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Lecture 13

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McGill University
PHGY 313
Sebastien Breau

Studying Neurotransmitter Systems: • Certain criteria must be met to distinguish a molecule as a neurotransmitter: o The molecule must be synthesized and stored in the presynaptic neuron o The molecule must be released by the presynaptic axon terminal upon stimulation o The 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: o Molecule is concentrated in the brain tissue o Application 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 o Once the neurotransmitter candidate has been chemically purified, it is injected into the bloodstream of an animal, where it stimulates an immune response o The 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 o This 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 o Complementary strand is called a probe o Process 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 o Since 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 o After 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 tra
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