Neuropharmacology Techniques PDF

**[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 electrophysio...

**[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 [Case story -- Otto] A person\'s face in a text Description automatically generated - Known for his discovery of acetylcholine  - Use of 2 frog hearts -- the hearts where placed in two separate dishes with liquid which were interconnected with a tube. - The vagus nerve was connected to one heart. - Stimulation of the nerve = decrease in heart rate in heart 1. - The decrease from heart 1 transmitted to heart 2 causing a heart rate decrease in 2. - Conclusion = stimulation of the nerve releases a substance which can transmit from one to another + provoke a response/action -- substance must have been mediating the action. - Substance = acetylcholine [Neuropharmacology research + techniques:] A diagram of a medical procedure Description automatically generated [Neuropharmacological testing:] Ligand testing involves a number of steps: 1. Choice of test system / preparation (in vitro, in situ or in vivo) a. In vitro = outside the living organism b. In vivo = within the living organism c. In situ = to examine the phenomenon exactly where it occurs 2. Route of ligand administration / delivery (will be system dependent, but may include microiontophoresis, pressure ejection, bath application, etc.) - elecrodes 3. Equilibration (penetration, distribution) -- how deep the drug can reach the system 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) -- need a control so if we apply a drug + see its effect, the drug needs to be washed out to show the effect produced goes away. [The technique of microiontophoresis:] - Goal = to deliver a charged substance i.e. acetylcholine to a discrete site within the experimental preparation. 1. Prepare micropipettes from glass capillary tubes using a horizontal micropipette puller -- 5 mm taper to provide stiffness -- longer tapers = more flexible. 2. Backfill the pipettes with acetylcholine (or test solution) -- make sure no bubbles are present a. To remove air bubbles -- hold pipette with tip down and gently click the micropipette. 3. Secure micropipette into a micropipette holder + then onto a micromanipulator 4. Connect the positive + negative wires to microiontophoresis programmer. 5. Micropipette tip is positioned adjacent to site of interest with a negative retaining current is set to prevent leakage of acetylcholine. 6. Silver wire is secured to edge of the preparation to serve as a reference electrode. 7. Negative wire is connected to reference electrode. Positive is connected to the external pin of the micropipette holder. 8. Acetylcholine is ejected from the micropipette by applying a positive current at a defined intensity + duration whilst recording fluorescence and diameter responses in the micro-circulation. 9. The signals measure calcium fluorescence which underlies endothelium dependent vasodilation of arterials controlling tissue blood flow. What can this method be used for? - Provides insight into the cellular signalling events that underlie blood flow control and arterials - Can be applied to neurophysiology to study synaptic transmission and activation of ion channels. The effectiveness of this method is determined by: - Internal diameter of the tip - Concentration of agonist in micropipette - Intensity + duration of the ejection current [Electrophysiological recording:]  - micropipette = sharp electrodes - electrodes used are very thin + sharp - can penetrate through a neurone or membrane + measure membrane potential A collage of diagrams and diagrams Description automatically generated - A = squid axon with an electrode inside -- allows for measurement of action potentials + understanding of sodium and potassium current which defines the shape of an action potential. - D = shows an **amplifier** on both sides measuring the potential difference across the membrane. Voltage clamp technique:  - Most simple form of this technique: - one electrode inside and one outside - use a voltmeter to measure the voltage difference across the membrane. - More complex amplifier: - Can set the membrane voltage to a specific number i.e. -70mv - Amplifier reads the output of neurones. - Can use another circuit to inject current into the perforation -- by adding or removing current, it would keep membrane voltage constant. - Keeping voltage constant allows us to put in voltage steps i.e. open + close certain channels and measure the activities. - Uses a feedback + control of the voltage of the membrane consciously [Electrophysiology kit:] A collage of several scientific equipment Description automatically generated - The tv have been replaced by computer screens in modern day -- allows us to visualise the cell + identify neurones + to guide the sharp/patch electrodes to desired location to measure the currents. [Sharp electrode recoding from motor neurones:] - Used to record neurotransmitters intracellularly + used to prove neurotransmission was occurring chemically and not electrically in most of the synapses in the CNS. [Intracellular recording of EPSPs and IPSPs]  - Potential = measure of voltage difference if measured intracellularly - Positive response = EPSP - Due to sodium influx depolarisation - Negative response = IPSP - Due to influx of chloride ions - NOTE: not in all cases as it depends on concentration gradient + in some cases i.e. chloride ions may not come in therefore leaving a positive response instead of a negative one. - Current = IPSC or EPSC [Extracellular vs intracellular recording:] - Can't clamp extracellularly as electrodes are placed in the extracellular fluid -- can still stick in electrodes to measure membrane potential. - Extracellular -- hippocampal slice - Negative response = the sodium ions are disappearing extracellularly as they enter within the neurones which is why we see a negative instead of a positive response. - B = inhibitory axon coming in -- terminate in this location as they can control action potential firing - Inhibition has a massive effect to prevent firing. [Patch-clamp techniques + study of ion channels:]  1. Place large pipette of 20 µm (or smaller depending on neurone size) -- the electrode is made up of polished glass. 2. The glass glues to the fat on the membrane and once pressure is applied, you can retract pieces of debris + measure function of channels using the membrane piece. 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. [Patch-clamp technique + the study of ion channels:] A diagram of a cell membrane Description automatically generated with medium confidence Patch-clamp varieties:\ a) cell attached\ b) inside-out patch -- rip out a patch -- the inside of the cell is outside -- can see how drugs are activating internal components of receptor transporter systems.\ c) whole-cell mode -- membrane below pipette is ruptured allowing access into the cell + measure/control the membrane voltage and current flow into the cell.\ d) outside-out patch -- if you pull the electrode up, the membrane will pull up and eventually fuse to make a bubble -- the outside of the membrane is one side and the inside of the membrane is on the other.\ (also, perforated patch, loose patch, etc.) [Choices of the system -- in vitro:]  - can insert DNA into eggs + produce channels -- can use patch-clamp or voltage techniques. - Eggs are quite large -- can insert 2 electrodes + control/measure the voltage across the membrane quite precisely. - HEK cells are often used -- measure fluorescence signals. A diagram of a plant life cycle Description automatically generated - i.e. how the GABAergic system has been studied - use of human brain or tissue 1. homogenize the tissue + produce RNA and microinject into oocytes 2. homogenize the tissue + collect vesicles and microinject into oocytes. - Eggs would express channel - Addition of GABA would produce a response - Can construct a concentration response curves  - Can plot a sigmoidal graph based off the responses + use it to determine the potency of GABA in these neurones. - Can do this from brain membranes and look at different neurotransmitters. [Choices of the system -- in vitro *(HEK cells)*] A diagram of a graph Description automatically generated with medium confidence - Can test the effect of antagonists on the receptors to inhibit the calcium signal. - Determines selectivity of different drugs on different receptor types  - Can be applied to real neurones -- can release an agonist to the synapses + study the inhibition of the antagonists. - Can see if the real neurones match responses from expression system [Choice of the system - in situ (slices):] A diagram of a graph Description automatically generated with medium confidence - AP5 is applied to inhibit STP + LTP -- applied AP5 locally - After washing of the drug STP + LTP are induced again. Choice of the system - in vivo (behaving animals)  - AP5 induced -- animal can't escape - Control rat can escape. - Principle = drug deliver to a specific site [Choice of the system - in vivo (behaving animals)] A diagram of a training process Description automatically generated with medium confidence - ZIP. Infuses the rats forget about the shock zone in red