Lecture 3 Cellular Neuro methods.pptx

Methods of research in cellular neuroscience Andy Trevelyan 2024 CELLULAR AND NETWORK NEUROSCIENCE: BMS2011 - Lectures 3 [email protected]  Textbook resources Excellent entry level...

Methods of research in cellular neuroscience Andy Trevelyan 2024 CELLULAR AND NETWORK NEUROSCIENCE: BMS2011 - Lectures 3 [email protected]  Textbook resources Excellent entry level Popular with students More hard core, but outstanding on cellular neuroscience  The squid giant axon https://www.youtube.com/watch? v=CXCGqwdtJ78 Below link to book companion to understanding the Hodgkin Huxley papers https://physoc.onlinelibrary.wiley.com/pb-assets/assets/14697793/classics/A%20compa nion%20guide%20to%20the%20Hodgkin-Huxley%20papers.pdf Link below to all the original Hodgkin Huxley papers and the amended versions compiled by Angus Brown https://physoc.onlinelibrary.wiley.com/hub/journal/14697793/classicsupdated  Voltage clamp technique unveils voltage- dependent currents Hodgkin & Huxley (1952a-d) J.Physiology; Nobel Prize, 1963  Voltage clamp technique unveils voltage- dependent currents Hodgkin & Huxley (1952a-d) J.Physiology; Nobel Prize, 1963  Voltage clamp technique unveils voltage- dependent currents Hodgkin & Huxley (1952a-d) J.Physiology; Nobel Prize, 1963 Voltage clamp – analysis of voltage- dependent conductances I K+ Na+ “Active” gK V EK ENa “Passive” gK  Voltage clamp technique unveils voltage- dependent currents Hodgkin & Huxley (1952a-d) J.Physiology; Nobel Prize, 1963 th Analysis of firing patterns using current clamp Nav1.1 mutations cause reduced excitability in cortical interneurons, leading to epilepsy  Cellular electrophysiology techniques Voltage clamp – fixing the voltage at different levels to identify how voltage affects membrane conductance – voltage-dependent conductance – aka “active” or “non-linear” conductance Current clamp – a closer simulation of normal neuronal behaviour – analysis of firing patterns Patch clamping: Sakmann and Neher Video of Sakmann talking about his work is at http://jp.physoc.org/site/misc/editorinterviews.xhtml#bertsakmann The whole cell response is equivalent to the average of many individual channels Voltage clamp comman Individual trials Ensemble mean adapted from Hille, Original research: Patllak & Ortz, 19  Open times b = phenobarbitone c = pentobarbitone Close times PB = pentobarbitone Barbiturates: “locks” GABA receptors in the open state Similar analysis of benzodiazepines: eases opening of receptors Both enhance GABA action, but do so by different mechanisms.  Extracellular recordings V Na+ K+ +50mV -70mV Extracellular recordings V Na+ K+ +50mV -70mV Extracellular recordings V Na+ K+ +50mV -70mV Extracellular recordings distance = speed delay V Na+ K+ +50mV -70mV  Extracellular recordings Distant reference electrode V Na+ K+ +50mV 100µm -70mV aki et al (2012) The origin of extracellular fields and currents – EEG, ECoG, LFPs and spikes Nature Ne  Extracellular recordings aki et al (2012) The origin of extracellular fields and currents – EEG, ECoG, LFPs and spikes Nature Ne oger Tsien, Nobel Prize 2008 The brainbow mouse Ca2+ imaging: single cell resolution imaging of activity patterns  General Neuronal Cell division Transduces electrical into Cell death chemical signals Cell structure Axon and dendritic elaboration and retraction Cell motility Synaptic vesicle release Organelle motility Synaptic plasticity Gene expression Why is Ca2+ doing everything?. Ca2+ is a common ion. Easy to construct proteins which bind Ca2+ and change sha 2-photon microscopy allows imaging deep into living tissue double single photon photon ptogenetics: using light to control the bra https://www.ibiology.org/neuroscience/optogenetics/  “Optogenetics” Controlling neural activity with light Excitatory Channelrhodopsin ChR: non-specific cation channel – excitatory Inhibitory Halorhodopsin NpHR: electrogenic Cl- pump – drives chloride into cells Archaerhodopsin Arch: electrogenic H+ pump – drives protons out of cells Zhang, Deisseroth et al. (2007) Nature 446, 633-639  Optical dissection of Parksinonian neural Gradinaru et al (2009) Science circuitry eep brain stimulation of subthalamic nucleus (STN)  good control of intention tremor oes this work by stimulating local neurons or fibers of passage? (OH)DA injection – kills neurons in substantia nigra  Parkinsonian symptoms 1. Halorhodopsin in local neurons 2. Halorhodopsin in local axons  Chemogenetics: DREADDs Designer Receptor Exclusively Activated by Designer Drugs human M3 muscarinic DREADD – coupled to Gq signalling pathway – activates neurons - excitatory human M4 muscarinic DREADD – coupled to Gi signalling pathway – opens K+ channels and silences neurons - inhibitory Activated by clozapine-N-oxide (CNO) – CNO has no native activity  Cell-specific targeting Different cell classes express different sets of protei stribution patterns of GluRA3 (left) and GluRA4 (right) Cortical chandelier cell Monyer, Seeburg, Wisden (1991) Neuron parvalbumin-positive Cell-specific promoters are like the cell’s IP address They can be used to target these classes of  Summary Neurons communicate using electrical signals – earliest studies of brain activity utilized this fact Latterly many new techniques have been developed – each typically allowing scientific breakthroughs by enabling experimentation Optical studies bring many advantages These different techniques have different advantages These can be complementary, and the choice of technique needs to be matched to the question. Artificial proteins can be targeted to very precise locations, so that optical read-out or manipulation only occurs at that site. Key issues are considerations of accessibility, and temporal and spatial resolution Non-invasive recording and stimulation now possible