Studying Neurotransmitter SystemsImmunocytochemistrySlide 3Slide 4In Situ HybridizationSlide 6Slide 7Slide 8Genetic ModificationsGain-of-Function/Loss-of-Function ExperimentsSlide 11SummaryStudying Neurotransmitter Systems•How do we study neurotransmitter systems?–Electrophysiological recordings–Pharmacology–Staining•Immunocytochemistry•In situ hybridization–Genetic manipulations•What is a gain-of-function/loss-of-function experiment?ImmunocytochemistryAntigenYou have antibodies for all the enzymes involved in the synthesis of all catecholamines. Using immunocytochemistry, how would you identify dopaminergic neurons (and distinguish them from noradrenergic and adrenergic neurons)?a) Stain for THb) Stain for DBH and PNMTc) Stain for TH and DBHd) Stain for TH and Dopa decarboxylasee) Stain for PNMT and Dopa decarboxylaseIn Situ HybridizationTo identify serotonergic neurons using in situ hybridization you would stain with a probe that is antisense to:1. The serotonin gene2. The VMAT (vesicular monoamine transporter) gene3. The tryptophan gene4. The tryptophan hydroxylase gene5. The gene for the serotonin receptorWould you be able to determine where a protein is localized using in situ hybridization?a) Yesb) NoGenetic Modifications•Knockout•Knockdown•Overexpression•Exogenous ExpressionGain-of-Function/Loss-of-Function ExperimentsYou have found a membrane protein that you think interacts with SNARE proteins to make the SNARE complex more sensitive to calcium and therefore the neuron should release more vesicles per action potential. Design a gain-of-function and a loss-of-function experiment to test your hypothesis.Summary•We study neurotransmitter systems using:–Electrophysiological recordings–Pharmacology–Staining•Immunocytochemistry uses fluorescently tagged antibodies to label proteins•In situ hybridization uses fluorescently tagged antisense probes to label mRNA–Genetic modifications can change the amount of mRNA and therefore protein a cell expresses•GOF and LOF experiments are used to test the hypothesized function of a molecule by either adding it to or removing it from the appropriate context to see if it correlates with a gain or loss of the hypothesized
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