NEUR306 - Exam 2

Who discovered the concept of synapses? How did he discover them and what did he call it?

Santiago Ramon y Cajal

Golgi Staining

Neuron Doctrine

Who coined the term "synapse"? How did they describe synapses?

Charles Sherrington

Specialized sites of contact where information transfer occurs

What is the sparker vs. souper debate over?

synaptic transmisstion

What did sparkers believe?

Electrical current flows from one neuron to the next

What did soupers believe?

Chemical substances transfer information from one neuron to the next at the synapse

Who was correct about synaptic transmission?

BOTH sparkers and soupers

What are some characteristics of the electrical synapse?

3 nm wide gap

Bi - directional

Fast w/ little to no delay

Reliable

Synchronize electrical activity

rare

Where are electrical synapses found?

Invertebrate escape circuits, pacemakers, and liver cells

What are some characteristics of chemical synapses (6)?

20 - 50 nm wide

Unidirectional

Common

Quantal Transmission

Ca2+ dependent NT release

Molecular Mechanisms of vesicle recycling

What is the structure of an electrical synapse?

Gap Junctions --> 2 hemi - channels aligned which allow ions and small proteins to go through

What is a hemi - channel made up of?

6 connexins

What was Otto Loewi's experiment for the Vagusstoff?

Taking 2 hearts, he put one in one container and the other in another one. He then connects the solution the first heart was in to the the second heart's container

He then stimulated the vagus nerve of the first heart and measured the contraction of both over time

What were the results/conclusion of Otto Loewi's experiment for the Vagusstoff?

Results: Stimulation of heart 1 slowed contractions. When the fluid was transferred to heart 2, it too slowed down contractions

Conclusion: Soluble stuff, not electrical impulses, mediate the vagus nerve effect

What was lucky about Otto Loewi's experiment?

The experiment would have failed if at a higher temp, as the enzymes for the neurotransmitter he discovered would have been broken down

What 3 criteria are used to define neurotransmitters?

1. Must be synthesized and stored in the PRE - synaptic neuron

2. Must be released by the pre - synaptic terminal following action potentials

3. The molecule (or agonists) must produce a responses in the post - synaptic neuron that mimics the one in the pre (and agtagonists block it)

What was the first major neurophysiological experiment to describe neurotransmitter release?

Synaptic transmission at a frog's neuromuscular junction

By Bernard Katz

Stimulate the axon before the junction and record the post - synaptic membrane potential at the muscle cell

What events did Katz find that occurred at the neuromuscular junction?

Evoked End Plate Potentials which occurred after stimulation

Spontaneous miniature EPPs which just occurred every now and then

What were the characteristics of the MEPPs?

Similar in size

When in low Ca2+, the motor neuron evokes smaller EPPs (which are usually double or triple the size of MEPPs --> integer multiples)

What the conclusion of Katz's Experiment?

Synaptic transmission is quantal (in discrete packets) of 0.4 mV

What revealed the presences of synaptic vesicles in the pre - synaptic terminal?

Electron Microscopy

What was the hypothesis about neurotransmitter release in reference to vesicles?

Each vesicle contains a fixed amount of neurotransmitter (quantum), which matches the amount needed to elicit one MEPP

Each vesicle then releases its entire content during synaptic transmission through fusion with the pre - synaptic membrane

What was the experiment to test neurotransmitter release in reference to vesicles?

Record vesicle fusions using freeze - fracture electron microscopy AND correlate with post -synaptic EPPs

What were the results of the experiment to test neurotransmitter release in reference to vesicles?

When stimulated, there are "pits" present (vesicles in the act of fusing

Amount of pits correlate with EPP size

What was the conclusion of the experiment to test neurotransmitter release in reference to vesicles?

Neurotransmitter release is quantal through the fusion of discrete synaptic vesicles with the pre - synaptic membrane (exocytosis)

What happens to all the vesicle membrane that fuses to the pre - synaptic membrane?

it gets reused !

What is the synaptic vesicle cycle?

Retrieved vesicle membrane passes through several intracellular components and is eventually used to make new synaptic vesicles

How were researchers able to determine the synaptic vesicle cycle?

Using HRP coated pits

What is the full synaptic recycling of vesicles steps?

1. Budding from endosome

2. Exocytosis --> docking, priming, fusion

3. Endocytosis --> budding off membrane

How does synaptic transmission operate in normal calcium levels? What about low levels?

Normal: EPPs and Pits in the membrane

Low: Subthreshold EPP and No pits in the membrane

How are calcium levels in both the inside and outside of the neuron?

WAYY more outside than inside

Why are the intracellular levels of calcium kept low?

Since it is important for NT release, low levels ensure that there is none freely flowing around to trigger release where there is no signal

How can we test whether calcium enters the terminal?

Using Calcium Imaging --> Calcium sensitive dyes that are viewed under a fluorescence microscope

What was the result of the calcium imaging?

A direct observance of a rise in calcium concentration at the pre - synaptic terminal following a chain of action potential in the pre - synaptic neuron

How can we test if calcium influx causes neurotransmitter release? What do they show?

1. Apply Calcium blockers --> if necessary

2. Inject Calcium directly --> if sufficent

What was the result of applying the calcium blockers into the cell?

Blocking calcium influx prevents the rise of EPSP --> proves calcium is NECESSARY for synaptic transmission.

Cd2+ is the blocker for calcium, this experiment requires voltage clamp as well as TTX and TEA in the control group to remove the Na+ and K+ currents

What was the result of injecting calcium directly into the cell (pre - synaptic terminal)?

Injecting calcium alone triggers a rise of postsynaptic membrane potential --> shows that presence of calcium alone is SUFFICIENT for synaptic transmission

What are the five (important) molecules involved with vesicle trafficking?

1. Synapsins

2. SNAREs

3. Synaptotagmins

4. Clathrin

5. Dynamin

What do synapsins do?

protein which reversibly binds to vesicles and is phosphorylated by CaMKII in order to move vesicles closer to the plasma membrane (tells vesicle to move to the active zone)

What do the SNAREs proteins do?

Involved with Docking and Priming

A protein complex which spans the 2 membranes to promote fusion while holding each other to hold the vesicles in place

What does SNAP - 25 and Syntaxin do?

plasma membrane SNARE proteins which form helical SNARE complex

What does Synaptobrevin do?

Vesicular SNARE protein which forms helical SNARE complex

What does synaptotagmin do?

Calcium Sensor that triggers the fusion of the two membranes (and the release of NTs)

What is the role of Clathrin? Dynamin?

Clathrin - attaches to membrane to be retrieved

Dynamin - pinches off membrane

What are the 12 basic steps of neurotransmission in chemical synapses?

1. NT synthesized and stored in vesicles

2. Action Potentials envade terminal

3. Depolarization opens VG Ca2+ channels

4. Influx of Ca2+

5. Ca2+ causes vesicles fusion

6. NT released via exocytosis into cleft

7. Removal of NT by reuptake or enzyme degradation

8. Retrieval of vesicle membrane and recycling

9. NT binds to post - synapse receptors

10. Open/closing of post - synapse channels

11. Current is either EPSP or IPSP

12. Summation determines whether an AP occurs or not

What is the visual pathway?

retina -> optic nerve -> optic chiasm -> optic tracts -> lateral geniculate nucleus (LGN) of thalamus -> visual radiations -> visual cortex

Eye anatomy

Pupil

Lens

Retina

Fovea

What 3 ways is light converted to electrical signal?

1. Pupil

2. Lens

3. Fovea

How does the pupil work?

Opens and closes to let the correct amount of light in through iris muscles

(Opens in low light, Closes in high light)

How does the lens work?

It changes shape, from more to less convex, so that light can be properly focussed on the retina through accommodations from ciliary muscle

Further objects = flatter and thinner

Closer objects = rounder and thicker

How does the fovea work?

aids in focusing the image onto the retina

What is the structural makeup of the retina?

Top (back) layer - Rods and Cones

Other (front) layers - retinal gangelion cells

What 5 cells are present in the retina?

1. Photoreceptor cells

2. Horizontal Cell

3. Bipolar cell

4. Amacrine cell

5. Ganglion cell

Why do we have a blind spot?

the point at which the optic nerves leave the eye, creating a "blind" spot because no photo receptor cells are located there (at the optic disc)

Can photoreceptor cells perform action potentials?

NO - ONLY ganglion cells

Where are photo - pigments located in rods and cones?

Discs in the outer segment

What do see when light is flashed onto photoreceptor cells in terms of membrane potential?

Hyper-polarization of MP

Graded Response which correspond with change in the rate of transmitter released onto the post - synap. neuron

What occurs at a rod photoreceptor when in the dark?

Vm at - 40 mV (depolarization)

INWARD current of Na+/Ca+ through cGMP - gated channels as a result of cGMP binding

OUTWARD current of K+ through leak channels

Continuous NT release

What light sensing mechanisms exist in microorganisms? other animals?

Microorganisms - light activated ion channels

Other animals - light activated metabotropic receptors

What occurs at a rod photoreceptor when in light?

1. Light Absorption leads to configuration change of retinal

2. Activated rhodopsin facilitates GTP binding to transducin

3. GTP bound alpha subunit dissociates and activated PDE

4. Activated PDE hydrolyzes cGMP into GMP, reducing its concentration

5. cGMP gated cation channels close in the membrane and Na+/Ca+ can't enter the cell

What is photopigment composed of?

Opsin protein and retinal (chromophore/cofactor)

What configuration change does retinal undergo when hit by light?

11 - cis to all - trans

What does Rhodopsin represent? What about Transducin?

Rhodopsin is the GPCR while Transducin is the G - protein

What is PDE?

phosphodiesterase -- 1st effector enzyme

Does light hyperpolarize or depolarize photoreceptors? What about dark?

Hyperpolarize for light, depolarize for dark

Where does amplification occur in phototransduction?

Activated Rhodopsin facilitating GTP binding on transducin

PDE hydrolyzes multiple cGMP

How can you bring the photoreceptor back to the "dark" phase?

1. Rhodopsin kinase + Arrestin -- binds + phosphorylation

2. Hydrolysis of GTP back to GDP

3. Guanylate cyclase to produce cGMP

Which between rods and cones is more sensitive to light?

Rods -- longer outer segment = more disks = more photopigments

What are the 3 different visual functions (in order from darkest to lightest)? What is mostly present at each?

1. Scotopic - ALL rods

2. Mesotopic - both rods and cones

3. Photopic - MAINLY cones

What is the cone threshold?

right above scotopic

What is the rod saturation?

right above mesotopic

What do we see in a patch clamp recording for an cGMP channel in rods? What does it tell us?

constant inward current in dark, increase in inward current after light stimulus which stays

rods have TEMPORAL SENSITIVITY

What do we see in a patch clamp recording for an cGMP channel in cones? What does it tell us?

constant inward current in dark, increase in inward current after light stimulus which goes down back to normal

cones have TEMPORAL RESOLUTION

What is the connectivity between rods and bipolar cells? What does that tell us?

Multiple rods to one bipolar cell - summation

Rods have SPATIAL SENSITIVITY

What is the connectivity between cones and bipolar cells? What does that tell us?

One cone to one bipolar cell

Cones have SPATIAL RESOLUTION (ACUITY)

What is mostly present in the fovea? Why?

All cones; higher resolution

What other specializations are there for cones?

Color Vision

What are the 3 pigments used for color vision in humans?

S pigment - blue (short)

M pigment - green (medium)

L pigment - red (long)

How does color vision work in humans?

trichromatic --> we require 2 bandwidths of light to match all colors

Why can't humans tell color with just one cone?

To see color, we compare; thus, we need more than one opsin type

What are the two types of chemical synapses?

Neuron to Neuron

Neuron to Muscle

What are the two types of vesicles? What do they carry?

Small - 40 - 60 nm; neurotransmitters; Ach, GABA, Glutamate

Dense - 90 - 250 nm; neuropeptides

Where are neurotransmitters synthesized?

Small NTs synthesized locally (in pre - synaptic terminal)

Where are neuropeptides synthesized?

Cell body then transported to terminal

What is the pathway of synthesis and storage for neuropeptides?

1. Precursor peptide in rough ER sent to golgi

2. Peptide becomes active and it package in dense core vesicles

How are neurotransmitters degraded?

Enzymes --> then metabolites are re - uptaked

How are neurotransmitters recycled?

Axonic Transporters which bring them back into the pre - synaptic terminal

What are the two kinds of neurotransmitter receptors?

Ionotropic: ligand - gated receptors

Metabotropic: G - protein coupled receptor

What are characteristics of the ionotropic receptor?

Fast --> NT binds directly to ion channels

What are characteristics of the metabotropic receptor?

Slow --> NT binding initiates signaling cascade

Are acetylcholine's receptors excitatory or inhibitory? Which one is which?

Both

nAChR is excitatory (I) - nicotinic acetylcholine receptor

mAChR is mixed (M) - muscarinic acetylcholine receptor

Are glutamates's receptors excitatory or inhibitory? Which one is which?

Both

AMPA, NMDA, Kainate are excitatory (I)

mGluR is mixed (M)

What is special about Glutamate?

major excitatory NT in the CNS and most prevalent one

nearly 60% of neurons in brain believed to use Glu as their receptor

What is special about the NMDA receptor?

Both ligand and voltage dependent ion channel

How does the NMDA receptor work?

lets in Na+ and Ca2+, lets out K+

has binding site for glutamate and glycine

magnesium binding site which blocks

When glutamate and glycine bind, as well as the membrane depolarizes (thus removing Mg2+), then the channels open and ions pass through

Are GABA's receptors excitatory or inhibitory? Which one is which?

Inhibitory

GABA(a) is ionotropic

GABA(b) is metabotropic

What is special about GABA?

Major inhibitory neurotransmitter

What did we see with the voltage clamping method at different potentials in squid axon?

With increasingly depolarized membrane potentials, the early inward current increase in size, then decreases, disappears, then reverses from inward to outward

What was the result of the external removal of Na+ experiment?

The early inward current is dependent on Na+

What was the scheme for voltage clamping a post - synaptic muscle fiber?

Stimulating the pre - synaptic axon and using a voltage clamp amplifier on the muscle fiber to see at what potential there will be zero current

What was the result for voltage clamping a post - synaptic muscle fiber?

At 0 mV, there was no current (the reversal potential - which is the equilibrium potential of the ion channel)