Techniques in Neuropharmacology 2024-2025 Lecture Notes PDF

Techniques in neuropharmacology @Arturas Volianskis Xenopus eggs...

Techniques in neuropharmacology @Arturas Volianskis Xenopus eggs tropicalis male Xenopus laevis female Shields R (2011) Breaking the Hybrid–Species Barrier. PLoS Biol 9(11): e1001201.  Techniques in neuropharmacology @Arturas Volianskis Xenopus eggs tropicalis male Xenopus laevis female Swammerdam (1637 – 1690) Shields R (2011) Breaking the Hybrid–Species Barrier. PLoS Biol 9(11): e1001201. Techniques in neuropharmacology Learning outcomes: 1. Introduce the (ultimate) neuropharmacology experiment and review the relation of neuropharmacology to the other neuroscience research areas 2. Introduce microiontophoresis; in vivo and in vitro techniques in electrophysiology 3. Discuss testing of ligands in expression systems in vitro or in situ and in vivo BI2432: Fundamental neuropharmacology Case story - Otto’s dream & the pharmacology experiment Otto Loewi “The night before Easter Sunday of that year (1873-1961) I awoke, turned on the light, and jotted down a few notes on a tiny slip of thin paper. Then I fell asleep again. It occurred to me at six o’clock in the morning that during the night I had written down something most important, but I was unable to decipher the scrawl. The next night, at three o’clock, the idea returned. It was the design of an experiment to determine whether or not the hypothesis of chemical transmission that I had uttered seventeen years ago was correct. I got up immediately, went to the laboratory, and performed a simple experiment on a frog heart according to the nocturnal design.” Otto Loewi BI2432: Fundamental neuropharmacology Case story - Otto’s dream & the pharmacology experiment BI2432: Fundamental neuropharmacology Neuropharmacology research & techniques Neuroscience neuroanatomy Medicinal chemistry neurophysiology Neuropharmacology cognitive sciences chemical methods, clinical disciplines pharmacodynamics & drug-receptor interaction, pharmacokinetics modelling & design in vitro in situ (ex vivo) in vivo Techniques may include: Neuropharmacology is a term describing the radioligand binding assays, study of drugs that affect the nervous autoradiography, system, whether they affect sensory microiontophoresis, perception, motor function, seizure activity, molecular biology, mood, higher cognitive function, or other electrophysiology, forms of nervous system functioning. imaging, But we are trying to limit ourselves to behavioural and cognitive testing, neurotransmitter pharmacology! clinical trials, etc. BI2432: Fundamental neuropharmacology Neuropharmacological testing Ligand testing involves a number of steps 1.Choice of test system / preparation (in vitro, in situ or in vivo) 2. Route of ligand administration / delivery (will be system dependent, but may include microiontophoresis, pressure ejection, bath application, etc.) 3. Equilibration (penetration, distribution) 4. Testing of pharmacological effects, establishing concentration (dose) / response relationships (electrophysiology and / or imaging) 5. Washout / reversal of pharmacological effects, or control experiments (specificity of the pharmacological effect) BI2432: Fundamental neuropharmacology The technique of microiontophoresis BI2432: Fundamental neuropharmacology Electrophysiological recording Extracellular electrodes - e.g. Intracellular (or sharp) microiontophoresis, but also recording electrode Scale-bar 5 μm ! BI2432: Fundamental neuropharmacology Electrophysiological recording BI2432: Fundamental neuropharmacology Electrophysiological recording https://youtu.be/3F-Uw1RZqAQ BI2432: Fundamental neuropharmacology Electrophysiological recording I=V/R I = current (A) Ohm’s law V=I*R V = voltage (V) R=V/I R = resistance (Ω) BI2432: Fundamental neuropharmacology Some electrophysiology kit Extracellular electrodes In-vivo electrophysiology in live and anaesthe=sed animals Combined imaging/electrophysiology Techniques for op=cal s=mula=on / excita=on BI2432: Fundamental neuropharmacology Sharp electrode recording from motorneurons Intracellular recording APs BI2432: Fundamental neuropharmacology The sound of action and synaptic potentials APs BI2432: Fundamental neuropharmacology Intracellular recording of EPSPs and IPSPs More Positive EPSP ions EPSP = excitatory postsynap=c poten=al ions More IPSP Negative IPSP = inhibitory postsynap=c poten=al APs BI2432: Fundamental neuropharmacology Extracellular vs intracellular recording Local field poten=al (LFP) Intracellular ions EPSP Membrane poten=al IPSP ions Extracellular (excitatory) 1 / 15 s S.r. responses f-EPSP 5s 0.5 mV CA1 5 ms CA3 BI2432: Fundamental neuropharmacology Patch-clamp technique & the study of ion channels Gigaseal: a. electrode touching the cell (left) and after formation of “gigaseal” and suction (right) b. recording of electrical activity before and after “gigaseal” formation. c. examples of channel openings, note the difference in noise levels. BI2432: Fundamental neuropharmacology Patch-clamp technique & the study of ion channels Patch-clamp varieties: a) cell attached b) inside-out patch c) whole-cell mode d) outside-out patch (also, perforated patch, loose patch, etc.) BI2432: Fundamental neuropharmacology Choice of the system - in vitro Testing of ligand function - in vitro Neurotransmitter receptors can be eggs expressed in a system that usually lacks that particular cell receptor type: e.g. Xenopus Xenopus oocytes or HEK293 cells tropicalis male Xenopus laevis (human embryonic kidney cells). female (Immortalised cell lines, permanently expressing receptors, might be also available for some receptor types.) Receptor function can be probed application of agonists and antagonists and responses can either be recorded electrophysiologically or using imaging techniques. (However, receptor properties and function might be different from in situ and in vivo, controls are needed.) BI2432: Fundamental neuropharmacology Choice of the system - in vitro (Xenopus oocytes) Diagram of the procedures used to transplant neurotransmitter receptors from the human brain to the oocyte plasma membrane by injecting either brain cell membranes (Upper) or brain mRNA (Lower) into Xenopus oocytes. Miledi R, et al. PNAS, 2002 https://doi.org/10.1073/pnas.192445299 BI2432: Fundamental neuropharmacology Choice of the system - in vitro (Xenopus oocytes) Voltage-clamp recording Time dependent expression Concentra=on-response rela=onship Miledi R, et al. PNAS, 2002 https://doi.org/10.1073/pnas.192445299 BI2432: Fundamental neuropharmacology Choice of the system - in vitro (Xenopus oocytes) Voltage-clamp recording Time dependent expression Concentra=on-response rela=onship Miledi R, et al. PNAS, 2002 https://doi.org/10.1073/pnas.192445299 BI2432: Fundamental neuropharmacology Choice of the system - in vitro (HEK293 cells) Fluorescence imaging Volianskis, et al. (2013) J Physiol 591, 955-972 BI2432: Fundamental neuropharmacology Choice of the system - in situ (slices) Voltage-clamp recording Hippocampal slices Volianskis, et al. (2013) J Physiol 591, 955-972 BI2432: Fundamental neuropharmacology Choice of the system - in situ (slices) Extracellular recording Hippocampal slices Microiontophoresis Specificity - washout STP LTP STP LTP PTP Collingridge, G.L., Kehl, S.J., McLennan, H.,. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. 1983 J. Physiol. (Lond.) 334, 33–46. Graham Collingridge Jeff Watkins BI2432: Fundamental neuropharmacology Choice of the system - in vivo (behaving animals) Behavioural tes=ng Intraventricular AP5 Specificity (L-AP5) BI2432: Fundamental neuropharmacology Choice of the system - in vivo (behaving animals) Behavioural tes=ng Intrahippocampal ZIP Specificity (saline) Pastalkova et al, 2006, Science, 313:1141-1144. BI2432: Fundamental neuropharmacology Neuropharmacological testing - summary Ligand testing involves a number of steps 1.Choice of test system / preparation (in vitro, in situ or in vivo) 2. Route of ligand administration / delivery (will be system dependent, but may include microiontophoresis, pressure ejection, bath application, etc.) 3. Equilibration (penetration, distribution) 4. Testing of pharmacological effects, establishing concentration (dose) / response relationships (electrophysiology and / or imaging) 5. Washout / reversal of pharmacological effects, or control experiments (specificity of the pharmacological effect) BI2432: Fundamental neuropharmacology Example question L4: Which of the experiments is performed in-situ? (A) Experiment in an anesthetised animal (B) Experiment in an acute tissue preparation (C) Experiment in a receptor expression system (D) Experiment in a tissue culture (E) Experiment in an organoid BI2432: Fundamental neuropharmacology Study materials: BI2432: Fundamental neuropharmacology Weekly schedule of the fundamental neuropharmacology Friday 29.11.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L1. Introduction to fundamental neuropharmacology Rang & Dale’s Pharmacology, L2. Basic principles of neuropharmacology I & lecture materials Friday 06.12.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L3. Basic principles of neuropharmacology II Rang & Dale’s Pharmacology L4. Techniques in neuropharmacology & lecture materials Friday 10.12.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L5. Acetylcholine and Glutamate (and a bit of Glycine) Rang & Dale’s Pharmacology L6. Pharmacological dissection of field responses The Hippocampus Book pages 27-30 & lecture materials Tuesday 07.01.2025 (13:10-14:00);C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L7. GABA and Glycine Rang & Dale’s Pharmacology & lecture materials Friday 10.01.2025 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L8. Catecholamines Rang & Dale’s Pharmacology L9. Serotonin & lecture materials Friday 27.01.2025 (13:00-13:45 & 14:00-14:45); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L10. Neuropharmacology of drug dependence and addiction I Rang & Dale’s Pharmacology L11. Neuropharmacology of drug dependence and addiction II & lecture materials Tuesday 21.01.2025 (13:10-14:00); C/-1.04 Tuesday 23.01.2025 Neuroanatomy L12. Exam preparation 2 and Neuropharmacology ICA BI2432: Fundamental neuropharmacology