - Synaptic Transmission

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Neuro - Synaptic Transmission

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Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.

Vesicles and synaptic transmission

Packaging of neurotransmitters into vesicles

Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu

Dynamics of synaptic vesicle release

Synaptic Transmission 1-4-10 Dr. Clare Bergson cbergson@mail.mcg.edu

The molecular machinery of vesicle fusion

Ligand-Gated and G-protein coupled Postsynaptic Receptors

Shutting off synaptic transmission

Phase I: January 4, 2010 Lecturer: Clare Bergson cbergson@mail.mcg.edu

Chemical transmission at neuronal synapses

Synaptic Release is Quantal

quantal = energy of neurotransmission

Fatt and Katz (1952): Spontaneous mEPPs

Physiological evidence for release of synaptic quanta*

** ** in low Ca++-containing media

*indivisible packet of neurotransmitter

Morphological evidence for synaptic quanta

Synaptic Vesicles Membrane-bound organelles 40-60 nm in size

Robertson (1956)1st EM of synaptic vesicles at the NMJ

Are vesicles the basis of quanta? Ceccarelli, Hurlbut..(1990)

shows: difference in end plate potentials

Myasthenia gravis: apparent effects on quantal size

antibodies against the receptors for ACh or nicotinic receptors

neostigmine = an AChE inhibitor

Two types of synaptic vesicles

Small clear core vesicles

prozac prevents reuptake of serotonin

Calcium channel structure ( 1 subunit)

Ca++ Channelopathies and Neurological Diseases

Auto-antibodies vs. P/Q-type Ca++ channels

Familial hemiplegic migraine (FHM)

Calcium influx triggers release (fusion) of docked vesicles

Ca2+ channels are situated close to the docked vesicles

Docked vesicles at ribbon synapses

tSNARE = target vSNARE = vesicle

What drives vesicle fusion?

Synaptobrevin (VAMP)=vSNARE Syntaxin=tSNAREon target SNAP25=tSNARE

SNAREs are targets of clostridial toxins

Synaptotagmin: a candidate Ca++ sensor

MEPP = mini end plate potential

How does calcium trigger vesicle fusion?

The reliability of release can be modulated

Synaptic vesicles are recycled

presynaptic summary (continued):

Neurotransmitter release is probabilistic

Short-term Plasticity (altered reliability) =f[previous history, stimulation pattern]

Point-to-point vs. diffuse synaptic transmission

LGICs: general properties

LGICs are clustered

NMDA Receptor Co-agonists

LGICs: Clinical Significance

targets of auto-immune disease

GPCRs: general properties

CB1 receptors and retrograde endocannabinoid signaling

Copyright 2009 American Physiological Society

Kano, M. et al. Physiol. Rev. 89: 309-380 2009;

GPCRs: clinical significance

GPCRs important for modulatory actions

Termination of transmission Neurotransmitter Removal

Catecholamine Transporters as Drug Targets

Depression Short term weight loss

Astrocytes are enriched in Glutamate Transporters

in low Ca++-containing media