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Chapter 5

Chapter 5 Notes.pdf

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PSYC 211
Yogita Chudasama

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PSYC211 Chapter 5 Notes Definitions: Experimental Ablation: The removal or destruction of a portion of the brain of a laboratory animal; presumably, the functions that can no longer be performed are the ones the region previously controlled Lesion Study: A synonym for experimental ablation Excitotoxic Lesion: A brain lesion produced by intracerebral injection of an excitatory animo acid, such as kainic acid Sham Lesion: A placebo procedure that duplicates all the steps of producing a brain lesion except the one that actually causes the brain damage Stereotaxic Surgery: Brain surgery using a stereotaxic apparatus to position an electrode or cannula in a specified position of the brain Bregma: The junction of the sagittal and coronal sutures of the skull; often used as a reference point for stereotaxic brain surgery Stereotaxic Atlas: A collection of drawings of sections of the brain of a particular animal with measurements that provide coordinates for stereotaxic surgery Stereotaxic Apparatus: A device that permits a surgeon to position an electrode or cannula into a specific part of the brain Fixative: A chemical such as formalin; used to prepare and preserve body tissue Formalin: The aqueous solution of formaldehyde gas; the most commonly used tissue fixative Perfusion: The process by which an animal’s blood is replaced by a fluid such as a saline solution or a fixative in preparing the brain for histological examination Microtome: An instrument that produces very thin slices of body tissues Transmission Electron Microscope: A microscope that passes a focused beam of electrons through thin slices of tissue to reveal extremely small details Scanning Electron Microscope: A microscope that provides three-dimensional information about the shape of the surface of a small object by scanning the object with a thin beam of electrons Confocal Laser Scanning Microscope: A microscope that provides high-resolution images of various depths of thick tissue that contains fluorescent molecules by scanning the tissue with light from a laser beam Anterograde Labeling Method: A histological method that labels the axons and terminal buttons of neurons whose cell bodies are located in a particular region PHA-L: Phaseolus vulgaris leukoagglutinin; a protein derived from kidney beans and used as an anterograde tracer; taken up by dendrites and cell bodies and carried to the ends of the axons Immunocytochemical Method: A histological method that uses radioactive antibodies or antibodies bound with a dye molecule to indicate the presence of particular proteins of peptides Retrograde Labeling Method: A histological method that labels cell bodies that give rise to a terminal buttons that forms synapses with cells in a particular region Fluorogold: A dye that serves as a retrograde label; taken up by terminal buttons and carried back to the cell bodies Pseudorabies Virus: A weakened form of a pig herpes virus used for retrograde transneuronal tracing, which labels a series of neurons that are interconnected synaptically Herpes Simplex Virus: A form of herpes virus used for anterograde transneuronal tracing, which labels a series of neurons that are interconnected synaptically Computerized Tomography (CT): The use of a device that employs a computer to analyze data obtained by a scanning beam of X-rays to produce a two-dimensional picture of a “slice” through the body Magnetic Resonance Imaging (MRI): A technique whereby the interior of the body can be accurately imaged; involves the interaction between radio waves and a strong magnetic field Diffusion Tensor Imaging (DTI): An imaging method that uses a modified MRI scanner to reveal bundles of myelinated axons in the living human brain Microelectrode: A very fine electrode, generally used to record activity of individual neurons Single-Unit Recording: Recording of the electrical activity of a single neuron Macroelectrode: An electrode used to record the electrical activity of large numbers of neurons in a particular region of the brain; much larger than a microelectrode Electroencephalogram (EEG): An electrical brain potential recorded by placing electrodes on in the scalp Magnetoencephalography: A procedure that detects group of synchronously activated neurons by means of the magnetic field induced by their electrical activity; uses an array of superconducting quantum interference devices or SQUIDs 2-deoxyglucose (2-DG): A sugar that enters cells along with glucose but is not metabolized Autoradiography: A procedure that locates radioactive substances in a slice of tissue; the radiation exposes a photographic emulsion or a piece of film that covers the tissue Fos: A protein produced in the nucleus of a neuron in response to synaptic stimulation Functional Imaging: A computerized method of detecting metabolic or chemical changes in particular regions of the brain Positron Emission Tomography (PET): A functional imaging method that reveals the localization of a radioactive tracer in a living brain Functional MRI (fMRI): A functional imaging method; a modification of the MRI procedure that permits the measurement of regional metabolism in the brian, usually detecting changes in blood oxygen level Transcranial Magnetic Stimulation: Stimulation of the cerebral cortex by means of magnetic fields produced by passing pulses of electricity through a coil of wire placed next to the skull; interferes with the functions of brain region that is stimulated In Situ Hybridization: The production of RNA that is complementary to a particular messenger RNA in order to detect the presence of the messenger RNA Microdialysis: A procedure for analyzing chemicals present in the interstitial fluid through a small piece of tubing made of a semipermeable membrane that is implanted in the brain Targeted Mutation: A mutated gene (also called a “knockout gene”) produced in the laboratory and inserted into the chromosomes of mice; fails to produce a functional protein Antisense Oligonucleotide: Modified strand of RNA or DNA that binds with a specific molecule of mRNA and prevents it from producing its protein Methods and Strategies of Research: The best conclusions about the physiology of behaviour are made not by any single experiment, but • by a program of research that enables us to compare the results of studies that approach the problem with different methods Experimental Ablation: • Experimental ablation is one of the most important research methods used to investigate brain functions by destroying part of the brain and evaluating the animal’s subsequent behaviour • In most cases, it does not involve the removal brain tissue but rather to destroy some tissue and leave it in its place Evaluating the Behavioural Effects of Brain Damage: • A lesion is a wound or injury A researcher who destroys a part of the brain calls this a brain lesion • • Lesion studies are where a part of an animal’s brain is destroyed to observe its subsequent functions. The rationale for lesion studies is that the function of an area of the brain can be inferred from the behaviours that the animal can no longer perform after the lesion • The distinction between brain function and behaviour is important. Circuits within the brain perform functions, not behaviours • Each part of the brain performs a function (or a set of functions) that contributes to performance of the behaviour • The interpretation of lesion studies is complicated by the fact that all regions of the brain are interconnected; thus, the functions we are interested in may actually be performed by neural circuits located elsewhere in the brain. Damage to structure X may simply interfere with the normal operation of the neural circuits in structure Y Producing Brain Lesions: • We typically accomplish tissue removal by sucking away brain tissue with a vacuum pump attached to the pipette • Brain lesions of subcortical regions (regions located beneath the cortex) are usually produced by passing electrical current through a stainless steel wire that is coated with an insulating varnish except for the very tip. We guide the wire stereotaxically so that its end reaches the appropriate location. Then we turn on a lesion-making device, which produces radio frequency which heats up and destroys that brain region • Lesions produced by RF currents destroy everything in the vicinity of the electrode tip, including neural cell bodies and the axons of neurons that pass through the region • A more selective method of producing brain lesions employs an excitatory amino acid such as kainic acid, which kills neurons by stimulating them to death. These are referred to as excitotoxic lesions. These lesions destroy neural cell bodies in the vicinity but spares axons that belong to different neurons that happen to pass nearby • The selectivity of excitotoxic lesions permits investigators to determine whether the behavioural effects of destroying a particular brain structure are caused by the death of neurons located there or by the destruction of axons that pass nearby • When we produce subcortical lesions by passing RF currents through an electrode or infusing a chemical through a cannula, we always cause additional damage to the brain because to get to our target, we inevitably cause a small amount of damage even before turning on the lesion maker or starting the infusion Sham lesions are used as control method to see whether just the initial damage to the brain, • without applying the actually lesion method will affect the functions • Investigators may not want to perform a permanent brain lesions; it may be more advantageous to disrupt the activity of a particular region of the brain temporarily. Thus, we inject local anesthetic or a drug called muscimol into the appropriate part of the brain • Muscimol is a drug that stimulates GABA receptors, inactivates a region of the brain by inhibiting the neurons located there Stereotaxic Surgery: • Stereotaxic surgery is how you get the tip of an electrode or cannula to a precise location in the depths of an animal’s brain • Stereotaxis literally means “solid arrangement”; more specifically, it means the ability to locate objects in space A stereotaxic apparatus contains a holder that fixes the animal’s head in a standard position • • The skull is composed of several bones that grow together and form sutures (seams) • The heads of newborn babies contain a soft spot at the junction of the coronal and sagittal sutures called the fontanelle. Once this gap closes, the junction is called bregma • We use a rat’s bregma as a reference point • A stereotaxic atlas contains photographs or drawings that correspond to frontal sections taken at various distances rostral and caudal to bregma • Each page of the stereotaxic atlas is labeled according to the distance of the section anterior or posterior to bregma • By finding a neural structure (which we cannot see in our animal) on one of the pages of a stereotaxic atlas, we can determine the structure’s location relative to bregma (which we can see) • The stereotaxic apparatus operates on simple principles. The device contains a head holder, which maintains the animals’ skull in the proper orientation, a holder for the electrode, and a calibrated mechanism that moves the electrode in three axes: anterior-posterior, dorsal-ventral, and lateral- medial • Stereotaxic surgery may be used for purposes other than lesion production. Wires placed in the brain can be used to stimulate neurons as well as to destroy them, and drugs can be injected that stimulate neurons or block specific receptors Histological Methods: • After performing a brain lesion and observing its effects on an animal’s behaviour, we must slice and stain the brain so that we can observe it under the microscope and see the location of the lesion • Brain lesions often miss the mark, so verification is required • The process of fix, slice, stain, and examination of the brain is known as histological methods • To study the tissue in the form it had at the time of the organism’s death, we must destroy the autolytic enzymes (“self-dissolving”) • The tissue must also be preserved to prevent its decomposition by bacteria or molds. To do so, we must place the neural tissue in a fixative such as formalin which halts autolysis, hardens the very soft and fragile brain and kills any microorganism that might destroy it • Before the brain is fixed, it is usually perfused where you remove the blood from the tissue and replace it with another fluid (typically dilute salt solution) • Once the brain has been fixed, we slice it into thin sections and stain various cellular structures to see anatomical details. Slicing is done with a microtome • Franz Nissl discovered that methylene blue would stain the cell bodies of brain tissue. The material that takes up the dye consists of RNA, DNA, and associated proteins located in the nucleus and scattered, in the form of granules, in the cytoplasm • The most frequently used stain is cresyl violet • The light microscope is limited in its ability to resolve extremely small details, thus to see small details, one must use a transmission electron microscope where a beam of electrons is passed through a thin slice of tissue to be examined • A scanning electron microscope provides less magnification than a standard transmission electron microscope, which transmits the electron beam through the tissue. However, it shows objects in three dimension • The advent of the confocal laser scanning microscope makes it possible to see details inside thick sections of tissue or even in slabs of tissue maintained in tissue cultures or in the upper layers of tissue in the exposed living brain. However, the cells or parts of cells of interest must be stained with a fluorescent dye Tracing Neural Methods: • In the example of the VMH, which is responsible for reproduction in mice, we want to be able to identify the paths followed by axons leaving the VHM; thus trace the efferent axons of this structure. To do so, we use an anterograde labeling method to trace these axons • Anterograde labeling method uses chemicals that are taken up by dendrites or cell bodies and are then transported through axons towards the terminal buttons • In the example of VMH, we inject PHA-L (a protein found in kidney beans) into that nucleus to see where it spreads over a few days Immunocytochemical methods take advantage of the immune reaction. The body’s immune system • has the ability to produce antibodies in response to antigens • Antigens are proteins (or peptides), such as those found on the surface of bacteria or viruses • Antibodies are also proteins, are produced by white blood cells to destroy invading organisms • Tracing efferent axons from the VMH will tell us only part of the story about the neural circuitry involved in female sexual behaviour: the part between the VMH and the motor neurons • To discover the part of the brain that are involved in the “upstream” components of the neural circuitry, we need to find the inputs of the VMH, its afferent connections; thus we will use retrograde labeling method • Retrograde labeling method uses chemicals that are taken up by terminal buttons and carried back through the axons toward the cell bodies • Using a chemical called fluorogold into the VMH, the chemical is taken up by terminal buttons and is transported back by means of retrograde axoplasmic transport to the cell bodies. We discover the medial amygdala is one of the regions that provides input to the VMH • Transneuronal tracing methods identify a series of two, three, or more neurons that form serial synaptic connections with each other • The most effective retrograde transneuronal tracing method uses a pseudorabies virus. For anterograde transneuronal tracing, a variety or the herpes simplex cirus is used • Together, anterograde and retrograde labeling methods - including transneuronal methods - enable us to discover circuits of interconnected neurons. Thus, these methods help to provide us with a “wiring diagram” of the brain Studying the Structure of the Living Human Brain: • CT scans were the first method to see the brain of a live patient. It required the patient to put his head in a large doughnut-shaped ring which contains an X-ray tube and directly opposite the patient’s head, an X-ray detector • The machine measure the amount of radioactivity that goes through the head and uses the figures to produce pictures of the skull and its contents • MRI provides a more detailed, high-resolution picture of what is inside a person’s head. Instead of using X-ray like CT, it passes an extremely strong magnetic field through the patient’s head. The nuclei of some atoms in molecules in the body spin with a particular orientation. If a radio frequency wave is then passed through the body, those nuclei emit radio waves of their own with different energy levels from hydrogen atoms, which is detected and converted into pictures • Unlike CT scans, which are generally limited to the horizontal plane, MRI scans can be taken in the sagittal or frontal planes as well • Diffusion tensor imaging (DTI) takes advantage of the fact that the movement of water molecules in bundles of white matter will not be random but will tend to be in a direction parallel the axons that make up the bundles. The MRI scanner uses information about the movement of water molecules to determine the location and orientation of bundles of axons in white matter Goal of Method Method Remarks Destroy or inactivate specific Radio frequency lesions Destroys all brain tissue near tip brain region of electrode Excitotoxic lesion; uses Destroys only cell bodies near excitatory amino acid such as tip of cannula; spares axons kainic acid passing through the region 6-HD lesion Destroys catecholaminergic neurons near tip of cannula Infusion of local anesthetic; Temporarily inactivates specific cryoloop brain region; animal can serve as its own control Place electrode or cannula in Stereotaxic surgery Consult stereotaxic atlas for specific region within brain coordinates Find location of lesion Perfuse brain; fix brain; slice brain; stain sections Identify axons leaving a Anterograde tracing method, particular region and the such as PHA-L terminal buttons of these axons Identify location of neurons Retrograde tracing method, whose axons terminate in a such as fluorogold particular region Goal of Method Method Remarks Identify chain of neurons that Transneuronal tracing method; are interconnected synaptically uses pseudorabies virus (for retrograde tracing) or herpes simplex virus (for anterograde tracing) Find location of lesion in living Computerized tomography (CT Slows “slice” of brain’ uses X- human brain Scanner) rays Magnetic Resonance Imagining Shows “slice” of brain; better (MRI Scanner) detail than CT scan; uses a magnetic field and radio waves Find location of fiber bundles in Diffusion tensor imaging (DTI) Shows bundles of myelinated living human brain axons; uses an MRI scanner Visualize details of cells in thick Confocal laser scanning Can be used to see “slices” of sections of tissue microscopy tissue in living brain; requires the presence of fluorescent molecu
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