PSYC 410 Lecture Notes - Lecture 7: Acetylcholine Receptor, Kainite, Skeletal Muscle

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26 Jan 2013
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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:
Immunocytochemistry- a method used to anatomically localize particular molecules to
particular cells
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
generated
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
molecules
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!
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