Module 1 Flashcards
“The Selfish Organ”
The brain consumes about 20% of the total bodily energy, leading it to get this nickname.
Human Genome Distribution to the Brain
Of the 20,000 total genes in the human genome, 14,000 of them are expressed in the brain, and 6,000 of them are unique to the brain showing its importance.
Ionic concentrations of blood
[Na+] = 145 mM
[K+] = 5 mM
[Ca2+] = 2 mM
These concentrations are meant to mimic those of seawater, as the blood became responsible for maintaining the appropriate ion gradients when life moved from the sea to land.
Visceral Motor System
The component of the motor system responsible for involuntary changes in the body.
This system acts on smooth muscles, cardiac muscles and glands.
Somatic motor system
The component of the motor system that controls motor nerves.
This system acts on skeletal muscles
Types of glia
1) Oligodendrocytes
2) Astrocytes
3) Microglia
4) Schwann Cells
Distribution of cell types in the brain
The brain is 10% neurons and 90% glial cells but by volume they’re about 50/50.
Space is 85% cells and 15% extracellular
What can be found inside a synaptic button?
Mitochondria and vesicles filled with neurotransmitters which contain the information that should be conveyed.
Axo-dendritic
Connections that have spines (excitatory) and shafts (inhibitory) where the synaptic buttons of the presynaptic cell attach to the shafts and spines of the dendrite of the postsynaptic cell.
Axo-somatic
Inhibitory connections that involve the terminal buttons of the presynaptic neuron connecting directly to the cell body of the postsynaptic neuron.
Axo-axonic
Modulatory connections that involve the synaptic button of the presynaptic neuron attaching to the axon of the postsynaptic neuron.
Dendro-dendritic
Very rare connections that involve the dendrite of the presynaptic cell attaching to the dendrite of the postsynaptic cell.
Actin filaments
6 nm diameter
Function: Structure, spines, growth cone
Microtubules
25 nm diameter- tubulin
Function: Movement of cargo
Neurofilaments
10 nm diameter, intermediate filaments
Function: Structure
Anteretrograde
Movement within the axon that goes from the cell body to the nerve terminal which is associated with axonal growth and delivery of synaptic vesicles.
Retrograde
Movement within the axon that goes from the axonal terminal to the cell body and involves “old” and “worn out” proteins and membranes which are transported to the lysozyme for degredation
Microtubule transport and polarity
Transport occurs along microtubules which are oriented in the + direction towards the distal end of the nerve
Kinesins
Anteretrograde axonal transport motor proteins that are due to polarity in the microtubules in axons which move from the - to the + end of the microtubule.
Dyneins
Retrograde axonal transport proteins which move from the + to the - end.
How do glia differ from neurons?
1) Do not form synapses
2) Have essentially only one type of process
3) Retain the ability to divide
4) Are less electrically excitable
Astrocytes
Glia that are mostly responsible for structural support and guidance. They also control K+ concentrations, regulate metabolism, and control transmitter concentrations in the glutamine-glutamate cycle.
Oligodendrocytes and Schwann Cells
Both of these glial cells insulate the axons of nerve cells.
Oligodendrocytes function in the CNS and myelinate approximately 50 neurons.
Schwann cells function in the PNS and myelinate only a single neuron.
Microglia
Glial cells that aren’t really glia because they are derived from and are part of the immune system. They are responsible for phagocytosis and are activated by tissue insult.
fMRI
A crude brain imaging technique which is based on the increase in blood flow to the local vasculature that accompanies neural activity in the brain.
fMRI is noninvasive and can record signals from all regions of the brain.
It is important to note that although blood oxygenation is linked to neural activity it is NOT a direct measure of neural activity.
Membrane potential
The electrical voltage different across the membrane which is due to differences in sodium and potassium ion concentration in and out of the cell.
Squid Giant Axon
The squid has an axon that has a diameter about 400x times larger than other mammalian axons (it’s 1 mm) as part of its neural circuitry to escape when a predator is nearby.
Studies of the giant squid axon have been essential to our modern understanding of how neurons function, as its large size has made a number of experiments possible that would not otherwise have been developed.
Voltage Clamp Technique
This technique is used to run tests about the membrane potential across an axonal membrane under certain conditions.
It involves putting an electrode in one end of an axon and a current-passing electrode in the other end. In this way, the current-passing electrode (Vc) can be used to maintain the voltage of the axon at a certain level and the membrane current (Vm) can be measured to see what the cell must do to move toward maintaining that voltage.
In this technique you clamp the voltage so that the voltage is constant and the current is altered and can be measured.
Passive electrical response
This is a response that almost any cell in the body would have to an electrical stimulant. The membrane potential merely moves momentarily in that direction and then returns to its normal levels.
C. Elegans
C. Elegans is a good model organism because it has a very rapid life cycle and a translucent body.
Each worm has only 959 cells and 302 are neurons. Each neuron has been ablated and the resulting behavior has been recorded to establish the purpose of each cell.
Fruit fly scientific name
Drosophilia melanogaster
Mouse scientific name
Mus Musculus
Knock-out approach
An approach to model organisms that involves delete a specific gene of interest and observing changes in phenotype.
Knock-in approach
An approach to model organism research that involves replacing a specific gene of interest with an exogenous gene.
Transgenic approach
An approach to model organism research that involves inserting a copy of an exogenous gene into the genome under control of the regulatory elements of a specific gene of interest.
Genetic tools researchers have when experimenting with model organisms
1) Labeling select neurons
2) Identifying neural networks
3) Activating neurons within a network
4) Inhibiting neurons within a network
Brainbow
A technique used in mapping brain circuits which labels each cell type with a different fluorescent reporter so that individual circuits can be followed distinctly from neighboring ones. This allows us to differentiate cells and follow individual circuits which was impossible before this approach.
Calcium imaging
A genetically encoded calcium sensor where a green fluorescent protein is fused to a calcium binding protein
Optogenetics
A technique developed that uses light to control behavior and activity of an organisms by exciting or inhibiting different neurons when specific wavelengths of light are exposed to the organism.
Channelrhodopsin activates neurons (blue light)
Halorhodopsin inhibits neurons (yellow light)
Relative levels of K+ and Na+ in and outside the cell
More K+ INSIDE the cell
More Na+ OUTSIDE the cell
Voltage
The potential to do work
Current
The flow of positively or negatively charged ions
Resistance
Something that impedes the flow of electrons
Ohms Law
V= IR
Hyperpolarized
More negatively charged (downward deflection of the voltage trace)
Depolarized
More positively charged (upward deflectino of the voltage trace)
Concentration Gradient
The gradient generated based on the principal of entropy by which ions want to move from the area of high concentration to that of low concentration.
Electrical Gradient
The gradient generated by the desire to stabilize electrical charges across the membrane.
Sodium’s equilibrium potential
+60 mv
Potassium’s equilibrium potential
-90 mV
Nernst Equation
The equation that allows us to calculate the equilibrium potential for each ion in equilibrium
(See notes for exact equation)
Key factors in resting potential determination
Resting potential is set by non-gated K+ channels (“leak” or “open” channels).
Note: These are distinct from the voltage-gated K+ channels that participate in the action potential.
What causes an action potential?
Action potentials arise from differential permeability to ions.
Equilibrium potential of a neuron
-60 mV
What does the GHK in GHK equation stand for?
Goldman-Hodkin and Katz
Rising Phase
This is the part of the action potential in which voltage-gated Na+ channels open. It is characterized by a sharp rise in the membrane potential toward the equilibrium potential of Na+, 40 mV.
Overshoot phase
The peak of the action potential before it falls.
Falling phase
This part of the action potential is characterized first by voltage-gated K+ channels opening and then by Na+ channels closing and inactivating.
Undershoot
This final part of the action potential goes below the resting membrane potential because voltage-gated K+ channels are still open but the Na+ channels remain inactive, so the membrane potential shoots toward the K+ equilibrium potential. This quickly goes back to resting potential.
This undershoot is part of the reason why action potentials will not propagate backward down an axon.
Capacitative Current
Capacitative current is the movement of charge near the membrane. At this point there is no ion flow across the membrane, the ions simply arrange themselves on either side of the membrane to prepare for movement across the membrane.
What are the two different types of voltage-dependent currents?
Early and late currents. We now know that the early current is caused by voltage-gated sodium channels and the late is caused by voltage-gated potassium channels.
Characteristics of the early current
Fast depolarization but then quickly back to resting membrane potential.
Characteristics of late current
Slowly builds up but lasts much longer than the early current.
What are the three-channel types in the axon of a neuron?
1) Non-gated K+
2) Voltage-gated Na+
3) Voltage-gated K+
Refractory Period
This refers to the period of time after the initiation of one action potential when it is impossible to initiate a second action potential no matter how much the cell is depolarized.
Why does voltage dissipate when it passively flows down an axon?
1) The cytoplasm has high resistance and is a poor conductor.
2) The membrane is not totally impermeable and some charge (ions) is lost due to leak.
Length constant
An indication of how far a potential will spread along an axon in response to subthreshold stimuli.
The larger the length constant, the more passive charge flow– we want a larger length constant.
It is dependent on the resistance of the intracellular cytoplasm, the resistance of the extracellular fluid and the resistance across the membrane.
Propagation is faster when the length constant is larger.
Represents until 2/3 of the signal decays, so there’s still enough to depolarize the threshold.
Time constant
A number that determines the time course for the change in membrane potential and is dependent on the membrane resistance and membrane capacitance.
Propagation is faster when the time constant is smaller.
Two ways to increase velocity of current
1) Increasing the diameter of the axon
2) Increasing membrane resistance (myelination)
Nodes of Ranvier
The space between the myelin sheaths were Na+ can get in and K+ can get out to start a new action potential.
Saltatory conduction
This refers to the way that an action potential hops along an axon when the axon is myelinated. The action potential is generated at each Node of Ranvier.
Negative current
The movement of positive ions into the cell or negative ions out of the cell.
Tetrodotoxin
A neurotoxin that blocks K+ ions and is often used in research to see the effects of blocking K+.
Conductance
The amount of ions crossing a membrane.
Ions that ion channels are generally selective for
K+, Na+, Ca2+ or Cl-
Types of ligand gated channels
1) Neurotransmitter receptor
2) Ca2+ activated K+ channel
3) Cyclic nucleotide gated channel
Three sources of energy for transporters
Electrochemical, concentration or electrical potential
Two types of ATPase Pumps
1) Sodium potassium pump
2) Calcium pump (coupled with H+ gradient)
Electrogenic
A term used to describe the fact that the sodium/potassium pump creates a net positive charge across a membrane leaving the cell negatively charged on the inside.
Ratio of sodium to potassium ions moved by the sodium/potassium pump
3 Na+ move out for every 2 K+ moved in
Uniporters
Transporters that transport single molecules down a gradient (glucose, amino acids).
Symporters
Transporters that couple tow molecules moving the same direction to move one against its concentration gradient and one down its gradient.
Antiporters
Transportesr that couple two molecules moving in opposite directions to move one against its concentration gradient and the other down its gradient.
Functions of transporters
1) Maintain ion gradient in vesicles
2) Take neurotransmitters and their precursors into the nerve or a vesicle via a transporter.
Transporters that move neurotransmitters and their precursors into a nerve or vesicle
1) Excitatory amino acid transporter (EAAT)
2) Vesicular Glutamate Transporter (VGLUT)
3) Vesicular Inhibitory Amino Acid Transporter (VIATT)
4) Glycine Transporter
Topology of a voltage-gated sodium channel
4 homologous domains each of which is made of 6 transmembrane domains and 1 P-segment.
Only one inactivating domain exists between the III and IV domains.
1 gene = Entire tetramer (4 units w/6 transmembrane domains)
There is a beta subunit on either side of the protein.
Consists of repeating motifs of six membrane-spanning regions related four times for a total of 24 transmembrane regions.
Topology of a voltage-gated potassium channel
6 transmembrane domains and 1 P-segment. The positive charges of the S4 act as the voltage sensor.
The n-terminal domain is the inactivating segment.
1 gene = 1 subunit with 6 transmembrane domains
One subunit made of 6 transmembrane components and a beta subunit on the intracellular side.
T = rm * cm
T = time for change in potential
rm = membrane resistance
cm = membrane capacitance
Decrease T to increase propagation speed
S4 domains
These components of voltage-gated ion channels are thought to be the voltage-sensors that make the receptors voltage-sensitive because they sense when positive charge shifts from one side of the membrane to the other.
Arginine is the positively charged amino acid that is used to sense these changes in voltage.
Oubain
A neurotoxin that blocks Sodium-Potassium ATPase.
When this is used, the resting potential degenerates slowly, showing that this pump is essential in maintaing the resting potential of a neuron.
How voltage is sensed across the membrane
The positively charged alpha helices sense the change in voltage and move toward the extracellular surface of the membrane which opens the gate.
Mechanisms of inactivation of voltage-gated Na+ channels
Inactivation is critical to the refractory period of the action potential.
The inactivation domain (a globular protein) plugs the pore of the channel (a ball attached to a chain) and remains there for a few ms so that nothing can pass through even if the pore is opened.
The length of the chain affects time until inactivation. A longer chain means longer time the channels are open. This can be manipulated by scientists on the genetic level.
What can we learn from the shaker channel in fruit flies?
Flies with a mutation in the K+ channel shake uncontrollably.
These mutant voltage-gated K+ channels have part of the amino acid terminus missing so the currents do not inactivate.
If a synthetic peptide corresponding to the N-terminus is added, the mutant channels do inactivate. When the chain is manipulated and shortened inactivation happens more quickly.
This all supports the current theories about how voltage-gated channels work.
How does ion channel selectivity work on the molecular basis (K+ channels specifically)?
The 4 p-segments from each part of the tetramer make up a selectivity filter that only allows specific ions to pass through the pore.
The ions enter the pore and become dehydrated and bind perfectly with the 8 carbonyl oxygens (2 from each p-segment). Dehydrated Na+ can not bind to the 8 carbonyl oxygens because it is too small and it is thus energetically unfavorable for it to pass through.
This being said, potassium is only 1,000 times more favorable than sodium, so for every 1,000 potassium ions that pass through the channel, one sodium ion does as well.
Evidence that the p segment controls selectivity in K+ channels
1) The Gly-Tyr-Gly sequence is similar amongst all K+ channels.
2) Mutations in the pore disrupt selectivity.
3) Species replacement does not alter selectivity.
How do ions pass through an ion channel?
Ions are always in position 1 and 3 or 2 and 4 (space in between). As a new ion moves into the pore, it bumps the other ions into the next position. The ions rehydrate themselves once they leave the channel, picking up 8 waters.
Thus, there are two states the channel can be in. In the first the ions are in positions 1 and 3 with the ion outside the channel holding 8 water molecules, and state 2 with ions in positions 2 and 4, and the ion outside the filter holding 4 water molecules as it moves toward in the inside of the ion channel.
Channelopathies
Genetic diseases that are a result of altered channel function.
Channelopathies that result from altered sodium channel function.
1) Myotonia: Excessive neural activity
2) Paralysis: Decreased activity
3) Generalized epilepsy with febrile seizures
4) Congenital insensitivity to pain
5) Paroxysmal extreme pain disorder (PEPD)
6) Erythermalgia
Channelopathies that result from altered calcium channel function
1) Familial hemiplegic migraine- several mutations in Ca2+ channels with no clear mechanism
2) Episodic ataxia type 2- abnormal limb movements and ataxia (can’t balance or walk well), channels are truncated
3) Night blindness (CSNB)- nonfunctional photoreceptors
Channelopathies that result from altered potassium channel function
1) Benign familial neonatal convulsion- K+ channel mutation which leads to hyperexcitability
2) Episodic ataxia 1
Channelopathies that result from altered sodium channel function
1) Myotonia- muscle contraction
Advantages of K+ channels being made of multiple subunits from multiple genes
Allows for more diversity and ability of the potassium channels.
Current Clamp Technique
An experimental technique in which nothing is clamped, but we just record the current
Rod MacKinnon
The scientist who won the Nobel Prize in 2003 for his work crystallizing the voltage-gated K+ channel and settling once and for all what the structure of it is.
Relationship between active transporters and ion channels
Active transporters create the concentration gradients that help drive ion fluxes through open ion channels
Properties that Hodgkin and Huxley determined that all ion channels must have
1) Capable of allowing ions to move across the membrane at high rates because the currents are quite large
2) Make use of the electrochemical gradient since the currents depend on the electrochemical gradient
3) Different channels types have to be able to discriminate between sodium and potassium, allowing only one of these ions to flow across the membrane under the relevant conditions
4) Sense changes in voltage, activating only when the voltage reaches a certain level
Patch clamping
A technique developed to measure the ion flow out of individual channels.
There are four types (each has it’s own flashcard):
1) Cell-attached patch clamp recording
2) Whole-cell recording
3) Inside-out recording
4) Outside-out recording
Cell-attached patch clamp recording
A patch clamp method that involves putting a recording pipette on a neuronal membrane and creating a seal that’s tight enough that everything the pipette sucks up must be from the single channel (signal is amplified by equipment in the pipette)
Whole-cell recording
A patch clamp method that involves sucking up with a strong pulse from the pipette so that the pipette becomes continuous with the cytoplasm and you can record measurements of electrical potentials and currents from the entire cell.
Inside-out recording
A patch clamp method that involves pulling a patch of the neuronal membrane off so that the intracellular component can be exposed to a different medium while maintaining the seal between the pipette and the membrane.
Outside-out recording
A patch clamp method that involves retracting the pipette while in the whole-cell configuration so that the extracellular membrane is exposed- especially useful when studying the effects of different extracellular conditions.
Microscopic currents
The currents flowing through single channels.
Macroscopic currents
The currents flowing through a large number of channels distributed over an extensive region of surface membrane.
Terodotoxin
A neurotoxin produced by a certain puffer fish and othe animals that process a potetnt and specific obstruction of the Na+ channels responsible for action potential generation, thereby paralyzing the animal who ingests it.
Saxitoxin
A chemical homologue of tetrodotoxin produced by dinoflagelllates which targets and obstructs Na+ channels.
Dendrotoxin
Peptide toxins that affect K+ channels by blocking them.
SCN genes
The 10+ genes in humans that code Na+ channels
CACNA genes
The 10 different calcium channel genes identified in humans.
Selectivity of voltage-gated v. ligand-gated channels
Ligand-gated channels tend to be less selective than voltage-gated channels.
Pore loop
A structure between the two helical membrane-spanning structures of a channel that inserts into the plasma membrane.
Ion movement definition of depolarization and hyperpolarization
Depolarization- movement of positive ions out of the cell
Hyperpolarization- movement of positive ions into the cell.
Gap junction
An intracellular specialization that physically connections two neurons to allow for the passive movement of electrical charge between neurons
Connexons
Precisely aligned, paired channels in a gap junction that allow for the passive movement of electrical charge in electrical synapses.
They’re present in the membranes of both pre and post synaptic neurons.
6 connexons join together to form a pore that connects the two cells.
Connexins
The ion channel proteins that compose connexons.
Different types are found in different cell types to give each gap junction a different physiological purpose.
Special properties of gap junctions
1) Can be bidirectional (some have specifications to block one direction, but in theory they’re all designed to have bidirectional capabilities
2) Very fast- no delay like chemical synapses
3) Synchronized neurons use electrical synapses to ensure that things are happening at essentially the same time.
4) Glial cells use gap junctions a lot to form large intracellular signaling networks to transfer second messengers and other molecules and communicate
Synaptic cleft
The space between the pre and post synaptic neuron which is much bigger in chemical than electrical signaling.
Synaptic Vesicles
Small membrane-bound organelles that are the key feature of all chemical synapses and are located in the presynaptic terminal.
Role of Calcium in synaptic transmission
When an action potential arrives at the end of a presynaptic neuron, it causes a release of calcium which is facilitated by the opening of voltage-gated calcium channels.
This release of calcium allows synaptic vesicles to fuse with the membrane of the presynaptic neuron allowing the contents of the vesicle to be released into the synaptic cleft.
Loewi’s Experiment
Otto Lowei performed an experiment using two cow hearts and the vagus nerve which proved that signals were transmitted on a molecular basis.
He placed one cow heart in a beaker and connected it to another beaker with a second cow heart that shared only the extracellular liquid. When he stimulated the vagus nerve on the first heart (which causes the heartbeat to slow), after a short delay the second heart, which hadn’t been stimulated, began to slow its beating too. This proved that the information was passed via chemical messengers that were in the shared extracellular fluid.
Types of functions small-molecule transmitters and neuropeptides regulate
Neuropeptides regulate slower, ongoing synaptic neuromodulatory functions, whereas small-molecule neurotransmitters mediate rapid synaptic actions.
Co-transmitters
When more than one transmitter is present within a nerve terminal, this term is used to refer to the molecules. The neurotransmitters do NOT have to be released simultaneously.
Slow-axonal Transport
The mechanism used to move enzymes produced in the cell body to the terminal ctyoplasm for small-molecule neurotransmitter synthesis.
Small-molecule neurotransmitter synthesis
Occurs locally within the presynaptic terminal.
Slow-axonal transport brings the necessary ingredients to the end of the axon of the presynaptic cell.
For some small-molecule neurotransmitters the finals steps of synthesis even take place in the synaptic vesicle itself.
They are packed in small clear-core vesicles
Small clear-core vesicles
40-60 nm in diameter
Centers appear clear in electron micrographs
Hold small-molecule neurotransmitters
Neuropeptide synthesis
Synthsized in the cell body of the neuron, so it has to travel far to be secreted.
Uses fast axonal transport to get the neuropeptides to the presynaptic terminal button where final modifications can be made.
Packed in large dense-core vesicle.
Fast axonal transport
The process by which peptide-filled vesicles are transported along an axon and down to the synaptic terminal for secretion.
Involves the use of kinesins for transport
Large dense-core vesicles
90-250 nm in diameter
Centers appear electron-dense in electron micrographs
Hold neuorpeptides
Processes that can take neurotransmitter out of the synaptic cleft
1) Diffusion away from the postsynaptic receptors
2) Reputake into nerve terminals or surrounding glial cells
3) Degredation by specific enzymes
4) Combination thereof
- -> Specific transporter proteins remove most small-molecule neurotransmitters and drive them back to the presynaptic terminal for reuse
Criteria that define a neurotransmitter
1) The substance must be present within the presynaptic neuron. (Some substances are amino acids that are used for protein synthesis and other processes, so their presence alone isn’t enough. Enzymes and precursors that facilitate synthesis must also be present)
2) The substance must be released in response to presynaptic depolarization, and the release must be calcium dependent.
3) Specific receptors must be present on the postsynaptic cell.
Agonist
A substance that facilitates a physiological action (in context = neurotransmitters)
Antagonist
A substance that inhibits or prevents a physiological action (in context = neurotransmitter)
End Plate Potential
An action potential in the presynaptic motor neuron which can be seen to elicit a transient depolarization of the postsynaptic muscle fiber
Miniature EPPs (MEPPs)
Spontaneous changes in the membrane potential that occur from random vesicles fusing with the membrane.
Very small with constant magnitude (-0.4 mV)
Evidence for quantal release of neurotransmitter
Freeze fracture analysis allows us to visualize how many vesicles there are and how many fuse, so when we compare how many vesicles we see fusing with the number of quanta released we see a linear correlation.
Experimental evidence for calcium’s role in transmitter release
1) Microinjection of calcium into presynaptic nerve terminals evokes release in the absence of an AP. (shows calcium is sufficient)
2) Injection of calcium buffer into the presynaptic neuron can block depolarization in postysnaptic neuron. (shows calcium is necessary)
Affect of frequency of stimulation on neurotransmitters released
Small clear-core vesicles are much closer to the synaptic cleft inside the presynaptic neuron, so low frequency stimulation causes the release of small-molecule neurotransmitters.
If the frequency of the stimulation is increased, the calcium diffuses far enough back into the presynaptic neuron to cause the large dense-core vesicles to come to the surface and release neuropeptides.
Experiment used as evidence for synaptic recycling
Horseradish peroxidase is put in the synaptic cleft so that it is taken into the vesicles that are recycled. It is then washed away and the neuron is stimulated again so that any HRP seen in the synaptic cleft is due to what was taken into the synaptic vesicles- did see it proving that the vesicles were recycled.
True or False: There aren’t many proteins involved in the synaptic vesicle cycle.
FALSE!!!! There are a crap ton of them!
Synaptobrevin
A SNARE protein that is in the membrane of synaptic vesicles.
Syntaxin
A SNARE protein found primarily in the plasma membrane that helps form the SNARE complex.
SNAP-25
A SNARE protein found primarily in the plasma membrane that helps form the SNARE complex.
This is what reacts to form the tight structure of the SNARE complex.
SNARE Complex
A protein complex that spans the membrane of the presynaptic cell and the synaptic vesicle to facilitate the fusion of synaptic vesicles.
Synaptotagmin
The calcium sensor for fusion of the SNARE complex.
Binds calcium at concentrations known to evoke fusion.
Mutations in synaptotagmin alter vesicle fusion and deletion is lethal in mice.
This is NOT part of the actual SNARE complex that holds the vesicle and plasma membrane together.
Clatherin
A plasma membrane protein that is critical in the process of endocytosis.
It forms a dome-like structure which confers stability to the vesicle as it is retrieved from the membrane.
Dynamin
The protein that facilitates the final pinching off of the vesicle from the membrane.
Clostridial Toxins
Proteases that cleave presynaptic SNARE proteins.
1) Tetanus toxin
2) Botulinum toxin
Ionotropic Receptor
A receptor that has an ion channel pore.
Metabotropic Receptor
A receptor that is indirectly linked to an ion channel via some signal cascade pathway (G-Protein coupled, etc.) –> requires second messengers
Ligand-gated ion channels
Ionotropic receptors that function quickly.
The signal is intramolecularly coupled to the opening of the ion channel happens immediately producing either depolarization or hyperpolarization
G-Protein Coupled Receptors
- A metabotropic receptor that signals intermolecularly by activating intracellular messengers that open or close ion channels. These intracellular messengers act on ionotropic receptors.
- These receptors funciton mostly in homeostasis and development (gene trasncription)
- Largest family of cell surface receptors (5% of the genes in C. elegans code for them)
- Work via altering the activity of the effector proteins (ion channels, enzymes, etc.)
- Activate trimeric G proteins
True or False: Each neurotransmitter works through one receptor type
False! Neurotransmitters can work through both receptors types to have even opposite effects
General architecture of ligand-gated receptors
Made of a number of subunits each having four transmembrane helices with the N and C terminus both on the extracellular side.
Structure of the nACh receptor/channel
- Pentameric protein that has different subunit composition depending on where it is expressed.
- At the neuromuscular junction two alpha subunits (each binds one ACh), beta gamma and delta subunit.
- Neuronal ACh receptors have 3 alpha subunits and 2 beta
- Non-selective cation channels (allow BOTH Na+ and K+ to pass)!!!!
Reversal Potential
The point at which there is no net flux of ion movement.
Structure of G-Protein Coupled Receptors
- Span the membrane seven times (serpentine receptors)
- N-terminus is extracellular
- C-terminus is intracellular (G-protein binding site)
Trimeric G Proteins
- Composed of 3 subunits (alpha, beta, gamma)
- During signalling, the beta and gamma subunits are attached and referred to as the Gby subunit
- In resting state Ga has a GDP bound and is associated with GBy
- Receptor activation binds to Ga and allows the release of GDP (GTP then binds)
Time-course of activation
Fluorescent techniques have measured the time course of G-protein activation (Fluorescence resonance energy transfer (FRET))
Which domain interacts with G-Proteins?
- Established through an experiment that swapped excitatory and inhibitory domains to find that S5 defines G-coupled protein receptor to be excitatory or inhibitory
- The C3 loop mediates the association with the G-proteins
Biogenic Amines (List)
SMALL MOLECULE NEUROTRANSMITTERS
1) Dopamine
2) Norepinephrine
3) Epinephrine
4) Serotonin
5) Histamine
Amino Acid Neurotransmitters (List)
SMALL MOLECULE NEUROTRANSMITTERS
1) GLutamate
2) Aspartate
3) GABA
4) Glycine
Major excitatory neurotransmitter
Neuromuscular junctions: ACh
Brain: Glutamate
Process of ACh production
- Mitochondria create acetyl CoA and choline which come together to form ACh in the postsynaptic cell
Reuptake of ACh
- Acetylcholine esterase is in the cleft to break acetylcholine into acetate and choline - some pass through and bind, but eventually quickly broken down after unbinding
- Enzymatic degredation!!!
Cholinergic Nerve Terminals
Nerve gases block acetylcholine esterase so that ACh stays in the synaptic cleft causing overstimulation.
Myasthenia Gravis
- Autoimmune disease that targets nicotine ACh receptors
- Reduced number of receptors
- Treatments with cholinesterase inhibitors (so that more ACh binds to make up for having fewer receptors)
- MEPPs are smaller blocks less receptors are activated so each vesicle has less of an effect
Ionotropic glutamate receptors
1) NMDA - Signal integration, synaptic plasticity
2) AMPA - Synaptic transmission, synaptic plasticity
3) Kainate - Synaptic transmission, presynaptic modulation (lesser known, not as important)
NMDA v. AMPA Receptors
- Both use glutamate as their neurotransmitter (distinguished by their agonist NMDA or AMPA)
- Functionally distinct due to a magnesium block of the NMDA receptor at rest (AMPA not blocked by magnesium)
- Opening of the NMDA channel requires glutamate and depolarization to relieve the magnesium block.
- Change in membrane potential drives the magnesium otu of the pore
- NMDA slower than AMPA, but they last longer
Glutamate cycling between neurons and glia
1) EATT: Excitatory amino acid transporter- Transports glutamate from the synaptic cleft into a glial cell for degredation.
2) Glutamine synthetase breaks down the glutamate into glutamine insdie the glial cell- then pushed back out
3) EATT takes the broken down glutamine into the presynaptic terminal so that glutaminase can synthesize it into glutamate
4) VGLUT: Vesicular glutamate transporter- Transports newly synthesized glutamate into the synaptic vesicles in the presynaptic terminal
Excitotoxicity
Definition: Excessive activation of glutamate receptors which leads to neuron death
- Only occurs in post synaptic cells (affects mostly dendrites, aons stay largely the same)
- Implicated in neuronal damage after strokes
- Decreased function of glutamate transporters removing it from the cleft leaves more glutamate in the synapse after injury
- Increased intracellular calcium levels cause oxidative damage
Proteolytic processing of pre-propeptides
Large polypeptides are cleaved by enzymes into smaller peptides. Some are even cleaved into single amino acids and then put together. The final result is a polypeptide that can function as a neurotransmitter.
Ionotropic GABA receptors
- Cl- selective channels
- Opening of the channel requires GABA (other sites are allosteric for GABA binding)
- Reversal potential for Cl- is low, making GABA a generally inhibitory receptor
Synthesis, release and reuptake of GABA
- Mitochondria synthesizes GABA from glucose and glutamate
- GAT transporters trasnport GABA from outside the cell (after leaving glial cell) back into the presynaptic terminal
- VIATT (Vesicular inhibitory amino acid trasnproter) puts the newly synthesized GABA in the presynaptic terminal into a synaptic vesicle
Endocannabinoids
- Endogenous signaling molecules that act on cannabinoid receptors in the brain (CB1 and CB2 are the targets for THC)
- Inhibit inhibition by controlling GABA release
- Increased calcium in post-synapti cell leads to endoC production and release from cells
- Diffuse back to the pre-synaptic cell to reduce release of GABA from the nerve terminal
- Result: Less inhibitory affect of the next AP - Does not use traditional neurotransmitter- a fatty substance that we’re not sure exactly how it works
GABA during development
- Excitatory neurotransmitter
- Sodium, potassium, chlorine transporter highly expressed in immature neuron which leads to higher intracellular concentration of chlorine
- Increased reversal potential of Cl- and thus GABA receptors - AS we mature, a potassium, chlorine co-transporter begins to be expressed which pumps out Cl- and lowers the intracellular concentration
Reversal/threshold and EPSP vs IPSP
If the reversal potential is below the threshold, then even if the AP depolarizes it is considered inhibitory, because it will weigh the total below the threshold since it will be highly energetically unfavorable to pass the reversal potential and get to the threshold.
If the reversal potential is above the threshold, the postsynaptic potential is considered excitatory even if it doesn’t hit threshold, because it is pushing the neuron toward threshold.
True or False: Postsynaptic potentials can be summed to determine an overall effect on a neuron
TRUE!
Most of the time an action potential is not induced from just one signal. There are constantly hundreds of signals coming into a neuron, so it’s a balance between how powerful the EPSPs and IPSPs are on a certain dendrite.
Three stages of cell signaling
1) Reception
2) Transduction
3) Response
Two types of cellular responses from cell signalling
1) Alteration of protein (enzyme) function
2) Altered gene transcription
Paracrine
Secreted molecules act locally and do not diffuse far. The range is short, but multiple cells are affected.
Endocrine
Hormone signalling. These act on target cells some distance away and are usually carried by the blood or other extracellular fluids.
Three classes of cell signalling molecules
1) Cell-impermeant molecules- Molecules must be released from cell and interact with proteins on the cell surface since they cannot pass through the membrane (polar)
2) Cell-permeant molecules- Molecules are released from one cell and can pass passively through the membrane of the second where they hit their targets inside the cell (nonpolar).
3) Cell-associated molecules- Two cells come in close contact so that signalling molecules protruding from one cell interact with receptors on the second.
Categories of Cellular Receptors
1) Channel-linked receptors: Binding of the ligand opens a channel
2) Enzyme-linked receptors: Signal binds to an inactive enzyme to active it and produce a product.
3) G-protein-coupled receptors: Signal binds to a receptor which causes a G-protein to activate and start a signal transduction cascade
4) Intracellular receptors: Receptors that lie inside the cell for cell-permeant molecules to bind to after they pass though the plasma membrane
Norepinephrine G-Protein-Coupled Receptor Pathway
- Associated with Gs protein
- Activation
Gs –> Adenylyl clyclase –> cAMP –> Protein kinase A –> Increase protein phosphorylation
Glutamate G-Protein-Coupled Receptor Pathway
- Associated with Gq protein
- Activation
Gq –> Phospholipase C – > diacylglycerol OR IP3 –> Protein Kinase A OR calcium release –> Increase protein phosphorylation and activate calcium-binding proteins
Dopamine G-Protein-Coupled Receptor Pathway
- Associated with Gi protein
- Inhibition
Gi –> Adenylyl cyclase –> cAMP –> Protein Kinase A –> Decrease protein phosphorylation
Receptor Tyrosine Kinase (RTK)
- Membrane receptor proteins that are involved in cell proliferation, differentiation and survival
- Survival and growth are two major effects
- Made up of three domains
1) Extracellular ligand binding domain
2) Single transmembrane domain
3) Cytosolic protein kinase domain (tyrosine kinase activity) - Trk A binds to two receptors and brings them together
- Tails are close together allowing for transduction pathways
Mechanism of Receptor Tyrosine Kinases
1) Ligand bind and the receptors dimerize allowing the two monomers to associate
2) Once a dimer, the kinase domain of one monomer phosphorylates the other
3) Kinase activity is enhanced and additional tyrosines on the receptors are phosphorylated
Can also lead to binding of other proteins or ATP
Calcium as a second messenger
- Stored in the ER or outside the cell, because the phosphates are poisonous to the cell if in the cytosol
- Removed by pumps that pump it out of the cell or into the ER or mitochondria (which may help move the calcium)
Secondary Messengers to Recognize
1) Cyclic AMP
2) Cyclic GMP
3) IP3
4) Diacylglycerol
5) Calcium
General mechanism of cyclic-nucleotide pathways
- An ATP interacts with an adenylyl cyclase or guanylyl cyclase to turn a cAMP or a cGMP into AMP or GMP
- The cAMP pathway releases PKA
- The cGMP pathway releases PKG
- Both can also open cyclic nucleotide-gated channels
IP3 Pathway
- PLC-cleavage of PIP2 activates the IP3 signaling pathway
- Increased IP3 actiavest the ER to release Ca2+ through an IP3 receptor channel
- Increased cytosolic Ca2+ activates PkC
DAG Pathway
- Activates PkC
- When DAG levels increase, PKC moves toward it and binds to promote phosphorylation
Protein Kinases
Phosphorylate
Phosphatases
Dephosphorylate- opposite of kinase action
Protein Kinase A
- A cAMP-dependent kinase
- Majority of cAMPs effects are mediated by PKA
Calcium/Calmodulin-dependent Kinase II (CaMKII)
- The most abundant kinase in the nervous system
- Calcium binds to calmodulin and displaces an inhibitory domain from a catalytic domain
- Regulates a larger number of intracellular signal transduction proteins and ion channels
Protein Kinase C
- Monomeric kinase that is activated by DAG and calcium ions
- DAG brings PKC to the membrane where calcium and phosphatidylserine bind
- During aging, PKC activity is reduced and is linked to Alzheimer’s Disease
Types of Protein Kinase signalling
1) PKA
2) CAMKII
3) PKC
Synaptic Plasticity
- The ability of a synapse to change in strength over time. This is thought to be the substrate of learning and memory over time.
- Operates over many time scales and on both the presynaptic and postsynaptic cells
- A change in the synaptic connection between two (or more) neurons
- Can be change for a larger OR smaller response
Synaptic Facilitation
Definition: A rapid increase in synaptic strength that can last for tens of milliseconds.
- Occurs when two or more APs reach the nerve terminal within a few milliseconds of each other
- Results in prolonged Ca2+ levels so calcium is able to build up in the nerve terminal leading to a stronger response the second time
- Subsequent APs will release more neurotransmitter resulting in an increase in the post-synaptic potential
- Unclear how increased calcium leads to facilitation, but might have to do with synaptotagmin activity
Synaptic Augmentation
Definition: An increase in synaptic strength that occurs over a few seconds.
- Due to residual calcium ions following previous activity; this leads to increased subsequent synaptic vesicle fusion (usually during conditions of low external calcium or low probability of release)
- Possible mechanism: Calcium-mediated protein kinases phosphorylating synapsin to increase available vesicles?
Synaptic Potentiation
Definition: An increase in synaptic strength that ccurs over tens of seconds to minutes to hours.
- Due to residual calcium ions following previous activity; this leads to increased subsequent synaptic vesicle fusion (usually during conditions of low external calcium or low probability of release)
Synaptic Depression
Definition: A decrease in synaptic strength
- During sustained synaptic activity the amount of neurotransmitter released declines
- Caused by a reduction in the amount of neurotransmitter released
- Lowering calcium concentration slows depression and reduces the number of quanta released
- More neurotransmitter release on the stimulus = more depression on the second
- If synapsin function is impaired, depression is increased because it becomes harder to release the vesicles
Short term synaptic plasticity
- Intrinsic part of presynaptic release machinery or postsynaptic neurotransmitter receptors- any synapse will show these changes
- Changes that last a few seconds or less
Vesicle depletion hypothesis
The progressive depletion of synaptic vesicles leads to synaptic depression.
Effects of elevating and lowering calcium levels outside the cell
Elevating calcium levels makes the synapse more likely to depress.
Lowering calcium levels make the synapse more likely to facilitate.
Reasons Eric Kandel chose the sea slug for his synaptic plasticity experiments
- Nervous system is made up of a small number of nerve cells
- Many of these nerve cells are very large
- Many nerve cells are uniquely identifiable
Mechanism of presynaptic enhancement underlying short-term sensitization (ie. why shocking the tail makes the gill response come back)
1) Interneuron (from tail) serotonin release acts on GPCRs in the presynaptic nerve terminal (sensory neuron)
2) GPCR is coupled to Gs and induces increased cAMP via adenlylyl cyclase.
3) Increased cAMP promotes PKA activity.
4) PKA phosphorylates a number of proteins (most likely K+ channels)
5) Phosphorylation of K+ channels reduces the probability that K+ channels will open during the AP leading to a prolonged AP and more calcium influx via voltage gated channels.
6) Enhanced calcium leads to increased transmitter release
Mechanism of presynaptic enhancement underlying long-term sensitization
- Serotonin-induced enhanced release leads to long-term changes
- Lasts weeks - Changes gene expression (protein synthesis)
- PKA-mediated
CREB
cAMP Response Element Binding Protein
- Transcriptional activator
- Normally bound to DNA
Long-Term Potentiation
Definition: Long-lasting increased synaptic strength
- Thought to be the substrate for active learning and memory in the brain
- Mechanism by which something we learn once is maintained over years
- Undergoing a short burst of activity that is maintained over 5-6 hours
- Very specific to a synapse- doesn’t change the entire cell, just that one synapse
- Occurs with brief, high frequency stimulation
- Fast, large rise in calcium
Hippocampus
Area of the brain critical for memory storage and retrieval.
- Damage to this part of the brain can prevent the formation of new memories
Long-Term Depression
Definition: Long-lasting decrease in synaptic strength
- Occurs with low frequency, long period stimulation
- Small, slow rise in calcium
LTP is state dependent
- Post-synaptic membrane potential determines if LTP will occur
- Post-synaptic depolarization must happen almost simultaneously with presynaptic neurotransmitter release
Four Major Properties of LTP
1) Specificity: If LTP is induced in one synapse, this dos not mean that it will occur in other synapses of the neuron (ie. some pathways can experience LTP while others will not)
2) Associativity: Strong activation paired with weak activation will still induce strengthening of both synapses because the two become associated.
3) Cooperativity: LTP can be either induced by strong tetanic stimulation of a single pathway to synapse, or cooperatively via the weaker stimulation of many.
4) Persistence: Lasts from several minutes to many months following a brief period of synaptic activity.
Molecular Mechanisms of LTP
- NMDA receptors open in response to both neurotransmitter release AND depolarization of the post-synaptic cell
- When NMDA receptors open they allow calcium entry into the cell which starts a phosphorylation cascade involving CamKII and PKC
- This causes an increase in the number of AMPA receptors on the post-synaptic terminal
- AMPA receptors are always open and allow sodium to rush into the cell, causing a stronger response
- The CREB cascade can then regulate transcription that leads to some cross-talk between cells and production of synapse growth proteins
How to block LTP
- Intracellular calcium buffer
Overall effects of LTP
1) Increased number of AMPA receptors on the postsynaptic cell
2) Larger spine size because of increased AMPA receptors
3) Can lead to creation of new synapses
Silent synapses
- Synapse is stimulated but there is no response
- Once you stimulate once you get a response - Only NMDA receptors, no AMPA receptors until after depolarization at which point it is no longer considered silent
Importance of protein synthesis changes for LTP
Without transcriptional changes, the LTP will eventually die down.
- We know this because in experiments that block the protein synthesis the LTP fades whereas in controls it does not
- Allows us to translate more proteins so that LTP continues (actual induction of LTP doesn’t require any new proteins because it uses AMPA receptors already premade in the cell
Three Phases of LTP
1) Short Term LTP (seconds to minutes): A still mysterious property, and almost nothing is known about how this early initial potentiation is produced
2) Early LTP (30 mins to 2 hours): Calcium and CamKII dependent process, is inhibited or abolished in presence of kinase inhibitors; decays after 2 hours without new protein transcription/translation
3) Late LTP (hours, days, months, etc.): First phase required translation, second phase requires transcription and translation. Series of signal transudction events including calcium, CREB, and protein kinase M zeta.
