Techniques for Investigating Brain Function PDF

Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons LECTURE 1: ELECTROPHYSIOLOGICAL METHODS IN ANIMALS All the electrophysiological methods have the same advantage, because they directly measure the el...

Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons LECTURE 1: ELECTROPHYSIOLOGICAL METHODS IN ANIMALS All the electrophysiological methods have the same advantage, because they directly measure the electrical ac/vity of neurons, hence why they have a good /me resolu/on (ms). The space resolu/on can be quite good, up to the synap/c channels. We can also use in vivo behaving experiments by placing electrodes in different brain regions in animals and follow the synap/c ac/vity – the only problem is the limited sampling because we cannot place thousands of electrodes. à it’s easy to go into the brain in animal but there is no much change between animals and humans A. HISTORY: THE FIRST NEURONAL RECORDINGS WERE MADE IN ANIMALS à the first recording was in animal not in humain Galien said that the seat of the soul is in the brain (especially the leN ventricle?), connected to the nerves that control the body through the circula/on of animal spirits. à Thing about ac/on poten/al 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons In the 18-19th century, scien/sts (Galvani, Volta, Aldini) observed that applying electricity to nerves led to muscles contrac4ons (they could see it, not record it). à This led to the crea/on of the Galvanometer, the first electric recording machine of current in nerves. Galvaniser à Donner de l’energie Volta à Donne les volt à il a inventé la baWerie Aldini à Donnait des chocs électriques aux cadavres à Donc probablement que l’électricité permet de contracter les muscles Probably élctricity is the way for the nervs to communic They (Loewi and Dale) also discovered that the Nervous System was working with electricity as well as with chemical compounds: Communica/on between neurons was electrochemical. A molecule slows frog heart – this molecule was acetylcholine, first NT discovered. Electrical signals travel through neurons but their communica/ons are electrochemical (Nobel 1936) OWo Loewi (1921): a molecule slows frog heart Henry Dale (1936): acetylcholine, first neurotransmiWer discovered Il a pris des cœurs de grenouilles car ils con/nuent à baWre même dehors du corps à Un deuxième cœur connecté à l’autre qui bat aussi donc il y a forcément une molécule qui permet de faire ça 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons The inside of the axon at rest is more nega/ve than the outside. It is traversed by fluctua/ons with poten/al reversal: the ac/on poten/al (AP) Hodgkin et Huxley: studies on the giant squid axon (Nobel 1963) LTP à Long terme poten/a/on of excitatory synapses in the rabbit hippocampus aNer s/mula/on of the perforant path. B. WHAT ARE THE PRINCIPLES UNDERLYING THE RECORDING OF NEURAL SIGNALS The membrane poten4al of neurons: the heart of the reactor. The difference in poten/al on either side of the membrane, a form of energy for cellular processes: We don’t have the same concentra/on of ions on either side of the membrane. 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons à Inside the cell you have more protein than outside, and this is why it is more - The membrane also is filled with different ion channels with selec4ve permeability (always open) which create flows of ions (electric currents) which depend on: - Concentra/on of ions = Osmo/c force - Of their charge and of the membrane poten/al = Electric force (electrical poten/al). - And their reversal (or equilibrium poten/al – Eion; Nernst equa/on), which is when the osmo4c and electric forces cancel each other out and reach a certain ion equilibrium where ions stop moving. This equa4on depends on the ion concentra4on. There also are leakage channels and gated channels, that open depending on: - voltage-gated (membrane voltage – open at a specific membrane poten/al) - Ligand-gated – will open when a specific molecule connects to them - S/mulus-gated – useful for sensory receptors All of these channels are specific to an ion. There also are pumps (energy dependent) that allow to maintain the membrane poten4al. When the channels open, the ions passively cross the membrane driven by osmo/c and electrical forces and according to their reversal poten/al 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Why Vm stabilises around -60/-70 mV? Let’s imagine, at the star/ng ion concentra/on a membrane with only potassium leakage channels: La membrane n’est pas imperméable, il y des fuites! The K+ comes out (osmo/c + electrical forces), Vm goes down to -90/- 100mV We add sodium leakage channels: Na+ enters (osmo/c + electrical forces), the membrane poten/al rises to -60/-70mV Ions travel, how concentra4ons do not change? Thanks to the sodium/potassium pump! Very energa/cally expensive Pump potassium sodium à Permet de garder un équilibre 1. SYNAPTIC POTENTIALS AND ACTION POTENTIALS These proper/es of the membrane give birth to the neuronal signals that we record à AP traveling across axons and ac/va/ng synapses à Synapses genera/ng Post Synap/c Poten/als: - Excitatory synapses (E; mostly open Na+ channels): Excitatory Post Synap/c Poten/al (EPSP) - Inhibitory synapses (I; mainly open Cl- channels): Inhibitory Post Synap/c Poten/al (IPSP) 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons The summa/on of EPSPs and IPSPs “decide” on the genera/on of the Ac/on Poten/als (AP) Pourquoi créer un poten/el d’ac/on ? à Parce que le neurone est très grand EPSP et IPSP va décroitre avec la distance mais pas les poten/els d’ac/on qui vont toujours avoir la même force 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons à L’électricité est plus rapide que les poten/els d’ac/on Pourquoi il va toujours dans le même sens ? à Parce qu’il y a un temps de réfrac/on et tu ne peux pas retourner en arrière C. ANIMALS MODELS: VALIDITY, ADVANTAGES, AND LIMITS If the ques/on is to understand our brain, how good is the animal model? Can we understand the human brain by looking at the animals’ – It is true, our brain differs in size, cor/ces and circumvolu/ons compared to other animal’s brain. The morphology of our brain is not totally iden4cal to one of a mouse, but they are preOy similar, yet, humans and rodents brains share fundamental physiological, historical and anatomical characteris4cs (cf. different slides comparisons). In conclusion, animals models are very useful for a variety of reasons. 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Humans and rodents brains share fundamental physiological, histological and anatomical characteris/cs Fundamental neuroanatomy: à Same embryologyc divisions: rhombencephalon (medulla, pons, cerebellum), mesencephalon (midbrain), prosencephalon (di+telencephalon) à Same cor/cal types: paleocortex, archicortex, neocortex Humans and rodents brains share fundamental physiological, histological and anatomical characteris/cs Fundamental units are neurons and astrocytes - Human brain= 100 billion neurons - Human cortex= 12 billion - Rat cortex= 15-30 million - Mouse cortex= 4 million à In both species, neurons make about 10.000 synapses each à No specific neuronal types in human? 6-layered neocortex, layers defined by cytoarchitecture and connec/vity 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons A good model: - Very similar to the human one - Similar fundamental physiological and histological characteris/cs - Similar fundamental neuroanatomy: mostly crossed, brainstem and thalamic relays before cortex, cortex - Cortex divided in similar func/onal areas - Similar func/onal and anatomical organiza/on: re/notopic, tonotopic and somatotopy Advantages: - invasive electrophysiology - experimental lesions - environmental manipula/ons - pharmacological studies - gene/c manipula/ons to study molecular basis of networks func/on and pathologies: Knock-out, Optogene/c, Dreadd… Limits: - Behavior? - Consciousness? - Language? - Ethic? « the benefit to humans does not jus/fy the harm to animals » D. WHAT ARE THE MOST USED METHODS IN ANIMAL ELECTROPHYSIOLOGY? Acquisi4on methods vary in terms of spa4al scales (µm to cm) and neural signal types (AP, synap/c ac/vi/es, ion channels). Depending on where we put the electrodes, we will measure different types of signals: - Surface recordings: from pia (ECoG), bone or scalp (EEG); field poten/als “not so local" and signal sources not always directly localized near the electrodes. - Extracellular electrode recordings: By introduc/on an electrode inside the brain but outside of the neuron (extracellular space). Ac/on poten/als (APs) of single (SU) and mul/ple (MUA) units “locally”, Local field poten/als (LFP). - Intracellular electrode recordings: single neuron, electrode in the cell body. - Patch clamp: single neuron and individual ion channels – electrode at the surface of the neuron. 1. INTRACELLULAR RECORDINGS: POSTSYNAPTIC POTENTIALS AND APS. Usually done in culture or slices prepara/ons because difficult to do in vivo. A glass micropipeWe filled with electrolyte (e.g. 3mol/l KCl) is inserted into the neuron à we then record the membrane poten/al inside the neuron - we also need a microelectrode amplifier to be able to record the signal. APs can be recorded, as well as EPSPs. 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons 2. PATCH CLAMP RECORDINGS Animals must not move – hence why also done in culture or slices prepara/ons. We use a glass electrode, to the surface of the cell to record the neuron’s ac/vity. The goals are to inves4gate the electrophysiological proper4es of ion channels and neurons: (1) membrane capacitance, conductance, input resistance, (2) Ions channels proper/es, including receptors, (3) EPSPs and IPSPs, (4) Firing frequency and paWerns of APs. There exist 4 different patch clamp configura/ons by going to the surface of the cell (neuron). - Cell-aOached: We just put the electrode at the membrane, we exert a suc/on that will aWach a small part of the membrane to the /p - Inside out/outside out: Removing the patch that we aWached during the cell-aWached – isola/ng of small patch of membrane to manipulate the concentra/on of liquid around this membrane (e.g. modifying the concentra/on inside and outside the membrane isolated. - Whole-cell: Most commonly used in patch-clamp. (1) we approach the part of the cell we are interested in, by pu}ng some mild posi/ve pressure. (2) Seal-forma4on: Applying a mild suc/on to aWach the neuron to the pipeWe, (3)Whole-Cell configura4on: brief and stronger suc/on to 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons break the membrane to be in direct contact with the intracellular matrix of the neuron (can record EPSPs and IPSPs of all channels at the same /me). We also have different modes: - Voltage-clamp mode: keeps the voltage constant to study ionic currents – measuring the current, measuring ionic currents from specific synap/c receptor channels (voltage or ligand ac/vated) to study synap/c excitability. - Current-clamp mode: Inducing currents to study the varia/ons of the Vm – study of the intrinsic excitability of neurons, the membrane poten4al varia4ons. Evoked poten/als and study the different paWerns of APs. What are the main contributors to the LFPs signals and what does represent LFP? Summa/on of all EPSPs and IPSPs happening around the neuron – the synap/c ac/vi/es more than APs because they easily sum up. 3. EXTRACELLULAR RECORDINGS Extracellular recordings: APs and LFP 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Separate APs and LFP Illustra/on: Duplicate the signals and apply different filters - 1-150 for LFP - 8000-6KHz for APs - Average responses across s/muli What are the main contributors to the LFPs signals? What does represent LFP? LFP contributors? … synap/c ac/vi/es more than APs; see preceding lessons about EEG sources And for those that are interested: Nature Reviews (2012) “The origin of extracellular fields and currents — EEG, ECoG, LFP and spikes” György Buzsáki, Costas A. Anastassiou and Christof Koch 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Intracellular vs extracellular APs: same events, inverse polarity How many recording sites can we target into the brain? 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Extracellular recordings: APs and LFP Careful with extracellular recording because the shape of APs is different – upside down. Can we isolate APs from specific neurons ? Yes, with the extracellular recordings Can we determine the iden44es of these neurons ? More difficult but feasible by knowing the shape of EPSPs or IPSPs we can connect it to a type of neuron (iden/ty). Sampling rate issues ? We need lots of filters and amplifiers either manually or using algorithms (clustering algorithm) to isolate APs when the threshold has been reached. 4. JUXTACELLULAR There’s no neuron altera/on because we are going close to the neuron but are not applying suc/on, which allows an extracellular APs recording. It is possible in vivo and allows staining of the neuron using bio/n. Once the neuron has been recorded for a while (and the animal dead), we can recognize the type of neuron it was (post-hoc iden/fica/on of the neurons by staining). Between patch and extracellular: Juxtacellular - Close to the neuron but no suc/on - Record extracellular Aps - Possible in vivo and allow staining of the neuron Example of a juxtacellular in vivo recording study combined with: Simultaneous surface - EEG and EMG - Post-hoc Iden/fica/on of the neurons by staining 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Schema/c ascending pathway 1. Trigeminal nucleus à Easy to study sensory processing aWhe neuronal level in vivo 2. Thalamus 3. Cortex 2ème Master 2024/2025  Alexis Hervais-Adelman Techniques for inves/ga/ng brain func/ons Illustra4on: combined intra/surface recordings in an epilep4c mouse 2ème Master 2024/2025