Methods and Strategies of Research

Transcription

1 Methods and Strategies of Research 1

2 Destroying part of the brain and evaluating the animal s subsequent behavior. Lesion studies. Example: If after part of the brain is destroyed, an animal can no longer perform tasks that require vision, we can conclude that the animal is blind and that the damaged area plays some role in vision. 2

3 The task of he researcher is to understand the functions that are required for performing a particular behavior and to determine what circuits of neurons in the brain are responsible for each of these functions. 3

4 Parietal lobe damage disrupts people s ability to follow or draw maps, to remember the locations of objects that they have just seen. Also they have difficulty performing arihmetic calculations*. *Try to multiply 55 by 12 without using pencil and paper. 4

5 The interpretation of lesion studies is complicated by the fact that all regions of the brain are interconnected. Example: Septum-Maternal Behavior in mice. Septum turns some of the functions of hippocampus on or off. 5

6 Producing Brain Lesions 1. Vacuuming a part of the brain 2. Radio frequency (RF) lesion 3. Excitotoxic lesion. Use of an excitatory amino acid such as kainic acid. Kills only cell bodies spears axons. Excitotoxic lesion. Radio frequency lesion. 6

7 Producing Brain Lesions 4. 6-hydroxydopamine (6-HD). Because of its resemblance to the norepinephrine and dopamine, it is taken up by transporter molecules in axons and terminal buttons of dopaminergic and noradrenergic neurons. Then the chemical poisons and kills the neurons. Sham Lesions Excitotoxic lesion. Radio frequency lesion. 7

8 Permanent vs Reversible Brain Lesions Local Anesthetics Cooling brain tissue by cryode Stereotaxic Surgey Stereotaxic apparatus Stereotaxic atlas A cryode. 8

9 Stereotaxic atlas Sutures First fontanelle, then bregma A sample page from a stereotaxic atlas of the rat brain (fornix). Relation of the skull sutures to a rat s brain and the location of a target for an electrode placement 9

10 Stereotaxic Apparatus A stereotaxic apparatus for performing brain surgery on rats. 10

11 Histological Methods Brain lesions often miss the mark, so we have to verify the precise location of the brain damage after testing the animal behaviorally. To do so, we must fix, slice, stain, and examine the brain. 1. Fixation and Sectioning 2. Staining 3. Electron microscopy A stereotaxic apparatus for performing brain surgery on rats. 11

12 Histological Methods Fixation and Sectioning First perfuse the tissue (cleaning out of the blood by replacing the contents of vessel with the diluted salt) Use fixative (e.g. formalin) to a. destroy the autolytic enzymes b. preserve the tissue to prevent its decomposition by bacteria or molds. 12

13 Histological Methods Fixation and Sectioning Once the brain has been fixed, we must slice it into thin sections and stain various cellular structures in order to see anatomical details. Microtome (sectioning generally µm in thickness) After the tissue is cut the section is attached to glass microscope slides. Than the section is stained and covered by a very thing glass coverslip by using a mounting medium. A microtom. 13

14 Histological Methods Staining The study of microscopic neuroanatomy requires special histological stains. -Methylene blue (Nissle substance in the cytoplasm takes up the dye) (cell bodies) -Cresyl violet -The discovery of cell-body stains made it possible to identify nuclear masses in the brain. A frontal section of a cat brain, stained with crsyl violet, a cell-body stain. The arrowheads point to nuclei, or groups of cell bodies. 14

15 Histological Methods Electron Microscopy A scanning-electron micrograph of neurons and glia. An electron photomicrograph of a section through an axodendritic synapse. 15

16 Tracing Neural Connections Example: VMH lesion in female rats and sham operated group. Tracing Efferent Axons Anterograde labeling method Anterograde axoplasmic transport. -Inject a minute amount of PHA-L into the VMH. -Couple days later, by using a special immunocytochemical method make the PHA-L molecules visible and examine slides under a microscope. Once we know that a particular brain region is involved in a particular function, we may ask what structures provide inputs to the region and what structures receive outputs from it. 16

17 Tracing Neural Connections Tracing Efferent Axons Immunocytochemical method Antibody molecules are attached to various types of dye molecules. Some of these dyes react with other chemicals and stain the tissue a brown color. Others are flourescent. Antibodies attach thmeselves to their antigen. The rationale for the use of PHA-L to trace efferent axons. 17

18 Tracing Neural Connections Tracing Efferent Axons -PHA-L is injected to the VMH -Two days later, animal is killed -Slices of the brain are treated with an antibody to PHA-L, attached to a dye that stains the tissue a brown color. -The lectin fills nearby cell bodies and dendrites. You see the periaqueductal gray matter contains labeled axons and terminal buttons. -Then you should destroy these areas that receive information from the VMH. -Until you discover the relevant pathways from the VMH to the motor neurons whose activity is necessary for copulatory behavior, follow the same procedure. An anterograde labeling method. 18

19 Tracing Neural Connections Tracing Afferent Axons Retrograde labeling method. Retrograde axoplamic transport. What abaout the circuits before the VMH? Upstream components of the neural circuitry: Touch, sight, odor of the male? The activating effect of a female s sex hormones on her behavior act through the VMH? A retrograde labeling method. Fluorogold was injected in the VMH, where it was taken up by terminal buttons and transported back through the axons to their cell bodies. The picture shows these cell bodies, located in the medial amygdala. 19

20 Tracing Neural Connections Putting together One of the inputs to the VMH and one of the outputs, as revealed by anterograde and retrograde labeling methods. 20

21 Study of the Living Human Brain Computerized Tomography (CT) The patient s head is placed in a large doughnut-shaped ring. The ring contains an X-ray tube and, directly opposite it, an X-ray detector. A computerized tomography (CT) scanner. 21

22 Study of the Living Human Brain Computerized Tomography A lesion in the right occipitalparietal area (scan 5) is evident with white area (because the stroke was accompanied by bleeding; blood absorbs more radiation than the surrounding brain tissue). A series of CT scans. 22

23 Study of the Living Human Brain Magnetic Resonance Imaging (MRI) The MRI scanner resembles a CT scanner, but it does not use X-rays; instead, it passes an extremely strong magnetic field through the patient d head. The MRI scanner is tuned to detect the radiation from hydrogen molecules. Unlike CT scans, MRI scans can be taken not only horizontal but also in the saggital or forntal planes. A midsaggital MRI scan of a human brain. 23

24 Summing Up 24

25 Recording and Stimulating Neural Activity Recording Neural Activity Electrical activity of a particular region can be used to determine whether that region plays a role in various behaviors. Examples: during stimulus presentations, decision making, motor activities, etc. Recording Cronically Recording Acutely Recording with Microelectrodes Recording with Macroelectrodes 25

26 Recording and Stimulating Neural Activity Recording with Microelectrodes Single unit recording Preperation of glass electrodes by using heat source and microelectrode puller. Filling the electrodes with a conducting liquid (a solution of potassium chloride). Alternatively fine tungsten wires can be used. The tip of the wire is sharpened by etching them in an acid solution. Microelectrodes produced by heating the center portion of a length of glass capillary tubing and pulling the ends apart. 26

27 Recording and Stimulating Neural Activity Recording with Microelectrodes The electrical signals detected by microelectrodes must be amplified. These signals can be displayed on an oscilloscope and stored in the memory of a computer for analysis at a later time. Example: drugs that effect serotonergic and noradrenergic neurons also effect REM sleep. We wonder if the activity of serotonergic and noradrenergic neurons would vary during different stages of sleep. If we record the activity of noradrenergic and serotonergic neurons during various stages of sleep, we will find that the firing rate of these neurons falls almost to zero during REM sleep. Suggesting that these neurons have an inhibitory effect on REM sleep. That is, REM sleep cannot occur until these neurons stop firing. A permanently attached set of electrodes, with a connecting socket cemented to the skull. 27

28 Recording and Stimulating Neural Activity Recording with Macroelectrodes Recording the activity of a region of the brain as a whole, not the activity of individual neurons. Macroelectrodes do not detect the activity of individual neurons; rather, the records obtained with these devices represent the postsynaptic potentials of many thousands or millions of cells in the area of the electrode. A record from a ink-writing oscillograph generally called polygraph. 28

29 Recording and Stimulating Neural Activity Recording with Macroelectrodes Electroencephalograms (EEGs) EEG activity from the rat hippocampus, recorded during various behaviors. 29

30 Recording and Stimulating Neural Activity Recording the Brain s Metabolic and Synaptic Activity 2-DG Autoradiography Neural activity ~ Metabolic Activity The experimenter injects radioactive 2-deoxyglucose (2-DG) into the animal. 2-DG resembles glucose, however it cannot be metabolized, so it stays in the cell. Then the experimeter prepares the animal s brain for autoradiography. A 2-DG autoradiogram of a rat brain (frontal section) 30

31 Recording and Stimulating Neural Activity Recording the Brain s Metabolic and Synaptic Activity Fos Method When neurons are activated particular genes in the nucleus are turned on and particular proteins are produced. These proteins then bind with the chromosomes in the nucleus. Follow the procedure that stains Fos protein (remember the experiment outlined in the previous section). Localization of Fos protein. The photomicrograph was taken through the medial amygdala. The dark spots indicate the presence of Fos protein. 31

32 Recording and Stimulating Neural Activity Recording the Brain s Metabolic and Synaptic Activity Positron Emission Tomography (PET) 1. Patient receives an injection of radioactive 2-DG. 2. The computer determines which regions of the brain have taken up the radioactive substance. PET scans of a human brain. 32

33 Measuring the Brain s Secretions Sometimes we are interested in secretion of specific neurotransmitters or neuromodulators in these regions. Interested in the releation between REM sleep and muscular paralysis. We may measure the secretion of acetylcholine in a region of the medulla known to contain neurons that inhibit motor neurons in the spinal cord. To do so we use a procedure called microdialysis. This way we collect molecules from the extracellular fluid of the brain (pushed across the membrane by the force of diffusion. What they found was that there was an increase the amount of acetylcholine present in the exracellular fluid of the nucleus in the medulla during REM sleep. Microdialysis. 33

34 Stimulating Neural Activity Change the activity of a particular region of the brain then investigate the changes on the animal s behavior. Example: Female rats will copulate with males only if certain female sex hormones are present. We remove the rat s ovaries. Hormone levels fell to almost zero, and sexual behavior is abolished. What we found earlier was that VMH lesions disrupt copulatory behavior in female rats. Perhaps if we activate the VMH, we will make up for the lack of female sex hormones and the rats will copulate again. Then how do we activate neurons? We can chemically stimulate the neurons by an excitatory transmitter substance such as kainic acid or glutamic acid (glutamate). Disadvantage of chemical stimulation: Difficult Advantage of chemical stimulation: It activates cell bodies not axons. Effect of stimulation is more localized. In our example, VHM stimulation does substitute for female sex hormones. An intracranial cannula. 34

35 Stimulating Neural Activity Behavioral Effects of Electrical Brain Stimulation One of the more interesting uses of electrical stimulation of the brain was developed by Panfield & Jasper (1954). In an epileptic patient. An intracranial cannula. 35

36 Summing Up 36