Lecture 5 + 6: Neurotransmitters Flashcards
Why study neurotransmitters?
Becuase neurotransmitter systems are central building blocks of theories linking brain activity to cognition and behavior. Therefore, they are key to understand effects of disorders and psychoactive drugs on cognition and behavior.
What criteria must molecule fullfill to be called neurotransmitter?
-> must be synthesized and stored in presynaptic neuron
-> must be released by presynaptic axon terminal upon stimulation
-> when experimentally applied, must produce response in postsynaptic cell that mimicks the response produced by release of neurotransmitter
What are methods used to classify neurotransmitters?
immunocytochemistry - using labeled antibodies to identify location of molecules within cells
in situ hybridization - localizing mRNA transcripts for proteins
immunocytochemistry
neurotransmitter candidate is injected into bloodstream of animal, causing immune response of antibodies
then blood is withdrawn from animal and analysed
antibodies are extracted and applied to the brain tissue
those antibodies are used as labels for cells containing neurotransmitter candidates
therefore, it can be used to localize any molecule for which specific antibody can be generated
in situ hybridization
strands of mRNA consist of nucleotides arranged in specific sequence
each nucleotide with bind with specific nucleotide
therefore, it is possible to construct a probe (complementary strand) to mRNA strands
this probe will bind to mRNA (hybridization)
therefore, it is possible to examine whether mRNA for particular peptide is localized in the neuron
How to study transmitter release?
1) using alive brain slices and bathing them with high K+ concentration and presence of Ca -> this causes membrane depolarization and trigger transmitter release
2) optogenetics
Which criterium is hardest to satisfy in CNS neurotransmitter research?
2nd: must be released by presynaptic axon terminal upon stimulation
we are unsure whether molecules were released from axon terminals or are consequence of secondary synaptic activation
How to study synaptic mimicry? (criterium 3rd: when experimentally applied, must produce response in postsynaptic cell that mimicks the response produced by release of neurotransmitter)
microiontophoresis -> pipette is used to inject small amounts of electrical current and neurotransmitter candidate
microelectrode is used to check if neurotransmitter is producing effects
How to study receptors?
1) neuropharmacological analysis of synaptic transmission
2) ligand-binding methods
3) molecular analysis of receptor proteins
neuropharmacological analysis of synaptic transmission
different receptors can be distinguished by actions of different drugs
example: acetylcholine
nicotininc Ach receptor: agonist in skeletal muscles, but no effect on heart
muscarinic Ach receptor: no effect on skeletal muscles, but agonist in heart
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different drugs can be distinguished depending to which glutamate receptor they bind: AMPA vs NMDA vs kainate
ligand-binding methods
RQ: how opiates affect the brain?
testing: radioactively labelling opiates and applying them in small quantities to neuronal membranes
results: radioactive drugs labelled specific sites on membrane
Dale’s principle
idea that neuron has only one neurotransmitter
refuted: many peptide-containing neurons violate it, also there may be many co-transmitters released from one nerve terminal
ligand
any molecule that binds to receptor
what are characteristics of amino acid gated channels?
fastest synaptic transmission in CNS
-> pharmacology of binding sites describes how transmitters affect them
-> kinetics -> transmitter binding and channel gating determine duration of their effect
-> selectivity -> excitation vs inhibition
-> conductance -> determines magnitude of their effects
What are 2 types of glutamate gated channels?
AMPA and NMDA
AMPA receptors
fast excitatory transmission, permeable to both Na and K, most non permeable to Ca, enable rapid depolarization
NMDA receptors
fast excitatory transmission, admit excess of Na into cell causing depolarization, may also cause widespread and lasting changes due to Ca permeability
at resting potential get blocked by magnesium
How NMDA and AMPA receptors differ?
1) NMDA-gated channels are permeable to Ca
2) inward ioninc current through NMDA-gated channels is voltage dependent
What mediates most synaptic inhibition?
In CNS -> GABA
everywhere else -> glycine
How does binding of drugs to GABA receptors occur?
drugs by themselves do not open the channel, but they change the effect that GABA has when it binds to the channel at the same time as the drug
benzodiazepines -> increase frequency of channel opening
barbiturates -> increase duration of channel opening
Why synaptic inhibition must be tightly controlled in the brain?
too little inhibition -> seizure
too much inhibition -> coma
What is the basic structure of G-protein-coupled receptor?
7-membrane spanning alpha helices, transmitter binding side (extracellular) and G-protein binding side (intracellular)
What is the basic modus operandi of G-protein-coupled receptors?
1) transmitter binds to receptor
2) G proteins get activated
3) G protein complexes get released and travel to activate different effector proteins
What is the shortcut pathway? How does it relate to G-protein-coupled receptors?
- many neurotransmitters use shortcut pathway from receptor to G-protein to ion channel
- example: muscarininc receptors in the heart (Ach recptors)
-> G-proteins in heart muscles get activated by binding of ACh to muscarininc receptors
-> activated g protein complexes DIRECTLY cause potassium channels to open
What are second messenger cascades?
G-proteins can also exert their effects by directly activating certain enzymes
push and pull method - one stimulates biochemical processes, the other inhibits them
What are protein kinases?
kinase which selectively modifies other proteins by covalently adding phosphates to them
phosphorylation of ion channels → effect on whether they will open or close
What are protein phosphates?
remove phosphates from proteins
why? because proteins may become quickly saturated with phosphates
What is function of singal cascade? (G protein)
synaptic transmission G-protein-coupled receptors is complex and slow -> however, due to long chain of command you obtain signal amplification! (activation of many ion channels)
divergence
ability of one transmitter to activate more than one subtype of receptor
convergence
multiple neurotransmitters can converge to influence the same effector system
When equilibrium potential and reversal portential are equivalent to each other?
in single ion system
equilibrium potential
net ion flux at voltage is zero (outward and inward rates are the same = EQUILIBRIUM)
reversal potential
perturbation of membrane potential on either side of the equilibrium potential reverses the net direction of ion flux
What are types of neurotransmitters?
aminoacids
amines
peptides
What are characteristics of aminoacids?
small
huge concentration
made in synaps with help of enzymes made in cell body
inactivated largely through reuptake - recycling
short activity
What are characteristics of amines?
small
huge concentration
made in synaps with help of enzymes made in cell body
inactivated largely through reuptake
- recycling
short activity
What are characteristics of peptides?
short chains of 2 or more amino acids
low concentration
they are build in cell body and need to be transported to synapse (due to size)
no reuptake = longer duration
inactivated by breakdown and diffusion
What is cytoskeleton?
skaffolding within the neuron = so it doesn’t collapse
also within dendrites and axons
What is green staining on the neuron?
tubulin -> cell body, dendrites, proximal axon
What is red staining on the neuron?
tau - microtubulin binding protein -> distal axon
How does transport within neuron occur?
transport via cytoskeleton => through microtubuli
What is anterograde transport?
from cell body (soma) to synaps = orthodrome -> can be traced with HRP, PHL
What is retrograde transport?
from synaps to cell body (soma) - needed for long-term potentiation = antidrome -> can be traced with FG, CT, FB
How to study systems by injection to PFC?
Inject retrograde tracer into prefrontal cortex. Look to what areas cells project to. You can also inject anterograde injections to those sites. Examples: locus coeruleus and dorsal rape (reciprocal connections with PFC).
What are reciprocal connections?
connections sending signals both ways
How anterograde injection in ventrolateral prefrontal cortex enables you to study areas on the other hemisphere?
because there are connections between two hemispheres via corpus callosum
How tracing was used in attention research in rat brain?
Retrograde injection was made into prefrontal cortex. PFC is connected with basal forebrain (acetylcholine), ventral tegmentum (dopamine), dorsal raphe (serotonin), locus coerulues (noradrenaline) etc. With specific tracers you can unconver which areas are using which neurotransmitters.
How does indirect immunhistochemistry work?
Identify proteins in the tissue you want to stain. Then, inject primary antibody against the protein -> this antibody will always attach to your protein of interest (you can have nice visualization). Secondary antibody with HRPO can be then used to color areas where you find protein.
How can you use viruses in your studies?
Virus is able to quickly infect multiple cells. As the infection spreads, you can have map of the whole system.
What is fluorescent in situ hybridization?
laboratory technique used to detect and locate a specific DNA sequence on a chromosome
How does in fluorescent in situ hybridization work?
In this technique, the full set of chromosomes from an individual is affixed to a glass slide and then exposed to a “probe”—a small piece of purified DNA tagged with a fluorescent dye. The fluorescently labeled probe finds and then binds to its matching sequence within the set of chromosomes. With the use of a special microscope, the chromosome and sub-chromosomal location where the fluorescent probe bound can be seen.
What is CLARITY technique?
New technique which enables to make the whole brain transparent by removing lipids blocking the view. Molecular markers can be added to highlight specific structures.
How is cerebral cortex organized?
layer 2: send information to other cortical areas (ipsilateral)
layer 3: send information to other cortical areas, opposite hemisphere
layer 5: connected to sub-cortical structures (striatum, superior colliculus)
layer 6: connected to thalamus = sending information to layer 4 and receiving them back
What happens if you inject retrograde tracer in nucleus accumbens?
You can examine which layers of prefrontal cortex are connected to it. Layer 5! because it connects to subcortical areas (also layers 2 and 3 - because connections are within the same hemisphere and across hemispheres)
What areas are targeting nucleus accumbens?
On the basis of retrograde injection in NA:
- prefrontal cortex
- thalamus
- ventral tegmental area
- amygdala
- ventral hippocampus
- dorsal raphe
Why there are no drugs which target glutamate?
Because glutamate is used by all cortical areas. Too much of it would lead to overstimulation (epilepsy), blocking it would lead to death.
To what system acetylcholine belongs?
cholinergic system
What is good marker for cells using acetylcholine?
ChAT = specific enzyme required in acetylcholine synthesis
What does ChAT do?
transfers acetyl group from acetyl CoA to choline
What limits how much acetylcholine can be synthesized?
availability of choline
What degrades acetylcholine in synaptic cleft?
AChE - degrades acetylcholine into choline and acetic acid
(choline is recycled)
What receptors have acetylcholine?
nicotinic receptors
What are characteristics of acetylcholine?
- medium - sized neurotransmitter
- similar to monoamines
- class of its own
- part of diffuse modulatory systems
- production depends on 2 enzymes: choline acetyltransferase - making
acetylcholine esterase - recycling
How acetylcholine relates to Navalny poisioning?
Poison used blocked acetylcholine esterase - so transmitter could not be broken down. Therefore, there was too much acetylcholine in the system -> overactivation of muscles (spasms), then paralysis.
