Neuroscience Animal Experimentation Support Cours PDF

# Expérimentation animale en Neurosciences ## Objectifs du module Dr. Chalour ## Introduction Santiago Ramon y Cajal (1852-1934), the father of modern neuroscience in his anatomy laboratory. ### Introduction Ramon y Cajal 1894: the neuronal theory of the brain in opposition to the reticular th...

# Expérimentation animale en Neurosciences ## Objectifs du module Dr. Chalour ## Introduction Santiago Ramon y Cajal (1852-1934), the father of modern neuroscience in his anatomy laboratory. ### Introduction Ramon y Cajal 1894: the neuronal theory of the brain in opposition to the reticular theory. Silver staining of Camillo Golgi which stains a neuron completely (axon, cell body and dendrites). ### The beautiful brain Nobel Prize 1906 Ramon y Cajal and Camillo Golgi for their work on the structure of the central nervous system. ## Modeles animaux A model animal is developed in a given animal species by genetic modifications, induced changes in its environment or its physiology, in order to study a physiological or biological process or the mechanisms of a human pathology. - The relevance in the choice of the species depending on the homology and the expression of the gene studied in the species: for example, rodents (mammals with 90% genomic homology with humans) in the study of human pathologies, primates in the study of cognition and behavior, ... - The relevance in the choice of the species depending on the process studied: for example, zebrafish in the study of clonal lineage of embryonic stem cells. - Genetic modifications: transgenic models. - Environmental modifications: light-dark cycle, diet. ## Objectifs du module 1. Acquire knowledge about techniques used in neuroscience: principle, practical implementation, parameters measured, format and data interpretation. 2. Master reading and analysis of data and results from published studies in the form of articles. 3. Mobilization of acquired knowledge in order to solve a biological problem in the field of neuroscience. ## Méthodes d'études du SNC - **Histology**: specific stains on sections of fixed tissue followed by observation under light and electron microscopy >>> General organization of the tissue, morphological data. - **Immunohistochemistry**: principle of antibody-antigen interaction on sections of fixed tissue followed by observation under light or electron microscopy >>> Expression, localization and expression level of a given protein. - **Electrophysiology**: a technique that allows the recording of electrical activity (currents/potentials) either of individual neurons (patch clamp) or of a group of neurons (patch clamp) or even of the entire brain (EEG). - **Behavioral test**: tests put in place in order to evaluate brain functions such as memory, based on a set of objectively observable reactions of the animal. They are based on the measurement of quantifiable parameters. ## Evaluation des capacités d'apprentissage spatial ### Test de la piscine de Morris Morris Water Maze (MWM) - Rats are trained to find a submerged platform/PF refuge in a circular basin filled with opaque water: the rat swims up the PF. - With training, the rat finds the PF more quickly and reduces the distance swum randomly in the basin before finding the PF. - The normal rat builds a spatial representation of the space using visual cues placed either on the walls of the basin or in the experimental room. - What would happen to rats with hippocampal lesions... ## MWM: Variables dependantes - **Speed** - **Latency time** (time taken to find the PF). - **Distance traveled** to reach the platform. - **Latency time and Distance** highlight the involvement of the hippocampus responsible for spatial mapping and memory of information. - **Speed** relies on the perceptual and motor skills of the subject. ## Dispositif ## Résultats - **First trial** - **After 10 trials** **Control rat** **Rat with hippocampal lesions** ## Transplantation of Induced Pluripotent Stem Cells Improves Functional Recovery in Huntington’s Disease Rat Model ## Etudes utilisant le test MWM - Involvement of a brain region in the processes of spatial learning and memory (spatial navigation). - Role of a gene in spatial learning/spatial memory. - Effect of aging on spatial learning/spatial memory. - Effect of prolonged or chronic stress (release of glucocorticoides or cortisol) on spatial learning/spatial memory. - Evaluation of cognitive abilities in the context of a pathology (for example, to verify if diabetes affects the cognitive abilities of the CNS). - The effectiveness of a pharmacological molecule on the improvement or restoration of spatial learning/memory abilities in animal models (aging, AD or (test of new treatments). ## Electrophysiologie ### Patch clamp sur tranches de cerveau Dr Chalour ## Supports vidéos du test MWM - [Morris Water Maze Experiment](https://www.youtube.com/watch?v=leHLL4vcbCc&t=53s) - [Morris Water Maze and EthoVision XT](https://www.youtube.com/watch?v=MlkoQDT30ЕК) - [Mice with Alzheimer regain their memory](https://www.youtube.com/watch?v=fIHOHV4ZUO4) **IMPORTANT:** The videos are an integral part of the course; it is therefore essential to consult them and to assimilate their content. ## Notions importantes - **Potential**: opposite electrical charges at 2 points create an energy potential whose measurement is called the potential difference (voltage). This potential energy is at the origin of the physiological activity of excitable cells (neurons, muscle cells). - **Electrical current**: displacement of electrical charges from one point to another under the influence of potential energy. - **Ionic current**: displacement of ions through a semi-permeable membrane or the plasma membrane of the excitable cell (ionic channels). - **Resistance**: opposition to the flow of electrical or ionic current. - Substance with a high resistance = insulator. - Substance with a low resistance = conductor. - **Conductance**: capacity to let electrical or ionic current pass. It is the inverse of resistance. ## Loi d'Ohm - Definition: It is the relationship that describes the relationship between the 3 previous notions: potential, current and resistance and which characterizes an electrical conductor: - One can assimilate the plasma membrane of excitable cells to an electrical circuit: - Ions provide the electrical charges. - The plasma membrane which is "impermeable" to ions creates an electrical potential between the extracellular medium and the cytoplasm. - Ionic channels permeable to ions can be assimilated to conductors of electrical currents across the plasma membrane. They are the channels that generate electrical currents by allowing the passage of ions across the plasma membrane. ## Différents types de canaux - **Leakage ion канала** always open. - **Functional ion channels** open in response to: - **Modification of the membrane potential**: voltage-dependent ion channels (CIVD) - The binding of a ligand or neurotransmitter known as **chemically-dependent channels** (CICD) (Ligand-gated ion channels). In short, in light of Ohm's law U = R x I, the opening of active channels allows the rapid diffusion of ions across the membrane, which generates an electric current that results in the modification of the membrane potential. ## Potentiel membranaire de repos - **Origin**: differences in concentrations (unequal distribution) of Na+ and K+ ions between the external face of the membrane bathing in the extracellular medium and the internal face bathing in the cytoplasm. - The cytoplasm being less concentrated in positive charges is more negative than the extracellular medium which is more concentrated in positive charges. - It is - 65mV for neurons. - At rest, through passive channels, there is a net inward Na+ flux and a net outward K+ flux (2 unequal fluxes), maintained by: - Electrochemical gradients of the 2 ions - The difference in membrane permeability to these 2 ions (number of K+ channels >> number of Na+ channels), and - The Na+/K+ pump that maintains the unequal distribution of the 2 ions on either side of the plasma membrane: the expulsion of 3 Na+ ions and the entry of 2 K+ ions. ## Quels sont les rôles des modification du potentiel membranaire? - Local changes in ionic currents that modify the PM serve as signals for: - **Receptive and integration of information**, respectively PPS graded (variable amplitudes, short durations, the potential decreases with the distance traveled, EPSP or IPSP) and global. - **Transmission of information**, it is the action potential. ## 2 sortes de variations du Pm - **Depolarization**: this is the decrease in the PM on either side of the plasma membrane due to the entry of cations (most often Na+) or the exit of Cl-. - The membrane potential becomes less negative (EPSP) and may even reverse generating an AP. ## 2 sortes de variations du Pm - **Hyperpolarization**: this is the increase in the PM on either side of the plasma membrane due to the entry of anions (often of Cl-). - The membrane potential becomes more negative (IPSP) than the resting potential. - The binding of the neurotransmitter to the membrane receptor of the postsynaptic cell will create an excitatory or inhibitory postsynaptic potential (PSP). ## Le potentiel d'action - Transient depolarization of 2ms characterized by: - Unchanging amplitude. - It obeys the all-or-nothing law (notion of threshold). - Propagation without attenuation along the axon (unlike graded PPS). - Due to the opening of the CIVD Na+ and K+. - The frequency of APs gives the code (meaning) of the nervous message. ## Propagation du potentiel d'action ## Exemple d'enregistrement sur un neurone ## Equation de Nernest: potentiel d'équilibre d'un ion - The equilibrium potential is the potential of the membrane for which a given ion will have a zero net flux across its membrane = (the outward flux being equal to the inward flux of the ion >>> thermodynamic equilibrium). Predicts the PM for which the membrane is only permeable to a given ion (only one). ## Equation Goldman Hodgkin et Katz (potentiel de diffusion) - The equilibrium potential is the potential of the membrane for which the total flux of the different ions across the membrane is zero. - If we consider 2 ions K+ and Na+, this means that the outward flux of K+ is equal to the inward flux of Na+. - This is not a thermodynamic equilibrium because there is still a net flux for each ion taken individually. - The potential depends on the concentration gradient and the membrane permeability to a given ion. - The permeability of a given ion at a given time (opening or closing of the channels, their number, ...) affects the membrane potential of the cell. **Advantage:** The GHK equation takes into account the total movements of ions at once >>> Describes a more realistic situation. ## Implication de l'équation GHK ## Combinaison Nernest and GHK - When the permeability of a given ion increases significantly, the potential of the membrane at rest will tend towards the equilibrium potential of that ion. ## Potentiel de Donan - It is the contribution, to the membrane potential at rest, of the macromolecules intracellularly negatively charged such as proteins. ## Patch clamp sur tranches de cerveau - **Definition**: A reference technique in electrophysiology that allows the recording, using a microelectrode, of either: - **The activity of whole neurons** - **The activity of ionic channels present on a fragment (patch) of membrane isolated from the rest of the cell membrane**. - The technique was developed by Erwin Neher and Bert Sakmann in the late 1970s; Nobel Prize in Physiology 1991. - **2 modes of recording:** - **Ionic currents of the channels** ----- **Voltage clamp mode** - **The PM ** ------------ **Current clamp mode** ## Configurations du Patch clamp ## Résumé des principales étapes de l'enregistrement ## Le patch clamp, dispositif et protocole ## Protocole expérimental ## Dispositif ## Patcher un neurones ou une cellule gliale ## Patcher un neurone: image microscope ou camera ## Résultats - The result shown by the oscilloscope for the 3 stages of approach: - **Pipette offset in bath** - **Bath** - **Cell-attached ** - **GO seal** - **Whole-cell patch clamp** ## Introduction - **Results** ## Résultats: caractérisation des canaux ioniques ## Résultats: Exemple d'étude publiée ## Domaines d'utilisation de l'électrophysiologie - **Diagnostic biomedical**: EEG; ECG. - **Research in neuroscience**: - **In vivo**: single electrode or multi-electrode chip - **In vitro**: Brain slices, cell cultures ## Domaines d'utilisation de l'électrophysiologie (suite) - **Innovation and therapeutic**: - **Development of brain-machine interfaces**: for example, this quadriplegic woman who had her first sip of a coffee thanks to this innovation. - **Development of brain implants (multi-electrode)** for spinal cord injuries in order to regain mobility. - **Development of bionic limbs** ## La culture cellulaire ### Cultures neurones/cellules gliales Dr. Chalour ## Introduction - **Definition**: Cell culture is a biology technique used for the proliferation of cells outside of their tissue of origin and to keep them alive in a conditioned culture medium (adapted to their survival). **Interests**: - To overcome the complexity of cellular responses resulting from the interaction with the different cell types comprising a given tissue, or even with other tissues of the organism. - In addition, experimental conditions can be better controlled than in the organism itself. - Evaluation and screening of the therapeutic potential of a large number of molecules at a low cost. - Simplify the system of studying a given cell type or at most 2 cell types (co-cultures). ## Conditions de la culture cellulaire - Culture medium adapted to the cell type: basic medium supplemented with molecules and specific growth factors, proliferation/differentiation medium. ## Conditions de la culture cellulaire (suite) - **O2/CO2**: Cells must be maintained in an atmosphere mimicking the concentration of organisms in O2 and CO2: 95% and 5%, respectively. - **Temperature**: The culture must remain at a temperature equivalent to that of the organism of origin, generally 37°C for mammals. - **pH**: The pH of the culture medium must respect the neutral pH of the organism. A pH indicator is added to the medium (i.e. phenol red that changes color according to the pH). - **Humidity**: The atmosphere must be saturated with water vapor (85%) - A heated incubator maintained constantly and connected to an O2/CO2 source in the bottom of which water is placed (treated to avoid mushroom or bacteria) whose evaporation humidifies the atmosphere constantly. ## Conditions de la culture cellulaire (suite) - **Incubator for culture** allowing to maintain the temperature, humidity, O2 and CO2 saturation conditions. ## La condition de stérilité - To ensure sterility: - **Grow cells in sterile, closed containers** - **Culture takes place in a room isolated from the rest of the laboratory, dedicated to cell culture, with a decontamination airlock** - **Cell manipulation is performed under a laminar flow hood using an air filter. ** ## La condition de stérilité (suite) - **Laminar flow hood** allowing the manipulation of cells (e.g. changing the medium) under sterile conditions. ## Milieu de culture The basic medium must contain: - **H2O** - **Mineral ions** (NaCl, KCI, MgCl2 ...) (for maintaining osmolarity). - **Source of carbon and energy** (glucose or pyruvate). - **Source of nitrogen** (amino acids). - **Growth factors** - **Vitamins, essential amino acids, fatty acids.** - **Buffer solution** (PBS), pH 7.4 stable (contains a colorimetric pH indicator). - **Complements to the medium:** 10% fetal bovine serum (FBS) (decomplemented) + antibiotics and antifungals ## Milieu de culture (suite) - Culture media have been developed by researchers and adapted to different types of cells studied. ## 2 types de cultures - **Primary culture** - **Definition:** primary cell cultures are obtained by dissociation of cells from fresh tissues and their immediate culture on a suitable support and in the presence of a culture medium promoting their survival. - **Characteristics:** - The availability of these cells is limited in time >>> death by apoptosis. - Their proliferative capacity is limited >>> cellular senescence. - The phenotype of adult cells is preserved for a few passages and then modified rapidly. - They are used to confirm a result obtained *in vivo* or on organ explants. ## 2 types de cultures (suite) - **Cell line culture** - **Definition**: a cell line is a homogeneous population of cells, containing only one cell type (originating from the same strain and genetically identical). - **Characteristics**: - They are stable over time and over passages. - They are able to divide indefinitely (transformed or immortalized cells). - They maintain the same phenotype throughout passage, though this may differ from the phenotype within the tissue of origin. - **Disadvantage**: transformation can alter the physiology of the cells compared to those in the original organism. ## 2 types de cultures (suite) - The survival curves of the cells show 2 different profiles of proliferation between primary cultures and cell lines. ## Applications générales des cultures cellulaires - Cell cultures have become a formidable tool in order to address a multitude of questions. Here is a non-exhaustive list of their applications. ## Source et intérêt des culture de neurones - Neuronal cultures are obtained by dissociation of cells from nervous tissue (specific region) of an adult or at a different stage of development. - The most commonly used are cortical neuronal cultures or hippocampal neuronal cultures. - **Interest:** Neuronal cultures allow simplified manipulations and observations on a given type of neuron and independently of the complexity of local or long-distance neuronal networks. - Cultures of neurons from different organisms: rodents, fish, nematodes. - The question to be addressed determines the chosen organism and region at a suitable stage of development. - **Examples:** Cortical embryonic neuronal cultures are preferred to those of the hippocampus for studying neuronal maturation and synaptogenesis because these steps occur earlier in the case of cortical cultures. ## Protocole de la culture des neurones - **The steps of neuronal culture (JoVE)!!!!!** [https://www.youtube.com/watch?v=UqrWmZ8tL1A](https://www.youtube.com/watch?v=UqrWmZ8tL1A) - **How to prepare neuronal cell cultures:** [https://www.youtube.com/watch?v=eHDapIC6QvY](https://www.youtube.com/watch?v=eHDapIC6QvY) **IMPORTANT:** The videos are an integral part of the course; it is therefore essential to consult them and assimilate their content. ## Types d'études réalisée avec les culture de neurones - **Types of studies**: - Studies of intracellular mechanisms unique to a type of neuron (signal transduction, axonal transport...). - Studies of the neuronal response to a given substrate (neurotransmitter, drugs...). - Studies of the mechanisms (development) involved in neuronal differentiation, neuronal maturation, axonal guidance (chemotaxis), synaptogenesis and neuronal plasticity. - Differentiation of neurons from stem cells for therapeutic purposes - Study of the migration, during development, of neuronal progenitors along the radial glial matrix. - **Precautions to take when choosing the stage of sampling:** - Taking samples during the early stages will lead to better survival and yield. - Brain structures are easier to identify and dissect at later stages. - Samples taken at later stages of development give more homogeneous neuronal cultures..