NEU 490 Synapses & Synaptic Transmission

Question 1

Synaptic Transmission - Classical definition: ?

Neuronal synapse types:

Answer 1

Classical definition: specialized zone of contact at which one neuron communicates with another neuron or effector cell (skeletal muscles,vicera, internal organs)

Neuro-effector synapses (e.g., neuromuscular junction) – between neuron and a effector cell that is non-neuronal like a muscle cell

Electrical synapses

Chemical synapses → between a neuron and another neuron and include NT release

Question 2

Chemical: Def?

Distance between pre and postsynaptic cell membrane: ?

Cytoplasmic continuity between pre and postsynaptic cells: ?

Ultrastructural components: ?

Agent of transmission: ?

Synaptic delay: ?

Direction of transmission: ?

–
Electrical: def?

Distance between pre and postsynaptic cell membrane: ?

Cytoplasmic continuity between pre and postsynaptic cells: ?

Ultrastructural components: ?

Agent of transmission: ?

Synaptic delay: ?

Direction of transmission: ?

Answer 2

Chemical: Have a gap between the pre and post; Substance released from presynaptic cell diffuses across synaptic cleft and produces effect on postsynaptic cell → unidirectional from pre to post
– Distance between pre and postsynaptic cell membrane: 20-40 nm
– Cytoplasmic continuity between pre and postsynaptic cells: no
– Ultrastructural components: presynaptic vesicles and active zones with postsynaptic receptors
– Agent of transmission: chemical transmitter NT
– Synaptic delay: significant at least 0.3 ms, usually 1-5 ms or longer this is a delay compared to electrical
– Direction of transmission: unidirectional

Electrical: Direct flow of ions from one cell to another; hence direct influence of electric current from one to another with small gap/distance → bidirectional using gap junction channels and going AP to Coupling potential back and forth
– Distance between pre and postsynaptic cell membrane: 3.5 nm
– Cytoplasmic continuity between pre and postsynaptic cells: yes
– Ultrastructural components: gap-junction channels
– Agent of transmission: ion current
– Synaptic delay: virtually absent
Direction of transmission: usually bidirectional

Question 3

Electrical Synapses:

Why Electrical? Creates WHAT between two neighboring neurons and the gap junction is where WHAT are formed. Mix of electrical and chemical synapses and in human nervous system has more WHICH TYPE synapses

TYPE OF speed transmission?

Faithful transmission of WHAT TWO TYPES?

Can be WHICH DIRECTION?

Synchronized Electrical Activity - fast response
Important in WHAT

Conduct nerve impulses faster than chemical BUT not very flexible BC LACK WHAT

“Gap junction” is a term from electron microscopy; it refers to??

Gap junctions are WHAT FOUND WHERE IN WHO

Gap junctions are found in:

More than one gap junction channel at each connection so beneficial that they create these pores that allow ????

Common functions of gap junctions include:
– Conducting ?
– Sharing ?
– Coordinating ?
– Cell ?
– protein complex ?
– Some intracellular ?

Answer 3

Why Electrical? Creates functional junction between two neighboring neurons and the gap junction is where the synapses are formed. Mix of electrical and chemical synapses and in human nervous system has more chemical synapses

High speed transmission (separated by just 3.5 nm) with no delay and not directly touching but very close

Faithful transmission of subthreshold potentials not just AP but even graded potential can be sent from one cell to the other

Can be bidirectional and impulsive can travel in either direction

Synchronized Electrical Activity - fast response
Important in escape mechanisms like defensive and other processes that require quick responses - crayfish jet propulsion escape

lack gain meaning the signal in the post is the same or smaller than that of the originating neuron

“Gap junction” is a term from electron microscopy; it refers
a special type of intercellular connection - two cells that have a direct connection and gap are one of four broad categories that form connections

Gap junctions are ubiquitous found everywhere - across vertebrates, invertebrates, mammals, reptiles and other stuff across multiple cell types

Gap junctions are found in:
Cardiac muscle, Smooth muscle, Immature skeletal muscle, Neurons, Endocrine tissues, Epithelia and endothelia of all sorts, and Liver, lung, kidney, pancreas

More than one gap junction channel at each connection so beneficial that they create these pores that allow ions and some molecules to flow from one cell to the next which connects the cytoplasm between the two cells in question

Common functions of gap junctions include:
– Conducting electrical signals - ionic current flow between
– Sharing metabolic needs - intracell components needed for health of cell so coupling and exchange
– Coordinating cell development between multiple cells at once
– Cell adhesion for neuronal migration
– Hexameric protein complexes composed of connexins
– Some intracellular molecules can pass so not just ions

Question 4

Electrical Synapses: Gap Junction Structure
Gap junctions are densely packed channels that span the membranes of two cells

6 connexin protein =

Hemichannel =

2 connexons =

Many GJ channels =

One connexin =

There’s more than one connexin type! About 20 different connexin genes in mammals:
– Variety of GJ channels created with different ?
– Proper combination for a functional channel and many possible pathologies from?

Homomeric connexon =

Heteromeric connexon =

One connexon =

2 Homomeric connexon =

1 Homomeric connexon + 1 diff Homomeric connexon =

2 mixed Heteromeric connexon =

Answer 4

6 connexin protein = 1 connexon

Hemichannel 1/2 of a gap junction channel so takes 2 connexons to create a jab junction channel

2 connexons = 1 GJ channel

Many GJ channels = 1 gap junction connection between two cells

One connexin = 4 transmembrane domains

– Variety of GJ channels created with different functions, expression patterns, and permeability
– Proper combination for a functional channel and many possible pathologies from dysfunction of connexins

Homomeric connexon = 6 same connexin

Heteromeric connexon = 6 diff connexin

One connexon = 1/2 gab junction so 2 connexon = 1 gab junction channel

2 Homomeric connexon = homotypic gab junction channel

1 Homomeric connexon + 1 diff Homomeric connexon = heterotypic gab junction channel

2 mixed Heteromeric connexon = Heteromeric gab junction channel

Question 5

A connexon with multiple connexin subtypes is called:

Which of the following are potential molecules that can pass through gap junction channels, depending on their composition?

Electrical synapses bring membranes of pre and postsynaptic cells within BLANK nM of one another, whereas the closest that a chemical synapse can bring the membranes is BLANK nM.

Answer 5

A connexon with multiple connexin subtypes is called: heteromeric

Which of the following are potential molecules that can pass through gap junction channels, depending on their composition? Ions, intracellular molecule like cAMP, and IP3

Electrical synapses bring membranes of pre and postsynaptic cells within BLANK nM of one another, whereas the closest that a chemical synapse can bring the membranes is BLANK nM.
Answer: 3.5 and 20-40

Question 6

Permeability of Gap Junction Channels

– Most electrical synapses are WHAT 3?

– Gap junction channels are permeable to WHICH 3?
→ specific homotypic and heterotypic gap channels displayed different permeabilities
Gap junction channel permeability depends WHAT?

– Mammalian electrical synapses: there are ~20 connexin genes; ~half are expressed in the mammalian WHAT; the large majority of connexins in the brain are in WHAT → most in WHAT and different combos of connexins of have different with WHAT 3?

– WHAT has been most consistently implicated in CNS neuronal gap junctions - predicted molecular weight of 36 KD

– Electrical events of bidirectional EXPLAIN?

Where do we often find electrical synapses?
— WHERE of inhibitory neurons in the mammalian brain are often connected by gap junctions
— (sensory integration) ??
— Synchronous activity – inhibitory interneuron is ?

Hypothesized plug gating mechanism of gap junctions:
– Each hemichannel can regulate ?
– Some are “chemically gated” by things like ?
– Others are voltage gated
Not always open and selective permeability.Many time plug moved due to voltage which can be either sensitive to ???

Answer 6

Most electrical synapses are bidirectional, symmetrical, and fast

Gap junction channels are permeable to small signaling molecules, including ions and intracellular signals like cAMP or IP3 → specific homotypic and heterotypic gap channels displayed different permeabilities
Gap junction channel permeability depends on connexin composition

Mammalian electrical synapses: there are ~20 connexin genes; ~half are expressed in the mammalian brain; the large majority of connexins in the brain are in glia → most in astrocytes and different combos of connexins of have different with size of charge, pore selectivity anions or cations, and second messenger selectivity

– Cx36 has been most consistently implicated in CNS neuronal gap junctions - predicted molecular weight of 36 KD

Electrical events from neuron 1 are passed with little modification to Neuron 2 and vice versa bidirectional

Where do we often find electrical synapses?
— Dendrites of inhibitory neurons in the mammalian brain are often connected by gap junctions
— Thalamocortical is thalamus and cortex (sensory integration) - reticular nucleus - with in thalamus - main inhibitory source with in TRN they are connected with gap junctions → spindle rhymes
— Synchronous activity – inhibitory interneuron is GABAergic with electrical synapses

Hypothesized plug gating mechanism of gap junctions:
– Each hemichannel can regulate its channel activity autonomously
– Some are “chemically gated” by things like calcium or protons (H+)
– Others are voltage gated
Not always open and selective permeability. Many time plug moved due to voltage which can be either sensitive to membrane voltage (Vm) or to transjunctional voltage (Vj)

Question 7

Synchronous firing of neurons – what happens when they aren’t active?

Electrical synapses are very good at WHAT?
– When Cx36 is knocked out WHAT HAPPENS TO CELLS?
– What happens to function? IN OLIVARY ?
– Wildtype: ?
– CX36 Knockout: inferior olive ??

Synaptic Plasticity of Electrical Synapses (compared to Chemical Synapses)
– Chemical and electrical synapses can be modified by neural activity?
– Plasticity of chemical and electrical synapses share ?

Plasticity: ?

Short term: ?

Long term: ?

Answer 7

Electrical synapses are very good at synchronizing the activity of neurons they interconnect

– When Cx36 is knocked out, cells can no longer synchronize
– What happens to function? When olivary neurons can not synchronize the coordination of muscle contractions is impaired - ataxia
– Wildtype: inferior live neurons - (inferior olive neurons major input to cerebellum) synchrony
– CX36 Knockout: inferior olive and no synchrony

(i.e. LTP and LTD) - LTD depression decrease removal and LTP potentiation increase hemichannel insertion
– Plasticity of chemical and electrical synapses share some similar mechanisms
– Chemical and electrical synapses plasticity

Plasticity: ms, seconds, mins, hours, and days

Short term: modification of existing proteins

Long term: transcription and translation of new proteins

Question 8

Which connexin has the highest level of expression in neurons?

Which cell type in the nervous system has the highest expression of gap junction channels?

Answer 8

Which connexin has the highest level of expression in neurons? CX36

Which cell type in the nervous system has the highest expression of gap junction channels? Astrocytes

Question 9

Chemical Transfer of Information
Vast majority of synaptic contacts in the nervous system involve chemical information transfer. Why?
– Great flexibility ?
– Functional ?
– Amplification of Signal ?
– Numerous ?
– Even more NT receptors - high ?

Evidence of Chemical Transmission – Ach (muscarinic inhibitory)
Stimulate ?

Answer 9

– Great flexibility - variety of combinations of NTs and NT receptor and the exact connect to excite, excite connect to inhibit, inhibit connect to excite and multiple presynaptic of same or mixed type synapse onto a cell postsynaptic
– Functional Complexity
– Amplification of Signal - electrical same or less response in postsynaptic and that isn’t the case in a chemical synapse
– Numerous NT
– Even more NT receptors - high diversity in possible response

Evidence of Chemical Transmission – Ach (muscarinic inhibitory)
Stimulate vagus nerve of heart and solution transferred to heart and induce bradycardia which slows down heart rate

Question 10

Steps in Chemical Synaptic Transmission
STEPS 1-5?

AP arrives at axon terminal, VG Ca channels open WHAT 6 STEPS??

Answer 10

1. Synthesis of neurotransmitters
2. Storage/packaging of neurotransmitter; synaptic vesicles traffic these to axon terminal
3. Release of neurotransmitter into synaptic cleft, in a Ca++- dependent manner
4. Binding of neurotransmitter to postsynaptic receptors (followed by postsynaptic response)
5. Termination of neurotransmitter action

AP arrives at axon terminal, VG Ca channels open, Ca enters the presynaptic neuron, Ca signals to NT vesicles, vesicles move to the membrane and dock, NT released via exocytosis, NT bind to receptors, signal initiated in postsynaptic cell

Question 11

Termination of Neurotransmitter Effects - we need to avoid overexcitation or overinhibition of post cell

Within a few milliseconds neurotransmitter effect terminated in one of three ways:

Diffusion?

Enzymatic degradation?

“Reuptake” ?

Answer 11

Diffusion(move from high to low concentration) - The neurotransmitter molecules “float away” from the synaptic cleft.

Enzymatic degradation - Enzymes located within the synaptic cleft hydrolyze the neurotransmitter. e.g., acetylcholine esterase
AChE: a popular target of insecticides (organophosphates) and nerve gas agents (sarin) - breaks down ACH

“Reuptake” - Specific transporters in the plasma membrane of the presynaptic neuron bind to the neurotransmitter, or its hydrolyzed metabolites, and transport them back into the cell (mechanism for recycling components). - also in astrocytes and a goodway to reduce energetically the need to make more NT

Question 12

Below are traces from electrically coupled inferior olivary neurons (a part of your medulla). The WT have normal expression of gap junctions, and the Cx36 knockout have connexin-36, a neuron-specific gap junction component, knocked out. A: shows traces from two neurons recorded during stimulation in either WT or KO mice B: shows “correlelograms” – essentially graphs to show how much synchrony there is. What are your conclusions from the figures?

Answer 12

Cx36 gap junctions are essential for synchronizing the activity of inferior olivary neurons

Question 13

Transport Mechanisms:

Why do we need to transport proteins? 8 things

Important Components of Axonal Transport: Microtubules: ?
— Consist of heterodimers of?
— Have largely uniform morphology that dictates the?
- Plus end points?
- Minus end ?

Motor Proteins:
Kinesin (anterograde motors): ??
Dynein (retrograde motors): ??

Adaptor Proteins: ?
Dynactin: ?
Shared bidirectional adaptors: ?

Anterograde: toward ?
Relies on kinesin?

Retrograde: away from ?
Relies on dynein?

Fast axonal transport:
– ? mm/day
– Various cargo: ?
– For a 1 meter long motor neuron, fast axonal transport can move cargo between ???

Slow axonal transport - move local
– ? mm/day
– Mobilization of ??

Answer 13

1. Intracellular cargo trafficking is tightly and spatiotemporally regulated
2. Most proteins synthesized in cell body
3. Transport especially important in neurons due to extreme anatomical polarization and cell size
4. Transport is bidirectional, sent along axon microtubules
5. Diff spatiotemporal control, send things in both directions
6. Proteins synthesized in cell body
7. Happens throughout neurons entire life
8. Microtubule - the roads at our axons

Microtubules: parts of the cytoskeleton critical for long range intracellular transport
— Consist of heterodimers of alpha-tubulin and beta-tubulin
— Have largely uniform morphology that dictates the directionality of motor protein transport
- Plus end points towards axon terminals - growing end of microtubule is plus
- Minus end faces the cell body

Motor Proteins:
Kinesin(anterograde motors): diverse set of motor proteins towards the axon terminal towards plus end and there are multiple kinesin
Dynein (retrograde motors): towards the cell body toward minus end and are on term/type

Adaptor Proteins: Bind distinct cargoes and bind to the motor proteins
Dynactin: binds dynein and aligns the motors to activate processive movement
Shared bidirectional adaptors: possess overlapping kinesin and dynein interaction regions

Anterograde: toward terminal plus end kinesin
From cell body towards synapse/axon terminal
Relies on kinesin - motor domain hydrolysis ATP and other domains bind adaptor proteins
Superfamily of many kinesin subtypes
Hydrolyzes ATP to power the motor

Retrograde: away from terminal toward cell body minus end dynein, toward cell body
Relies on dynein - only one type in neurons
Hydrolyzes ATP to power the motor

Fast axonal transport:
– 50-400 mm/day
– Various cargo: vesicles of NTs and membrane-bound organelles
– For a 1 meter long motor neuron, fast axonal transport can move cargo between the cell body and the axon tip within a week

Slow axonal transport - move local
– 0.2-10 mm/day
– Mobilization of cytoskeletal proteins

Question 14

Dysfunction of Neuronal Transport
Alzheimer’s Disease - also other tauopathies

WHAT FIVE?

Answer 14

• Tau is a microtubule associated protein that helps to stabilize microtubule structures, it is enriched in axons
• Phosphorylation of tau is typical in normal physiological conditions, but in pathological conditions tau can become hyperphosphorylated
• Hyperphosphorylation leads to the self-aggregation and oligomerization of tau
• These aggregated tau proteins eventually get converted into neurofibrillary tangles
• This leads to loss of axonal transport and microtubule destabilization
• Tau/neurofibrillary tangles
• Amyloid-beta plaques

Question 15

In-Class Question: Rabies is a viral disease that is typically transmitted via the bite of an infected animal. Early symptoms can include fever and abnormal sensations at the site of exposure. These symptoms are followed by one or more of the following symptoms: nausea, vomiting, violent movements, uncontrolled excitement, fear of water, an inability to move parts of the body, confusion, and loss of consciousness. Once symptoms appear, the result is virtually always death, regardless of treatment. The time period between contracting the disease and the start of symptoms is usually one to three months but can vary from less than one week to more than one year. Research: how does the rabies virus travel from the area where the bite is to the CNS? Is this anterograde or retrograde transport? Why is the time period from contracting the disease to symptoms appearing so variable?

Answer 15

Viral load, innervation, distance from the brain

Retrograde transport: internalized via endosome in peripheral nerves and trafficked in and transposed to cell body

Question 16

Vesicle Subtypes - you can have SCCV and LDCV at some synapse but that’s not required

Small Clear Core Vesicles
Characteristics: WHICH 5?
Types of Neurotransmitter:
WHICH 3?

Large Dense Core Vesicles
Characteristics:
WHAT 3?
Types of Neurotransmitter:
WHICH 2?

Monoamines like dopamine or serotonin ??

Vesicle Filling:
Vesicular Transporters: 3 THINGS?

Norepinephrine, Dopamine, Histamine, Serotonin, Acetylcholine: ?

GABA and Glycine: ?

Glutamate: ?

Answer 16

Small Clear Core Vesicles
Characteristics:
Require less presynaptic activation to release contents
Small
Optically clear interior
Larger pools of them
40-60 nm in diameter
Types of Neurotransmitter:
Classical NT like GABA or glutamate
ACH

Large Dense Core Vesicles
Characteristics:
Larger like 100-250 nm in diameter
Optically opaque
Requires high frequency stimulation to induce release
Types of Neurotransmitter:
Neuropeptides like CGRP, substance P and multiple AA in length

Monoamines like dopamine or serotonin can be packaged in either!

Vesicle Filling:
Vesicular Transporters: ATP-dependent, Proton (H+) gradient, Quantal size variability (variation in the response to the release of a single synaptic vesicle)

Norepinephrine, Dopamine, Histamine, Serotonin, Acetylcholine: Inward Movement NT and Outward Movement 2H+

GABA and Glycine: Inward Movement NT and Outward Movement 1H+

Glutamate: Inward Movement NT + Cl and Outward Movement 1H+

Question 17

Vesicles in the nerve terminal are grouped into three pools:

Readily Releasable Pool:
membrane?
%?
stimulation?
other?

Recycling Pool:
membrane?
%?
stimulation?
Larger pool then ?
Rate of vesicle formation?

Reserve Pool:
membrane?
%?
stimulation?
Vesicles not ?

Answer 17

Readily Releasable Pool:
Detached to membrane
Less than 1%
First group of vesicle to be released upon stimulation
Small and easily exhausted

Recycling Pool:
Proximate to cell membrane
5-20%
Cycled at moderate stimulation
Larger pool then readily releasable pool
Rate of vesicle formation same as rate of release

Reserve Pool:
Inside membrane at cytoskeleton fibers for microtubules
80-90%
Vesicles not released under normal conditions
Requires high frequency stimulation it prolonged intense stimulation

located inside the cell membrane, specifically within the cytoplasm of the presynaptic terminal in a neuron, and are considered to be a part of the “reserve pool” which means they are not directly docked at the cell membrane and are only released under strong stimulation when other readily available vesicles are depleted

Question 18

In-Class Question: Presynaptic terminals contain hundreds to thousands of synaptic vesicles (SVs), specialized organelles that store neurotransmitters and fuse with the active zone plasma membrane for regulated transmitter release. Despite the fact that in electron micrographs these small clear vesicles appear morphologically similar, SVs are not all functionally identical: some vesicles are clearly more prone to be released than others. Why do you think that some of these vesicle pools are easier to release than others? Discuss possible contributors that might alter propensity to be released.

Answer 18

1. Different expression of synaptic machinery
2. Proximity to the membrane

Question 19

Calcium’s Role in Neurotransmitter Release:

Synaptic Transmission: 3 steps?
Neurotransmitter release is mediated by?
To support rapid and repeated rounds of release?
The focal point of the vesicle cycle?

exocytosis VS endocytosis

Answer 19

Neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. - axon terminal

To support rapid and repeated rounds of release, synaptic vesicles undergo a trafficking cycle. - stereotypical series of events to recycle vesicles

The focal point of the vesicle cycle is Ca2+-triggered exocytosis that is followed by different routes of endocytosis and recycling - AP arrives at presynaptic terminal and VG opens Ca channels

Exocytosis is the process where a cell releases substances from its interior by fusing vesicles with the cell membrane, while endocytosis is the process where a cell takes in substances from outside by engulfing them with its cell membrane, forming a vesicle to bring them inside; essentially, exocytosis is “exporting” materials, while endocytosis is “importing” them

Question 20

Voltage-gated Calcium Channels

High threshold (high voltage activated; HVA) - requires more WHAT TO OPEN???
– L type ??
— N Type ??
— P type ???

Low threshold (Low voltage activated; LVA):
type? ??

Calcium channels in intracellular organelles - IP3 and endoplasmic reticulum ROLE?
- ? receptors
- Ryanodine receptors: ?

Evidence for Importance of Calcium in Neurotransmitter Release:
– Gain of function experiment: ?
– Loss of function experiment: ?

Answer 20

High threshold (high voltage activated; HVA) - requires more depo to open:
– L type (intracellular signaling cascades):
Slow inactivation
Generates Ca2+ spikes in dendrites
— N Type - (primarily playing a role in NT release) MOST IMPORTANT:
Medium inactivation
Presynaptic terminal
— P type (smaller construction to NT release - cerebellum):
Slow inactivation
Presynaptic terminal
Purkinje cell dendrites

Low threshold (Low voltage activated; LVA):
T type
Fast inactivation
Inactivated at depolarized voltages

Calcium channels in intracellular organelles - IP3 open these channels and endoplasmic reticulum release intracellular Ca stores
- IP3 receptors
- Ryanodine receptors: calcium-gated ion channels that release calcium from endoplasmic reticulum (ER)

Evidence for Importance of Calcium in Neurotransmitter Release:
– Gain of function experiment: adding protein of interest to a cell that does not normally express that
– Loss of function experiment: remove a protein and see how target functions - knockout of VG Ca Channel so see no NT release in pre or post but normally will release NT in pre and be slightly delayed in post

Question 21

Diseases related to Calcium Channel Dysfunction: Lambert Eaton Myasthenic Syndrome

Etiology: ?

Signs/Symptoms: ?

Other Characteristics: ?

Treatment: ?

Answer 21

Diseases related to Calcium Channel Dysfunction: Lambert Eaton Myasthenic Syndrome

Etiology: Antibodies against presynaptic Ca channels at NMJ (neuromuscular junction)
Results in decreased Ach release following AP invasion of terminal
Decreased ACh

Signs/Symptoms: Muscle weakness that improves with repeated use

Other Characteristics:
Associated with malignancy occurring as paraneoplastic syndrome (small cell carcinoma)

Treatment:
3,4 – diaminopyridine: Lengthens time it takes to repolarize the membrane - prolongs axon term depo likelihood increase that VG Ca channels will open so increase likelihood of ACH release
Thereby increasing Ca2+ influx, leading to enhancement of ACh release

Question 22

In-Class Question:
What component of vesicle machinery acts as the calcium sensor?

What are some consequences of dysfunction in this specific protein?

Answer 22

What component of vesicle machinery acts as the calcium sensor? Synaptotagmin

What are some consequences of dysfunction in this specific protein? NT release issue like reduced synchrony and issues with recycling of vesicles

Question 23

Synaptic Machinery: Synaptic Fusion Complex

SNARE proteins: SNAP REceptors ??

Vesicular components (v-SNARE): ? types?

Membrane components (t-SNAREs):? type?

Calcium Sensor: ? type?

SNARE proteins: Synaptobrevin/VAMP2, SNAP25, and Syntaxin
– Conserved domains of ?
– Complex formation is accompanied by ?
– Extracellular loop of ?

Answer 23

SNARE proteins: SNAP REceptors
Mediate the fusion of vesicles with the target membrane to mediate exocytosis

Vesicular components (v-SNARE): stuck into the membrane of the vesicle — Synaptobrevin/VAMP

Membrane components (t-SNAREs): stuck in the axon terminal membrane at active zone — Syntaxin, SNAP-25

Calcium Sensor: stuck into membrane of our vesicle — Synaptotagmin

SNARE proteins: Synaptobrevin/VAMP2, SNAP25, and Syntaxin:
– Conserved domains of v- and t-SNAREs close to the membrane anchor bind each other.
– Complex formation is accompanied by changes in the protein structure which probably drives membrane fusion.
– Extracellular loop of VAMP

Question 24

Synaptic Machinery:
Calcium Sensor

Calcium sensors and their spatial coupling to VGCCs are crucial for WHAT - ?

Synaptotagmin:
- Multiple copies on ?
- Binds to a total of ?
- Has two domains?

Calcium binding changes its conformation and results in interactions with ?

All of this leads to fusion with ?

When calcium (Ca2+) binds to synaptotagmin, the protein undergoes a conformational change that triggers the release of neurotransmitters:
C2A domain: ?
C2B domain: ?

Exocytosis - release of vesicular contents to the exterior of the cell and release of NT in synaptic cleft:
– Zippering model for ?
– Three helices anchored in ?
– Inward force vector generated?

Answer 24

Calcium sensors and their spatial coupling to VGCCs are crucial for exocytosis - located nearby and Ca 2+ concentration drop rapidly over a few dozen nm from an open VG Ca channel and we need a Ca 2+ sensory nearby in order to respond

Synaptotagmin:
- Multiple copies on each vesicle surface
- Binds to a total of five calcium ions
- Has two domains, C2A and C2B

Calcium binding changes its conformation and results in interactions with SNARE proteins, and other proteins in the active zone - movement of vesicle

All of this leads to fusion with the membrane

When calcium (Ca2+) binds to synaptotagmin, the protein undergoes a conformational change that triggers the release of neurotransmitters:
C2A domain: Changes from an interaction with the synaptic vesicle (SV) membrane to an upright orientation at the vesicle membrane
C2B domain: Associates with the plasma membrane and enhances its interactions with the membrane

Exocytosis - release of vesicular contents to the exterior of the cell and release of NT in synaptic cleft:
– Zippering model for SNARE-catalyzed membrane fusion
– Three helices anchored in one membrane assemble with the fourth helix anchored to the vesicle to form trans-SNARE complexes
– Inward force vector generated that pulls the bilayers together

Question 25

Synaptic Machinery:

SNARE Proteins are targets of neurotoxins:
– Neurotoxins generally?
– Many neurotoxins are now widely used in ?

Tetanus toxin (product of Clostridium tetani ):
Causes muscle rigidity and spasms – ?

Mechanism:
- Taken up in lower motor neurons and transported retrogradely to ?
- Moves trans-synaptically into inhibitory nerve terminals, where it cleaves ?

Answer 25

SNARE Proteins are targets of neurotoxins:
– Neurotoxins generally inhibit or promote the release of neurotransmitters or bind to receptors that are located in the pre- or postsynaptic membranes
– Many neurotoxins are now widely used in clinical treatment and have demonstrated good therapeutic outcomes

Tetanus toxin (product of Clostridium tetani ):
Causes muscle rigidity and spasms – Often manifesting as painful contractions of jaw and neck (lock jaw), difficulty swallowing (dysphagia), severe hyperextension and spasticity of back, or rigidity and spasms of respiratory, laryngeal, and abdominal muscles, which could cause respiratory failure.

Mechanism:
- Taken up in lower motor neurons and transported retrogradely to the spinal cord and/or brainstem. - dynein
- Moves trans-synaptically into inhibitory nerve terminals, where it cleaves synaptobrevin/vesicle-associated membrane protein—VAMP-2, leading to disinhibition of lower motor neurons. - VAMPs from synapse to synapse

Question 26

Synaptic Machinery:

In-Class Question:

What is Botulinum toxin?

What are possible ways you might encounter it? (Think: food, cosmetics, medicine).

What is the mechanism of Botulinum toxin’s effects at synapses?

What is a neurotoxin that affects synaptic machinery that INCREASES the release of neurotransmitters?

Answer 26

What is Botulinum toxin? Bacterial toxin clostridium botulinum. Causes difficulty swallowing or speaking, weakness, and paralysis.

What are possible ways you might encounter it? (Think: food, cosmetics, medicine). Food like canned goods or botox like cosmetic or migraines

What is the mechanism of Botulinum toxin’s effects at synapses? Blocks ACH at NMJ and LMNS stays at nerve terminal and cleave syntaxin or SNAP-2s

What is a neurotoxin that affects synaptic machinery that INCREASES the release of neurotransmitters? Latrotoxin black widow cause increase in Ca 2+ and increase in synaptotagmin activity

Question 27

Synaptic Machinery:

Full Fusion vs. Kiss and Run:

Full fusion - WHICH VESICLE?
- Vesicle fully fuses and collapses into?
- Retrieved by ?
- Releases all ?

Kiss and run - WHICH VESICLE?
- Vesicle docks?
- Can release different amounts of?
- Vesicle can be ?

Post-synaptic responses of different release type: What is a quanta of neurotransmitters? - mini EPP=?

NTs are released into a synapse in packaged vesicles called quanta. One quantum generates a miniature end plate potential (mEPP), the ?

The size of mEPPs differs depending on the ?

Electron microscopy - calculation diff scenarios for like model neuron, stimulated ?

Answer 27

Full fusion - LDVC only undergo full fusion and clear core can also under go and not done under every condition bc requires intense stimulation and increase Ca 2+ influx so increase likelihood of full fusion
- Vesicle fully fuses and collapses into the plasma membrane
- Retrieved by a clathrin-coat dependent process
- Releases all NT – useful for large events but less energetically efficient

Kiss and run - clear core vesicles
- Vesicle docks to the membrane, transiently fuses to the presynaptic membrane and releases NTs
- Can release different amounts of NT, smaller amounts of NT can be used
- Vesicle can be reused

Post-synaptic responses of different release type: What is a quanta of neurotransmitters? - mini EPP=one vesicle of NT being released

NTs are released into a synapse in packaged vesicles called quanta. One quantum generates a miniature end plate potential (mEPP), the smallest amount of stimulation that one neuron can send to another neuron

The size of mEPPs differs depending on the amount of NT released, larger EPSC amplitude with full fusion → Full fusion mEPPs are larger than Kiss and Run mEPPs

Electron microscopy - calculation diff scenarios for like model neuron, stimulated mEPSC

Question 28

Synaptic Recycling: Models of Synaptic Recycling

Kiss and Run Model:
Vesicles transiently ?
After NT release the fusion pore is?

Clathrin-mediated Endocytosis:
A vesicle fuses and ?
A new vesicle that is ?

Ultrafast Endocytosis Model:
After a rapid internalization ?

Answer 28

Kiss and Run Model:
Vesicles transiently(for a very short time or temporarily) fuse with the plasma membrane
After NT release the fusion pore is closed and vesicles are recovered

Clathrin-mediated Endocytosis:
A vesicle fuses and collapses into the membrane
A new vesicle that is empty is formed in a region distant from fusion site

Ultrafast Endocytosis Model:
After a rapid internalization of the membrane via ultrafast endocytosis we deliver vesicle membrane to an endosome and clathrin mediated budding odd of endsome to more new vesicles

Question 29

In-Class Question: Experimental design: 10Hz field stimulation for 2 min Looking at the graph below of traces from two different presynaptic neurons releasing NT, which of events appear to be Kiss and run? Which looks like full fusion? What do you predict the ratio of Kiss and Run to Full fusion is over the course of the 2 minutes?

Answer 29

Over time axon term becomes more depo and more Ca2+ enters full fusion more likely - equal likelihood of either

Question 30

Neurotransmitter - Chemical that allows the movement of information from one neuron to an adjacent neuron

Must meet following criteria:
Synthesis: ?
Localization: ?
Release: ?
Inactivation: ?
Pharmacology: ?

Small Molecule Neurotransmitters: ?
Amino Acids: ?
Biogenic Amines: ?
Purines: ?
Peptide Neurotransmitters: ?

Answer 30

Synthesis: the molecule is synthesized in the presynaptic neurons
Localization: the molecule is present in the presynaptic terminal
Release: the molecule is released upon stimulation of the presynaptic neuron
Inactivation: a specific mechanism or a set thereof exists to remove the molecule from the synaptic cleft or to degrade it
Pharmacology: when applied exogenously (e.g., from a micropipette), in concentrations similar to those observed following stimulation of the presynaptic cell, the molecule (agonist) mimics the action of the endogenously released transmitter; Antagonists will block effect.

Small Molecule Neurotransmitters: Acetylcholine
Amino Acids: Glutamate, Glycine, GABA
Biogenic Amines: Serotonin, Dopamine, Norepinephrine, Epinephrine, Histamine
Purines: Adenosine, ATP
Peptide Neurotransmitters: Neuropeptide Y, Substance P, Enkephalin etc

Question 31

Postsynaptic Neuron: Receptors and Postsynaptic Potentials - Postsynaptic potentials:

So what is the nature of information transfer at synapses?
Same-signed (“excitatory”): ?
Opposite-signed (“inhibitory”): ?
Neurotransmitters can be “excitatory” or “inhibitory,” depending upon ?
Example: glutamate?

Potential ionic bases for same-signed and opposite-signed information transfer

Same-signed EPSP:
Ina
Ica
Ik

Opposite-signed IPSP:
Ina
Ica
Ik

Answer 31

Same-signed (“excitatory”): AP in presynaptic neuron leads to an increase in APs in the postsynaptic neuron. Elicit depo of the postsynaptic cell.

Opposite-signed (“inhibitory”): AP in presynaptic neuron leads to a decrease in APs in the postsynaptic neuron.

Neurotransmitters can be “excitatory” or “inhibitory,” depending upon the postsynaptic receptor mechanisms (more on that later).
Example: glutamate is primarily excitatory and NMDAR AMPAR inotropic NT receptors
mGluR inhibitory and on-bipolar cells in retina

Same-signed EPSP:
Depolarize cell; make Vm less negative; cell gets closer to threshold, so greater probability of firing AP.
Increase Ina, Increase Ica
Decrease Ik

Opposite-signed IPSP:
Hyperpolarize cell; make Vm more negative; cell gets farther from threshold, so lower probability of firing AP
Decrease Ina, Decrease Ica
Increase Ik
Increase Icl

Ina: Represents the inward sodium current ( 𝐼 Na I Na ​ ), which involves the flow of sodium ions ( 𝑁 𝑎 + Na + ) into the neuron. An increase in 𝐼 Na I Na ​ leads to depolarization, bringing the cell’s membrane potential closer to the threshold for firing an action potential.

Ica: Refers to the inward calcium current ( 𝐼 Ca I Ca ​ ), which involves the flow of calcium ions ( 𝐶 𝑎 2 + Ca 2+ ) into the neuron. An increase in 𝐼 Ca I Ca ​ also contributes to depolarization and can support action potential firing, as well as triggering other cellular processes.

Ik: Stands for the outward potassium current ( 𝐼 K I K ​ ), involving potassium ions ( 𝐾 + K + ) leaving the cell. When 𝐼 K I K ​ increases, it generally causes hyperpolarization (making the membrane potential more negative) and moves the cell away from the action potential threshold.

In summary, increasing 𝐼 Na I Na ​ or 𝐼 Ca I Ca ​ tends to make the cell more likely to fire (excitatory), while increasing 𝐼 K I K ​ tends to make it less likely (inhibitory).

Question 32

Postsynaptic Neuron: Receptors and Postsynaptic Potentials - Postsynaptic potentials:

Classical example of same-signed information transfer: the neuromuscular junction (NMJ)
– AP in motor neuron axon produces AP and then?
– End plate potentials?
– At NMJ only ?

Same Neurotransmitter – Different Effect: Metabotropic acetylcholine receptor
- Highly expressed in WHICH Nervous System
Postganglionic parasympathetic neurons release NT onto ?
- M2-type is ?
- Muscarinic Synapse
Inhibitory M2-type: hyperpolarization of K leaves cell and reduce activity of ?
Excitatory M1/M3 type: inducing release of?

Classical example of opposite-signed information transfer: Metabotropic opioid receptors
Binding of mu-opioid activates WHICH proteins:
– Closes WHICH channel
– Opens WHICH channel
– Leads to??

Answer 32

Classical example of same-signed information transfer: the neuromuscular junction (NMJ)
– AP in motor neuron axon produces AP and then contraction in muscle - ACH nicotinic ACH receptors ionotropic excitatory
– End plate potentials – graded postsynaptic potentials due to transmitter/receptor/ channel - same as an EPSP happening in the CNS
– At NMJ only nicotinic ACH receptors

Same Neurotransmitter – Different Effect: Metabotropic acetylcholine receptor
- Highly expressed in Autonomic Nervous System
Postganglionic parasympathetic neurons release NT onto muscarinic receptors located on the target organ
- M2-type is Gi coupled
Inhibitory postsynaptic effects
- Muscarinic Synapse
Inhibitory M2-type: hyperpolarization of K leaves cell and reduce activity of ACH reducing levels of CAMP cases reduction of phosphorylation also Gi g-protein open K channels
Excitatory M1/M3 type: inducing release of Ca from ER

Classical example of opposite-signed information transfer: Metabotropic opioid receptors
Binding of mu-opioid activates g-proteins - Gi/o family of g-proteins:
– Closes VG Calcium channel
– Opens Potassium channel
– Leads to hyperpolarization of the cell reduction of likelihood of sending AP

Question 33

In-Class Question: What is the smallest amount of response that you can measure from a postsynaptic neuron? (What is it called?) mini end plate potential mEPPS. Research: Can vesicle release be spontaneous? Why or why not?

Answer 33

Yes bc neurons cultured without astrocytes are electrically silent and non mini EPSPS

Question 34

mEPPS: How did we discover individual vesicle release?

Katz recorded from the neuromuscular junction. He upped the gain of the recording device and saw an interesting random phenomenon. There were spontaneous, small depolarizations happening in the postsynaptic cell.

These small deflections had interesting properties:

First: they occurred in the ?

Second: they were small with an average amplitude of about 0.5 mV. The distribution could be fit by a single?

Third: these events could only be recorded in the vicinity of the synaptic junction.

Fourth: they were blocked by curare = ?

Fifth: they were enhanced by?

Sixth: their frequency was sensitive to ?

Answer 34

Katz recorded from the neuromuscular junction. He upped the gain of the recording device and saw an interesting random phenomenon. There were spontaneous, small depolarizations happening in the postsynaptic cell.

These small deflections had interesting properties:

First: they occurred in the absence of any stimulus.

Second: they were small with an average amplitude of about 0.5 mV. The distribution could be fit by a single gaussian function, indicating that the events arose from a common underlying process. - the average size was always the same so very little variability

Third: these events could only be recorded in the vicinity of the synaptic junction.

Fourth: they were blocked by curare = nicotinic ACHR antagonist. Events are mediated by NT receptor binding and record from areas of post cell close to NMJ these depo didn’t ravel far

Fifth: they were enhanced by neostigmine - increases the frequency of miniature end-plate potentials

Sixth: their frequency was sensitive to basal levels of intracellular Ca2+ - neostigmine inhibits ACH and increase amounts of time ACH can bind to receptor

Effect
When neostigmine is applied through a pressure ejector, mEPPs appear at high frequencies during the plateau, repolarization phase, and a period after the sustained depolarization (SD).

Mechanism
Neostigmine is an anticholinesterase that increases the amount of acetylcholine in the synapse. This allows more acetylcholine to bind to the receptors in myasthenia gravis, which helps trigger muscular contraction.

Question 35

mEPPS: Quantal Hypothesis: MEPPs are the building blocks of EPPs

mEPPs: ?

EPPs: ?

Why do we discuss this concept using EPP instead of EPSP?

Quantal size = size of 1 quanta (a.k.a. vesicle), measured as ?

Quantal content = ?

QC = EQUATION

Answer 35

mEPPs: Single, spontaneous events (mEPSP in CNS) - release of one vesicle of NT

EPPs: Action potential dependent – evoked - synaptic potentials (EPSPs in CNS) - AP happens in presynaptic neuron and many NT vesicles being release

Why do we discuss this concept using EPP instead of EPSP?
This was discovered at the NMJ first, so vocabulary has continued – also the NMJ is a great synapse to study this at due to its location in the periphery

Quantal size = size of 1 quanta (a.k.a. vesicle), measured as the smallest postsynaptic depolarization (mEPP) or the post-synaptic response to the release of a single vesicle. - 1 NT filled vesicle= a quanta

Quantal content = number of quanta (a.k.a. vesicles) that are released to make up the end-plate potential (EPP) - quantal content of 5=5 vesicles of NT released

QC = EPP/mEPP - N 0.4-0.5
Example: 2mv/0.5mv= QC of 4

MEPPs are small, with an amplitude of about 0.5 mV, while EPPs have an amplitude of about 50 mV. MEPPs are caused by the random release of a single quantum of neurotransmitter, while EPPs are produced by the summation of many MEPPs. MEPPs occur randomly, not rhythmically
Threshold: A normal EPP causes the postsynaptic neuron to reach its threshold of excitation and elicit an action potential.
Vesicles: MEPPs are caused by the release of a few presynaptic vesicles containing acetylcholine.

Question 36

mEPPs are spontaneous
At other synapses (nonNMJ) they can be called?
Quantum of NT?

EPPs are evoked
Similarly, EPSPs are evoked
Multiple vesicles of ?
Can be subthreshold or can reach?

Distribution of EPP size: Statistical analysis of mEPPs supports the hypothesis that they represent the fundamental electrical unit of ?
– Quantized nature of EPPs WHICH units
– 0.4 mV = ?

How much ACh binds per quanta?

Independent occurrence of unitary events:
Distribution predicted by Poisson statistics, Poisson distribution provides ?

Perturbing mEPPs vs. EPPs size:
Extracellular calcium concentration?

Postsynaptic receptor number?

Amount of NT packaged in each vesicle?

Botulinum toxin?

Tetrodotoxin (TTX)?

Answer 36

mEPPs are spontaneous
At other synapses (nonNMJ) they can be called mEPSPs
Quantum of NT is released

EPPs are evoked
Similarly, EPSPs are evoked
Multiple vesicles of NT released
Can be subthreshold or can reach threshold(-55mv) - less than 15 mv depo

Distribution of EPP size: Statistical analysis of mEPPs supports the hypothesis that they represent the fundamental electrical unit of synaptic communication
– Quantized nature of EPPs (0.4 mv) units
– 0.4 mV = one mEPP = one vesicle of NT

How much ACh binds per quanta?
1000s – each ACh binding would lead to 0.4 uV of depo

Independent occurrence of unitary events:
Distribution predicted by Poisson statistics, Poisson distribution provides a good estimate of the observed EPP amplitude distribution

Perturbing mEPPs vs. EPPs size:

Extracellular calcium concentration? Inracese Ca 2+ concentration. No change in mEPPs size and EPP likely increase in amplitude

Postsynaptic receptor number? Increase mEPPs size and increase EPP size

Amount of NT packaged in each vesicle? Increase mEPPs size and increase EPP size

Botulinum toxin? Inhibit synaptic machinery. Size of mEPPs will be same and EPPs decrease so likelihood of either is reduced

Tetrodotoxin (TTX)? Inhibit VG Na channel. No effect on mEPPs or EPPs size