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Chapter 5: Methods and Strategies of Research The Means of Research 1. Experimental Ablation 2. Neurochemical Methods (including pharmacology) 3. Recording and Stimulating Neural Activity 4. Genetic Methods Experimental Ablation (a.k.a. a lesion study)  Evaluating the behavioral effects of brain damage o Ex: Phineas Gage  Phineas Gage was a railroad worker that had an iron rod propelled through Gage’s skull, entering through his left cheekbone and exiting through the top of his head. Gage survived and recovered, but his injury had an impact on his mental condition. This allowed scientists to study the behavioral effects of his brain damage.  Experimental ablation involves destroying part of the brain (referred to as a lesion) and evaluating the animal’s subsequent behavior  Goal: to discover what functions are performed by different regions of the brain and to understand how these functions are combined to accomplish particular behaviors  Producing brain legions 1. Electrolytic (radio frequency) lesion  A brain lesion produced by passing an electrical current across an electrode placed in a specific brain area  This lesion making device produces an alternating current of very high frequency  Damages cells (because heat is produced near the tip of the electrode) and damages fibers of passage (because inserting an electrode into the brain damages whatever it comes into contact with) 2. Excitotoxic lesion  A brain lesion produced by intracerebral injection of an excitatory amino acid, such as kainic acid, which kills neurons by stimulating them to death.  Injections are made via a cannula, which is a small metal tube  Damages cells, but spares fibers of passage 3. Toxic chemicals  Can attach toxic chemicals to antibodies that will bind with particular proteins found only on certain types of neurons in the brain  The toxic chemicals kill the cells to which the proteins are attached 4. Sham lesion  Very important control  A “placebo” procedure that duplicates all the steps of producing a brain lesion except for the one that actually causes the brain damage  But, how do you know where?! o Skull is composed of several bones that grow together and form sutures (seams). At first, the junction of the coronal and sagittal sutures is called the fontanelle, but

once it closes, the junction is called bregma. Bregma serves as a convenient reference point. o Stereotaxic atlas contains photographs and drawings that guide you where to go based on bregma. o A stereotaxic apparatus contains a holder that fixes the animal’s head in a standard position and a carrier that moves an electrode or cannula through measured distances in all three axes of space.  There are also stereotaxic apparatuses for humans Neurochemical Methods (including pharmacology)  Histological methods o To verify the precise location of brain damage, one must fix, slice, stain, and examine the brain o Use a fixative in order to prevent autolytic enzymes from turning tissue into mush and to prevent decomposition by bacteria or molds  Most common fixative is formalin  Formalin is an aqueous solution of formaldehyde o After fixation, brain is sliced into thin sections  Usually accomplished using a microtome (rotary, sliding/sledge) or a cryostat o In order to see the anatomical details in the various cellular structures and the specific substances within and outside of cells, one must use special histological stains  Simplest: a cell body stain (methylene blue, cresyl violet)  Must use an electron microscope to see small structures  Immunocytochemical methods o Use antibodies to show us where or how much protein there is  uses radioactive antibodies or antibodies bound with a dye molecule to indicate the presence of particular proteins of peptides o Immunohistochemistry is a type of immunocytochemical method. Immunohistochemistry uses antibodies (immuno) in tissue sections (histo) to generate a reaction (chemistry) where the target of interest is.  In situ hybridization and antisense oligonucleotides o Antisense oligonucleotides: uses a modified strand of RNA or DNA that binds with a specific molecule of mRNA and prevents it from producing its particular protein o In situ hybridization: Antisense strands of RNA, or single stranded DNA are used for in situ, but they are tagged so they can be seen. In situ hybridization lets us see where in the brain the message (mRNA) is, because it's done in tissue sections (in situ, means in the situation...as in, in the brain).  Tracing neural connections o Anterograde tracers (“moving forward”)  Used to trace efferent axons  Move from soma to the axon terminal of a cell

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Chemicals, such as PHA-L, are taken up by dendrites or cell bodies and are then transported through the axons toward the terminal buttons o Retrograde tracers (“moving backward”)  Used to trace afferent axons  Move from the terminal toward the soma of a cell  Chemicals, such as fluorogold, are taken up by the terminal buttons and are carried back through axons toward cell bodies o Transneuronal tracing  Used to identify a series of neurons that form serial synaptic connections with each other  Uses various strains of weakened rabies virus or herpes virus. Infection is transmitted in an anterograde or retrograde direction Recording and Stimulating Neural Activity  Neurophysiology: recording from a brain area with an electrode o Electrode records electrical events from action potentials and postsynaptic potentials o Single-unit recording  Recording electrical activity of a single neuron  Uses a microelectrode, a very fine electrode o Macroelectrodes record electrical activity from lots of neurons in a specific brain area  Studying structures in living people o Computerized tomography (CT scan): x-ray beam passes through patient’s head and x-ray detector measures the amount of radioactivity that gets through o Magnetic resonance imaging (MRI scanner): passes an extremely strong magnetic field through patient’s head, which causes nuclei of some atoms in molecules to spin with a particular orientation. If a radio frequency is then passed through the body, these nuclei emit radio waves of their own. The MRI detects the radiation emitted from hydrogen atoms. o Diffusion tensor imaging (DTI scan): modified MRI scanner used to reveal bundles of myelinated axons  Autoradiography o 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 o Ex: radioactive 2-DG  2-DG resembles glucose and will be taken into cells. Unlike normal glucose, 2-DG cannot be metabolized, so it will stay in the cell. The radioactivity of 2-DG can then be seen using autoradiography  Fos o When neurons are activated, particular genes in the nucleus (called immediate early genes) are turned on and particular proteins are produced. Proteins then bind with the chromosomes in the nucleus. The presence of these proteins indicates that the neuron has just been activated

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Fos is one of the nuclear proteins produced during neural activation. Need to stain with an antibody to observe Fos Measuring metabolic activity o Functional imaging: computerized method of detecting metabolic or chemical changes within brain  Ex: positron emission tomography (PET scan): reveals the location of a radioactive tracer  Ex: functional MRI (fMRI): measures brain activity by detecting levels of oxygen in the brain’s blood vessels

Genetic Methods  Optogenetic methods o The use of a genetically modified virus to insert light-sensitive ion channels into the membrane of particular neurons in the brain; can depolarize or hyperpolarize the neurons when light of the appropriate wavelength is applied o Scientists combine opsin gene with an element called a promoter that will cause the gene to be active only in a specific type of cell. The modified gene is inserted into a virus, which can then be injected into the brain. The virus infects many nerve cells, but because of the promoter, only one type of neuron makes the opsin protein. Fiber-optic probes inserted into animal’s brain can flash light on brain to control specific patterns of neural activity  Ex: When blue light strikes ChR photosensitive protein, channel opens and rush of positively charged sodium and calcium ions depolarizes the membrane, causing excitation. When yellow light strikes HR photosensitive protein, a transporter moves chloride ions into the cell, causing an inhibition  Twin studies o Estimates the influence of genetic factors on a particular trait o Compares the concordance rate for this trait in pairs of monozygotic (identical) and dizygotic (fraternal) twins  Adoption studies o Estimates the influence of genetic and environmental factors on a particular trait o Compares people who were adopted early in life with their biological and adoptive parents...