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NROD66H3 Lecture Notes - Acetyl Group, Retrograde Signaling, Choline

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Chapter 6- Neurotransmitter Systems
Three major classes of neurotransmitters: amino acids, amines, and peptides
First molecule identified as a neurotransmitter was acetylcholine, Ach
Cholinergic- cells that produce and release Ach
Noradrenergic- neurons that use the amine neurotransmitter norepinephrine (NE)
Glutamatergic- synapses that use glutamate
GABAergic- synapses that use GABA
Peptidergic- synapses that use peptides
Ach and all the molecular machinery associated with it are collectively called cholinergic system
Studying Neurotransmitter Systems:
Certain criteria must be met to distinguish a molecule as a neurotransmitter:
oThe molecule must be synthesized and stored in the presynaptic neuron
oThe molecule must be released by the presynaptic axon terminal upon stimulation
oThe molecule must produce a response in the postsynaptic cell
Localization of Transmitters and Transmitter-Synthesizing Enzymes:
Hints that a particular molecule may be a neurotransmitter:
oMolecule is concentrated in the brain tissue
oApplication of the molecule to certain neurons alters their action potential firing rate
To confirm the molecule is a neurotransmitter, the molecule must be localized in and synthesized
by particular neurons
Two techniques used are immunocytochemistry and in situ hybridization
Immunocytochemistry- a method used to anatomically localize particular molecules to particular
oOnce the neurotransmitter candidate has been chemically purified, it is injected into the
bloodstream of an animal, where it stimulates an immune response
oThe response is the generation of large proteins called antibodies
Antibodies can bind tightly to specific sites on the foreign molecule such as the
transmitter candidate
Best antibodies for this method bind very tightly to the transmitter of interest, and
bind very little or not at all to other chemicals in the brain
oThis method can be used to localize any molecule for which a specific antibody can be
In Situ Hybridization:
Is also useful for confirming that a cell synthesizes a particular protein or peptide
Recall: proteins are assembled by the ribosomes according to instructions from specific mRNA
A unique mRNA molecule for every polypeptide is synthesized by a neuron
If the sequence of nucleic acids in a strand of mRNA is known, it is possible to construct in the lab
a complementary strand that will stick to the mRNA molecule
oComplementary strand is called a probe
oProcess by which the probe bonds to the mRNA molecule is called hybridization
In order to see if the mRNA for a particular peptide is localized in a neuron, we chemically label the
appropriate probe so it can be detected, apply it to a section of brain tissue, allow time for the
probes to stick to any complementary mRNA strands, then wash away all the extra probes that
have not stuck; finally we search for neurons that contain the label
In situ hybridization, probes are usually labelled by making them radioactive
oSince we cannot see radioactivity, hybridized probes are detected by laying the brain tissue
on a sheet of special film that is sensitive to radioactive emissions
oAfter exposure to the tissue, the film is developed like a photograph, and negative images
of the radioactive cells are visible as clusters of small dots
This technique for viewing the distribution of radioactivity is called autoradiography
Immunocytochemistry is a method for viewing the location of specific molecules, including
proteins, in sections of brain tissue
In situ hybridization is a method for localizing specific mRNA transcripts for proteins
Both methods put together, enable us to see whether a neuron contains and synthesizes a
transmitter candidate
Studying Transmitter Release:
Most regions of the outer central nervous system (CNS) contain a diverse mixture of intermingled
synapses using different neurotransmitters
Read Pg. 137-138!
Studying Synaptic Mimicry:
Knowing that a molecule is localized in, synthesized by, and released from a neuron is still not
sufficient to qualify it as a neurotransmitter
A 3rd criterion must be met:
oThe molecule must evoke the same response as that produced by the release of naturally
occurring neurotransmitter from the presynaptic neuron
To asses the postsynaptic actions of a transmitter candidate a method called microionophoresis is
oMicroionophoresis- a method of applying drugs and neurotransmitters in very small
quantities to cells
Read this section on Pg. 138
Studying Receptors:
Each neurotransmitter exerts its postsynaptic effects by binding to specific receptors
oAs a rule no two neurotransmitters bind to the same receptor; but one neurotransmitter can
bind to many different receptors
Each of the different receptors a neurotransmitter binds to is called a receptor subtype
Ach acts on two different cholinergic receptor subtypes: one type is present in skeletal muscle and
the other is in heart muscle
oBoth subtypes are also present in many other organs and within the CNS
3 Approaches to study the different receptor subtypes have been useful:
oNeuropharmacological analysis of synaptic transmission
oLigand-binding methods
oMolecular analysis of receptor proteins
Neuropharmacological Analysis:
Skeletal muscle and heart muscle respond differently to various cholinergic drugs
Nicotine (derived from tobacco plant), is a receptor agonist in skeletal muscle but has no effect in
the heart
oThe receptor is called nicotinic ACh receptors in skeletal muscle
Muscarine (derived from poisonous species of mushroom), has little or no effect on skeletal muscle
but is an agonist at the cholinergic receptor subtype in the heart
oThe receptor is called muscarine ACh receptors in the heart
ACh slows the heart rate
Muscarine is poisonous because it causes a precipitous drop in heart rate and blood pressure
Nicotinic and muscarinic receptors also exist in the brain
Glutamate receptors mediate much of the synaptic excitation in the CNS
o3 subtypes of glutamate receptors are:
AMPA receptors
NMDA receptors
Kainite receptors
Each named for a diff. chemical agonist
oThe neurotransmitter glutamate activates all 3 receptor subtypes
But AMPA acts only at the AMPA receptor, NMDA acts only at the NMDA receptor and
so on
Pharmacological analyses were used to split the receptors into two subtypes:
oNE receptors into alpha and beta
oGABA receptors into GABAA and GABAB
Ligand-Binding Methods:
Any chemical compound that binds to a specific site on a receptor is called a ligand for that
Ligand-binding method- technique of studying receptors using radioactively labelled ligands
oA ligand for a receptor can be an agonist, an antagonist, or the chemical neurotransmitter
Molecular Analysis:
Read Pg. 141
Neurotransmitter Chemistry
Most of the known neurotransmitter molecules are either:
oAmino acids
oAmines derived from amino acids
oPeptides constructed from amino acids
ACh is an exception; but it is derived from acetyl CoA,
Choline which is important for fat metabolism throughout the body
Amino acid and amine transmitters are generally each stored in and released by separate sets of
Dale’s principle- idea that a neuron has only one neurotransmitter
Many peptide containing neurons violate Dale’s principle because these cells usually release more
than one neurotransmitter: an amino acid or amine and a peptide
Co-transmitters- two or more transmitters released from one nerve terminal
But still most neurons release only a single amino acid or amine neurotransmitters
Cholinergic Neurons:
Acetylcholine (ACh)- is the neurotransmitter at the neuromuscular junction and therefore is
synthesized by all the motor neurons in the spinal cord and brain stem
ACh synthesis requires a specific enzyme, choline acetyltransferase (ChAT)
ChAT is manufactured in the soma and transported to the axon terminal like all presynaptic
Only cholinergic neurons contain ChAT, therefore this enzyme is a good marker to identify cells
that use ACh as it’s neurotransmitter
ChAT synthesizes ACh in the cytosol of the axon terminal, and the neurotransmitter is
concentrated in synaptic vesicles by the actions of an ACh transporter
oChAT transfers an acetyl group from acetyl CoA to choline
oSource of choline is the extracellular fluid, where it exists in low micromolar concentrations
oCholine is taken up by the cholinergic axon terminals via specific transporter
oBecause the availability of choline limits how much ACh can be synthesized in the axon
terminal, transport of choline into the neuron is said to be the rate-limiting step in ACh
Rate-limiting Step- in a biochemical reaction that leads to the production of a chemical, the one
step that limits the rate of synthesis.
Cholinergic neurons also manufacture the ACh degradative enzyme acetylcholinesterase (AChE)
oAChE is secreted into the synaptic cleft and is associated with cholinergic axon terminal
oAChE is also manufactured by some noncholinergic neurons, so this enzyme is not as useful
a marker for cholinergic synapses as ChAT
AChE degrades ACh into choline and acetic acid
oThis happens very quickly because AChE has one of the fastest catalytic rates among all
known enzymes
Inhibition of AChE prevents the breakdown of ACh, disrupting transmission at cholinergic synapses
on skeletal muscle and heart muscle