Exam II Flashcards
2 types of synaptic transmission
Electrical
Chemical
Compare/contrast the 2 types of synaptic transmission
Electrical
- distinct minority
- faster (no delay)
- simpler
- physically connected
- maintained through gap junctions (connexons)
- direct + passive flow of current
- EPSP and IPSP
Chemical
- majority
- slower (slight delay)
- more complex
- mediated through NTs
- imp: so many molecules, lots of room for things to go wrong (many neurological disorders)
Plasticity
Synaptic transmission is plastic – can be increased/decreased, allows us to memorize/learn
Gap junction
Specialized intercellular contacts formed by connexon channels that directly connect the cytoplasm of 2 cells
- 1.5-2 nm diam (5-6x wider)
The close connection of 2 adjacent membranes
Most of the time open, only close under certain conditions
Connected via connexon channels Connexons made of 6 connexin subunits 1 connexon = hemichannel 2 connexons = form channel Connexons connect 2 cells and allow direct passive current to flow
Connexons
Connected VIA connexon channels Connexons made of 6 connexin subunits 1 connexon = hemichannel 2 connexons = form channel Connexons connect 2 cells and allow direct passive current to flow
Pore of connexon = 1 nm (much larger)
Connexons are highly conserved in extracellular short groups (bc that’s where they link)
Highly variable in cytoplasmic regions (bc different ones are regulated in different ways
Electrical coupling
Direct passage of signal from one neuron to the next
Allows for synchronized firing
Directionality of electrical transmission
Bidirectional – meaning that current can flow in either direction depending on which member of the coupled pair is invaded by an AP
What is the molecular basis for electrical transmission?
Gap junctions between 2 electrical cells
What can go through gap junctions
All physiologically important molecules
(Ca2+, Mg2+, Na+, K+, Cl-, bicarbonate, phosphate)
And small metabolic/signaling molecules
(AAs, glucose, ATP and 2nd messengers (i.e., cAMP, cGMP, IP3)
How can we quickly and easily determine whether cells are connected through gap junctions?
Dye-coupling experiment
You COULD stick in electrode and see if electrically coupled…but TEDIOUS
EASIER WAY: dye-coupling
Insert fluorescent dye into one cell, small enough to diffuse in 10-20 mins
GFP too big (protein), only small peptides can fit through
How are gap junctions closed?
Closed by intracellular ↑ in Ca2+ or lower pH (more H)
The junctions will be closed if exposed to acidic conditions (lower pH, more H added) or high levels of calcium
The gap junction closure by calcium ions and protons is a self-saving mechanism of the cell to protect/seal normal cells from injured or dying cells near by
Increase in Ca2+ and acidity (lower pH, higher [proton]) occurs when cells are damaged
Key properties of electrical synapses
- rapid signalling
- reliable
- synchronous activity
- direct transfer of key small molecules
- more gap junctions present during neural dev (help them find common pways)
- more common in NS of invertebrates
RRSDDI
RRISDD
Importance of electrical coupling shown through…
The heart ! - best example of the importance of electrical coupling
- Highly synchronized
- ♡beat triggered by spontaneous pacemaking in the node – travels through fibres
For the ♡ to pump so rapidly and synchronously, you need gap junctions to allow for direct passage of current
Key properties of chemical transmission
- Much more abundant
- Much more versatile – fast, slow (direct, indirect);
excitatory, inhibitory; short-term and long-term
regulation; etc. - No direct flow of current between the cells
- No direct physical connection between the cells
Otto Lewi experiment
Existence of NTs –
Synaptic cleft length
50-60 nm wide
Chemical synaptic transmission steps
- NTs synthesized and packed into vesicles
- AP invades presynaptic terminal
- Depolarization causes Ca2+ channels to open
- Influx of Ca2+ into cell
- Ca2+ causes vesicles to fuse with pre-membrane
- NTs released via exocytosis into synaptic cleft
- NT binds to Rs on postsynaptic membrane
- Channels open/close - thus changing ion flow across membrane
- The resulting NT-induced current flow alters membrane potential and post-synaptic cell conductance (⇅probability of cell firing)
Synthesis AP DC - depol/Ca channels Influx of Ca Fusion Exocytosis Receptor binding Channels open/close
S A Dc I F E R - C
How do NTs affect current?
The resulting NT-induced current flow alters membrane potential and post-synaptic cell conductance (⇅probability of cell firing)
Fast synapse steps vs. slow chemical synapses steps
FAST - ionotropic
- Opening of iontropic R
- Generates postsynaptic current (ions flowing in/out)
- EPSP or IPSP
- ⇅ of AP firing
SLOW - metabotropic
- Activation of metabotropic R (usally GCPR)
- G protein activated
- 2nd messenger activated (Ca2+, cAMP)
- Protein kinase activated
- Regulation of ion channels
- ⇅ of AP firing
NMJ ballpark #s
7,000 ACh molecules/vessel
Synaptic cleft = 50-60 nm wide
Active zone
Regions where vesicles fuse
Why is the NMJ the best studied chemical synapse?
Simple, large, and peripherally located
great for studying chemical transmission
NMJ chemical transmission steps
- AP propagates to nerve terminals
- Voltage-gated Ca2+ channels open, increasing Ca2+ in terminal
- Exocytosis of ACh
- ACh diffuses across the synaptic cleft
- Binds to postsynaptic nicotinic ACh receptors (nAChRs)
- Na/K flow generate EPC that produces EPP
- If the EPP is above the action potential threshold, one or more APs will be fired in the muscle fibers
- Acetylcholinesterase (AChE) degrades the ACh and terminates signaling
*6. Na+ and K+ ions flow through nAChR channels, generating an inward end-plate current (EPC) into the postsynaptic cell and producing an end-plate potential (EPP)
How is a current defined
By the flow of positive ions
Basic metabolism of ACh in NMJ
- ACh is made up of choline and acetylCoA
- In synaptic cleft, ACh is rapidly broken down by AChE
- Choline is transported back into presyn terminal (via Na+/choline cotransporter)
- AChE limits amount of tiem ACh is in synapse
ACh R and enzyme blockers
n ACh R:
- nicotinic
- blocked by curare, βbungarotoxin
AChE
- blocked by insecticides, nerve gas, neostigmine, physostigmine
==> ALLOWS ACh to remain longer in the cleft, enhanced effect of synaptic transmission at NMJ
Why were Katz recordings were done in partially curarized NMJ?
Curare will block ACh receptors, keeping EPP subthreshold
So that way electrode is not kicked out/dislodged
EPP experiment results
- Fast rise to the peak in ~2-3 ms
- Amp is largest near the endplates and decrease with distance – it’s a graded potential and propagates passively
- EPP is produced by a brief surge of current at the endplate
- EPP triggers APs when it’s large enough to reach the firing threshold
Katz
synaptic transmission pioneer
What produces EPP?
EPC - inward current that produces EPP
When you superimposed EPC and EPP, what do you find?
What contributes to the rise/fall of each?
EPC produces EPP
- this is why current rises faster than voltage
Voltage decays more slowly than EPC
- due to large capacitance of muscle membrane that slows down voltage
What causes the EPC current to decay?
- Desensitization of ACh Rs
1. Mean open time of nACh Rs
2. AChE (hydrolrzes ACh in cleft, which will stop current)
3. Diffusion of ACh (~10-15ms)
How to test effect of AChE?
Use AChE inhibitor (ex: neostigmine)
Stimulate axon – EPC response will decay much more slowly
Since it still does decay, implies that other molecules are involved
EPC
the macroscopic current resulting from the summed opening of many ion channels in the NMJ
Bc the EPC is normally inward, it causes the postsynaptic membrane to depolarize
Conductance underlying EPC
Return to
Structure of nAChR channel
Pentamer
With pore in middle (nonselective, bigger than Na/K)
Each subunit has 4 transmembrane domains
What happens at NMJ?
How to measure what happens?
- Use voltage clamp to find reversal potential of NT R channel
=> Then can infer what ions can go through - Use ion substitution to confirm
- Patch clamp
- Can do single-channel recording
Experimentation - ion identification
Return to
Why is transmission in CNS much harder to study?
Many inputs (100s/1000s), EPSP+IPSP, many NT types, Ionic and metabo Rs, small presyn terminals(hard to record), tiny postsyn responses (0.2-0.4 mV PSP), spatial+temporal integration
Hundreds to thousands of inputs
Excitatory and inhibitory inputs
Many different types of neurotransmitters
Different types of receptors – ionotropic vs. metabotropic
Small presynaptic terminals – hard to record
Tiny postsynaptic responses – 0.2-0.4 mV PSP
Extensive spatial and temporal integration
Excitatory v. inhibitory synapse
features
EPSP:
- occurs in dendritic spines
- large (1-2 μm^2) terminal
- NTs: Glutamate, ACh, 5-HT, ATP
- functional effect: more APs
IPSP:
- occurs in soma
- small (less than 1 μm^2) terminal
- NTs: GABA, glycine
- functional effect: less APs
Synapse markers
PSD-95: abundantly expressed in postsynaptic density
Synapsin: presynaptic marker
Where are excitatory synapses typically found in the CNS?
Dendritic spines
GABA
Inhib NT
Glycine
Inhib NT
Glutamate
Excit NT
ACh
Excit NT
Ionotropic glutamate Rs
NMDA Rs
AMPA Rs
Kainate Rs
Both glutamate-gated cation channels allow Na/K passage, always produce EPSP
Most central excitatory synapses express both
How to determine role of glutamate Rs given that nearly all have both NMDA and AMPA Rs?
Selective antagonist blockage…
=> NMDA currents slower + last longer
=> AMPA Rs = largest amp, therefore known as primary mediators for excitatory transmission
AMPA R experimentation
- Express AMPA Rs (on xenoput oocytes) without dealing with other channels
- Do voltage clamp, then apply current
- Add glutamate on cell and record inward current
- Current will rise + decay quickly, then desensitize
If you add CAQX (AMPA antagonist), you mainly conduct inward Na+ current
Mainly conducts inward Na+ under resting conditions
CNQX
Competitive AMPA/kainate R antagonist
NMDA R experimentation
- Express NMDA Rs
- Clamp at -70
- Apply glutamate
- We do not see response
- Could be that rev. potential is -70…so we clamp at a different level - Clamp to very low potential
….still no response…we only start seeing current when become less negative
current rev. potential = 0
Researchers found that the current-voltage relationship is caused by Mg2+
– The lack of current at negative voltages is due to Mg2+ channel blockage, voltage-dependent
WHY MG2+ DIP???
NMDA R properties
Common: nonselective cation channel, permeable to Na/L
Unique:
- Permeable to Ca2+
- Votage-dependent block by extracellular Mg2+
- Requires glycine as a cofactor
- Slow opening and desensitization
- Blocked by AP5
AP5
Competitive NMDA R antagonist
How does the Mg2+ block work?
(voltage-dependent)
AA lining of NMDA pore = negative
Mg2+ (being pos) gets sucked into pore
The more negative the voltage, the stronger the pull on Mg2+ to block
When voltage is less negative, the pull becomes less strong –> bigger and bigger current due to less+less negative block
Degradation eventually occurs due to the driving force - as membrane potential approaches reversal potential
How would you prove that large response due to both Rs, and how to dissect each?
Synapse b/w Ca3 axon and Ca1 dendrites (glutamatergic)
- stick electrode in Ca1 cell body
Apply blockers to see which Rs involved
- Apply AP5 (NMDA R antagonist)
- Apply CNQX (AMPA R antagonist)
If you use both antags, no response
If you use AMPA antag (CNQX), you get no response
If you use NMDA antag (AP5), you get lessened response
Conduction effects at different stimulation
At strong stimulation, Mg2+ block is relieved
At low stimulation, Mg2+ exerts its effects – ∴ small current bc only one R conducting
NMDA R channel structure
Most of channel is outside in extracellular domain
Contains glutamate, glycine, etc. binding sites
Fast inhibitory synaptic transmission experimentation
First Stimulate neuron
Produces hyperpolarization, 15-20 ms lasting
- Do voltage clamp
Outward current produces hyp.
Find rev. potential -75 mV
….Could indicate involvement of K+ or Cl-….
- Ion substitution
Proves K+ not involved, it’s Cl-
….∴ Rs are Cl- channels….(GABAa Rs)
Cl- flows into cells when channels open (outward current bc current defined by flow of positive ions)
Causes hyper. –> IPSP
GABAa Rs
Ag/Antag
Ligand-gated Cl- channels
Zolpidem/ambiem = GABAa agonist Flumazenil = GABAa antagonist, antidote for OD
Zolpidem/ambiem
GABAa agonist
Flumazenil
GABAa antagonist
antidote for OD
Fast v. slow synaptic transmission time comparison
Fast (10s of ms)
Slow (100s of ms - seconds)
Fast v. slow synaptic transmission time comparison
Fast: Direct NT binding ∴ syn. transmission quick (10s of ms) Direct opening of ion channels EPSP or IPSP Can trigger AP
Slow:
Slower onset (10-100s of ms) and lasts longer (10s of ms to secs)
Indirect opening of ion channels -Mediated by metabo Rs
Usually small EPSP, mostly IPSP
Usually does not trigger AP
∴ regulates neuronal excitability
Slow synaptic transmission steps
- NT binds to metabo. R
- Activates to G protein
- Activates primary effector
- 2nd messenger activated
- Activates secondary effectors
- (P) to produce effect
Primary effectors
Enzymes that can change the 2nd messenger levels inside a cell; either produce or break down 2nd messengers
The heart - what is it an example of?
Ex of slow synaptic transmission
♡ innervated by PS and S
S innervation: AP much faster, ♡faster, when excited
PS/vagus innervation: Less AP firing, ♡beat slower
BOTH responses caused by slow syn. transmission
S system: speeds it up
- vagus nerve releases ACh
- binds to m Ach R
- dissociates trimeric G protein
- Gβγ binds to GIRK channel and activates it
- efflux of K+ creates hyperpolarization
PS system: slows it down
- PS nerve releases E
- Pacemaker cells have βadrinergic Rs
- Activates G protein
- Activates AC
- ↑ cAMP production
- (P) to many Ca2+ channels
Efflux of K+ would be…
Outward current
Influx of Cl- would be….
Cl- flows into cells when channels open (outward current bc current defined by flow of positive ions)
cAMP pathway, IP3 pathway
Know steps, know how to dissect
What does it mean the “fastest” slow syn. transmission?
G protein modulation of ion channels
ACh I Activation of Muscarinic ACh receptor (mAChR) I Activation of G proteins I Direct activation of Kir channels by G I Hyperpolarization
The phosphoinosital (PI) system - IP3 pathway
Many NTs activate this system, ex: ACh
PLC: effector/enzyme that breaks down PIP2
IP3 then ↑ [Ca2+] in cytosol
Shortcut: PIP2 can directly bind/activate some channels
You stimulate a presynaptic terminal and elicit a fast EPSP, followed by a slow one. Explain how.
Fast EPSP elicited by fast synaptic transmission. Slow EPSP is by slow synaptic transmission.
Fast produced by N ACh Rs, can prove with curare or snake toxin block (wouldn’t affect slow response)
Slow produced by M ACh Rs - metabotropic cascade causing delay, can prove with hyocine block (wouldn’t affect fast)
Ion channels are regulated by (in slow syn. transmission)
(P)
2nd messengers
G proteins
Intermediate signalling molecules
Nicotinic vs. Muscarinic Rs
Nicotinic - ionotropic, fast chemical transmission
Muscarinic - metabotropic, slow chemical transmission
Amplification
*GCPR can activate many G proteins
G protein can only bind 1:1
*Primary effector can bind many 2nd msngers
2nd msngr can only bind 1:1
*2nd effector can bind to many kinases
YOU CAN ALSO HAVE CROSS TALK TO AMPLIFY SIGNALING PATHWAY
Properties of slow synaptic transmission
Mediated by metabotropic receptors Slow on and slow off Amplification Alteration of the biochemical (metabolic) state Crosstalk between signaling pathways No direct opening of ion channels Indirect opening or closing of ion channels Modulation of neuronal excitability
