Task 4 - Methods PDF

Task 4 - Methods Learning Goals What brain imaging methods are there and when do we use them? What are the advantages and disadvantages of each method? Experimental ablation Experimental ablation - destroying part of the brain and evaluating an animal's subsequent behavior. It usually does not inv...

Task 4 - Methods Learning Goals What brain imaging methods are there and when do we use them? What are the advantages and disadvantages of each method? Experimental ablation Experimental ablation - destroying part of the brain and evaluating an animal's subsequent behavior. It usually does not involve removal of tissue, but instead damaging it to disrupt its functioning. Brain lesion - a wound or injury in the brain. Lesion studies - experiments in which part of the brain is damaged and the individual's behavior is subsequently observed. The function of a brain area can be inferred from the behaviors that the individual can no longer perform after the area has been damaged. All regions of the brain are interconnected. Damage to one structure may simply interfere with the normal operation of the ne ural circuits in a different structure. Producing brain lesions Aspiration lesions - cortical tissue is drawn off by suction through a handheld glass pipette. The underlying white matter is more resistant to suction, which makes it possible to peel off layers of cortical tissue without damaging the underlying white matter and major blood vessels. Radio frequency (RF) lesions of subcortical regions are usually produced by passing electrical current through a steel wire that is insulated except at its tip. 1. The wire is guided to a precise location within the brain. 2. A lesion-making device is activated, which produces a (RF) current - an alternating current of a very high frequency. 3. The passage of current through the brain tissue produces heat that kills cells in the region surrounding the tip of the elect rode. This way of producing lesions destroys cell bodies at the region and axons that pass through the region. Knife cuts - a cutting device is positioned stereotaxically (see stereotaxic surgery below) in the brain, then a blade swings out to make the necessary cut. It is used to eliminate conduction in a nerve/tract. Excitotoxic lesions - a more selective way of producing lesions, namely by injecting an excitatory amino acid into the brain through a cannula (a small metal tube). The amino acid stimulates the neurons in the region to death, leaving the axons that pass through the region alive. Excitotoxic lesions permits determining whether the behavioral effects of destroying a particular brain structure are caused by death of neurons located there or by the destruction of axons that pass nearby. RF and excitotoxic lesions produce additional brain damage because of the insertion of the electrode/cannula in the brain. This incidental damage to the brain regions above the lesion may actually be responsible for any observed behavioral deficits. This is avoided by including an additional (control) group of animals in a lesion study and produce sham lesions (in which they insert the electrode/cannula but do not produce the lesion, only the damage caused by the insertion itself). There are more selective methods of killing particular types of neurons. Attaching saporin (a toxic protein) and antibodies that bind with particular proteins only found on certain types of neurons in the brain is one such method. The antibodies target these proteins and the saporin killsthe cells to which the proteins are attached. Reversible brain lesion - disruption of brain activity of a particular region temporarily. Can be caused by injecting an anesthetic (which blocks axonpotential in axons leaving/entering a region) or muscimol (which stimulates GABA receptors and inactivates a brain region by inhibiting the neurons there) or cooling the target structure. Stereotaxic surgery An electrode or cannula is inserted to a precise location in an animal brain by the method of stereotaxic surgery. Stereotaxis - the ability to locate objects in space. Stereotaxic apparatus - an apparatus that contains a holder that keeps the animal's head in a standard position and an arm that moves an electrode/cannula through measured distances in 3 spatial dimensions (anterior - posterior, dorsal-ventral and lateral-medial). Stereotaxic atlas - a book/website/software that contains images of frontal sections of the brain taken at various distances rostral/caudal to the bregma (the junction of the sagittal and coronal sutures at the top of the skull). Each image of the stereotaxic atlas contains distance of the section anterior/posterior to the bregma and a grid, which indicates distances of brain structures ventral to the top of the skull and lateral to the midline. To place the tip of a wire in a brain structure S, one has to drill a hole above S and then lower the electrode through the hole until the tip is at the correct depth. Due to variations in different strains/ages of animals, the atlas gives only an approximate location, which may need to be verified by using other methods. The way of operating with a stereotaxic apparatus is the following: 1. The researcher obtains coordinates from a stereotaxic atlas. 2. The animal is anesthetized and placed in the apparatus. 3. The scalp is cut open and the bregma is located. 4. A hole is drilled through the skull. The appropriate coordinates are dialed and the electrode/cannula is lowered into the cor rect brain region. Deep brain stimulation - a procedure that utilizes a stereotaxic apparatus to implant a permanent electrode into the brain of a human. Rather than producing a Body and Behavior Page 1 Deep brain stimulation - a procedure that utilizes a stereotaxic apparatus to implant a permanent electrode into the brain of a human. Rather than producing a lesion, current is passed through the electrode to stimulate brain regions and reduce symptoms of chronic pain, movement disorders, epilepsy, depression, and OCD. Visualizing the structure of the living human brain Functional brain images - images of brain activity. Structural brain images - images of brain structure. X-Ray-based techniques Conventional x-ray photography - an x-ray beam is passed through an object and then onto a photographic plate. Each molecule that the beam has passed through absorbs some of the radiation => only the unabsorbed portions of the beam reach the photographic plate. Contrast x-ray techniques - a substance is injected in a compartment of the body that absorbs x-rays either less or more than the surrounding tissue. Conventional x-ray photography is not useful for visualizing the brain due to the high number of overlapping structures of the brain. Howeve r, contrast x-ray techniques are. Cerebral angiography - a contrast x-ray technique, in which a radio-opaque dye is infused into the cerebral artery to visualize the cerebral circulatory system during x-ray photography. Cerebral angiograms are most useful for localizing vascular damage (of vessels) and tumors (which are indicated by the displacement of blood vessels from their normal position). Computerized tomography (CT) - a computer-assisted x-ray procedure that can be used to visualize the brain and other internal structures of the living body. 1. The patient's head is placed in a large doughnut-shaped ring. The ring contains an X-ray tube and, directly opposite it (on the other side of the patient's head), an X-ray detector. 2. The X-ray beam passes through the patient's head, and the detector measures the amount of radioactivity that gets through it. 3. The beam scans the head from all angles, and a computer translates the received information from the detector into pictures of the skull and its contents. CT can detect differences in structure or tissue type, such as tumors or bleeding (blood appears white, because it absorbs more radiation than the surrounding brain tissue). Structural MRI Magnetic resonance imaging (MRI) is a structural brain-imaging procedure that provides a more detailed, high-resolution picture of the skull and the brain than CT. An MRI scanner looks like a CT scanner, but it does not use X- rays. Instead, it passes a strong magnetic field through the patient's head. 1. When a person's head is placed in the magnetic field the nuclei of the spinning H atoms align themselves to the magnetic field. 2. A pulse of radio frequency wave is passed through the brain, and the nuclei flip at a certain angle to the magnetic field, then they flip back to their original position at the end of the radio pulse. 3. When they flip back, they release energy that they absorbed from the radio pulse. The released energy is sense by a coil of wire that serves as a detector. 4. Different tissues contain different amounts of water (thus H atoms), so they emit different amounts of energy. The computer analyzes the signal and produces pictures of slices of the brain. MRI provides relatively high spatial resolution. MRI can also produce 3D pictures. Pictures from MRI scans distinguish between regions of gray and white matter, so major fiber bundles (e.g. the corpus callosum) can be seen. However, small fiber bundles are not visible on these scans. This is possible with the next method (see below). Diffusion tensor imaging (DTI) or diffusion tensor MRI is a modification of MRI that takes advantage of the fact that the movement of water molecules in bundles of white matter will not be random, but tend to be in a direction parallel to the axons that make up the bundles. Body and Behavior Page 2  parallel to the axons that make up the bundles. This modification of MRI permits the visualization of even small bundles of fibers and the tracing of fiber tracts. Recording and stimulating neural activity Recording neural activity Intracellular unit recording - recording of the moment-by-moment fluctuations in a neuron's membrane potential. Single-unit (or extracellular unit) recording - recording an individual unit (neuron) using microelectrodes placed in the extracellular fluid next to the neuron. Changes in the membrane potential, however, cannot be recorded. Microelectrodes - thin wires with a very fine tip, small enough to record the electrical activity of individual neurons. The microelectrodes are implanted in an animal's brain through stereotaxic surgery and they are bonded to an animal's skull, using plastics that are used in dental medicine. The electrical signals detected by microelectrodes are quite small and must be amplified. These signals are then displayed or saved on a computer. Multiple-unit recording - when it is needed to record the activity of a whole brain region (not of individual neurons), macroelectrodes are used. Macroelectrodes - devices, which obtain records that represent the PSPs of many thousands or millions of cells in the area of the electrode. Macroelectrodes are sometimes implanted into the brain or onto its surface, but many are temporarily attached to the human scalp with a special paste that conducts electricity. Recordings taken from the scalp represent the activity of many neurons, whose electrical signals pass through the meninges, skull, and scalp before reaching the electrodes. Human brain activity recorded by macroelectrodes is displayed on a polygraph, which plots the changes in voltage along a timeline. Electroencephalogram (EEG) - a polygraph of activity recorded from macroelectrodes attached to various locations on a person's scalp (see picture to the right). The power of EEG is not displaying exact neural activity. However, some EEG waves forms are associated with particular states of consciousness or types of cerebral pathology (e.g. epilepsy). Alpha waves - regular, 8- to 12-per-second, high-amplitude waves that are associated with relaxed wakefulness. EEG signals decrease in amplitude as they spread from their source, which is why a comparison of signals recorded from various sites on the scalp are required to deduce the origin of particular waves. Event-related potentials - EEG waves that accompany particular psychological events. Sensory evoked potential - the change in the cortical EEG signal elicited by the momentary presentation of a sensory stimulus. P300 wave - a positive wave that occurs ~300ms after a momentary stimulus that has meaning to the participant. Signal averaging - a method used to reduce the noise of the background EEG. Invasive EEG recording - recording EEG signals through implanted electrodes Body and Behavior Page 3  Invasive EEG recording - recording EEG signals through implanted electrodes rather than scalp electrodes. When electricity flows through a conductor, it produces a magnetic field => action potentials travelling down axons and PSPs travelling through dendrites/soma both create magnetic fields. These magnetic fields are quite small, but they can be detected with superconducting detectors (SQUIDs). Magnetoencephalography (MEG) is performed with neuromagnetometers (devices that contain several SQUIDs), whose output a computer can use to calculate the source of particular signals in the brain. MEG has much better spatial resolution and reliability than EEG. MEG has high temporal resolution. The image produced by means of MEG is acquired very rapidly and can reveal fast-moving events. Because of the small magnitude of the magnetic signals induced by neural activity, only those induced near the surface of the brain can be recorded from the scalp. MEG is very expensive and an MEG machine is very large. Patients must remain very still during recordings. Recording the brain's metabolic and synaptic activity If the neural activity of a particular brain region increases, then the metabolic rate of this region also increases (mostly due to operation of ion channels, which require more energy from the cell). This metabolic activity can be measured by injecting radioactive 2-deoxyglucose (2-DG) into an animal's bloodstream. The chemical resembles glucose (main food for the brain) => it is taken into cells => the most active cells will take up the highest concentrations of radioactive 2-DG. Unlike normal glucose, 2-DG cannot be metabolized, so it stays in the cell. After administering the 2-DG, the animal is euthanized, the brain is removed, sliced, and prepared for autoradiography. The result of autoradiography is that after several weeks the most active regions of the brain (which contain the most radioactivity) are visible on microscope sli des. When neurons are activated, particular genes in the nucleus called immediate early genes are turned on, and particular proteins are produced. The presence of these proteins indicates that the neuron has just been activated. They can be detected by euthanizing the animal, remove and slice the brain and stain it. Positron emission tomography (PET) is the first developed functional imaging method. 1. A person/animal receives an injection of radioactive 2-DG (a harmless dose that gradually leaves the cells). 2. The person's head is placed in a machine similar to a CT scanner. 3. When the radioactive molecules of 2-DG decay, they emit positrons, which meet nearby electrons. The particles annihilate each other and emit 2 photons, which travel in directly opposite paths. 4. Sensors around the person's head detect the photons, and the scanner plots the locations from which these photons are emitted. PET is very expensive because the radioactive chemicals are short-lived and need to be produced on site. PET has relatively low spatial resolution (blurriness) of the images. PET has relatively low temporal resolution (the emitted positrons need to be sampled for a long time, which means that rapid, short-lived events within the brain are likely to be missed). PET images are not images of the brain, but simply colored maps of amount of radioactivity in each voxel that composes the scan. The mapping of voxel onto a particular brain structure can be estimated only by superimposing the PET scan on a brain image. These disadvantages are not seen in functional MRI. However, PET scanners can measure the concentration of particular chemicals (e.g. neurotransmitters, receptors, transporters) in various parts of the brain (fMRI cannot do that). Different colors indicate different rates of uptake of 2-DG. Functional MRI (fMRI) is a functional imaging method that measures brain activity by detecting levels of oxygen in the brain's blood vessels. Increased brain activity stimulates blood flow to that region, which increases the local blood oxygen level. BOLD (blood oxygen level-dependent signal) is the formal name for this type of imaging. The BOLD signal indicates the parts of the brain that are active/inactive during a cognitive or behavioral test. Unlike PET, fMRI does not require injecting a substance into the patient. FMRI provides both structural and functional information in the same image. FMRI is the imaging method with the highest spatial resolution. FMRI pictures are NOT pictures of electrical brain activity. They are pictures of BOLD signals. The relationship between the BOLD signal and neural activity is complex. FMRI has poor temporal resolution. It takes 2-3ms to measure the BOLD signal, during which many neural responses (e.g. action potentials) happen. Body and Behavior Page 4 Functional ultrasound imaging (fUS) - a functional imaging method that uses ultrasound (sound waves of higher frequency than we can hear) to measure changes in blood volume in particular brain regions. When a brain region becomes active blood levels increase there, and alter the passage of ultrasound through that brain region. fUS is cheaper than PET and fMRI. fUS is highly portable. fUS can be used for imaging individuals who cannot undergo PET or fMRI (e.g. infants). Stimulating neural activity Brain imaging methods merely show correlation between brain activity and cognitive activity (e.g. experience of a pleasant emotion). In order to find evidence for causality, the following can be done: Cognitive activity can be assessed in people with brain damage Cognitive activity can be assessed after "turning off" a particular brain region (can be done with transcranial magnetic stim ulation - TMS). Cognitive activity can be assessed after "turning on" a particular brain region (can be done with transcranial electrical sti mulation - tES and transcranial ultrasound stimulation - tUS). Transcranial magnetic stimulation (TMS) uses a coil of wires, usually arranged in an 8- shape, to noninvasively stimulate neurons in the cerebral cortex. The stimulating coil is placed on top of the skull so that the crossing point in the middle of the 8 is located immediately above the region to be stimulated. Pulses of electricity send magnetic fields that activate neurons in the cortex. Depending on the strength and pattern of stimulation, TMS can either excite the region of the brain over which the coil is positioned or interfere with its functions. TMS is used to help determining causality between brain activity and cognitive activity. Transcranial electrical stimulation (tES) applies an electrical current through 2 electrodes placed directly on the scalp. The electrical stimulation temporarily increases activity in part of the brain. tES is used to stimulate ("turn on") an area of the cortex. Transcranial ultrasound stimulation (tUS) uses multiple sources of low-amplitude ultrasonic sound wave sources. Each of these sound sources is placed around the individual's head and is directed at the target brain structure. When the waves from each of those sources reach the target structure, they sum together, such that the amplitude of the sound waves at the target brain structure is sufficiently large to stimulate activity there. tUS is also used to activate particular brain structures. However, unlike tES and TMS, which can only be used to stimulate co rtical structures, tUS can also be used to active subcortical structures. tUS can also be used to make small permanent lesions to a brain structure, when the amplitude of each ultrasound source is la rge enough. Optogenetic methods can be used to stimulate/inhibit particular types of neurons in specific brain regions. Photosensitive proteins exist in many organisms. Some of them open ion channels when activated by light of specific wavelength. Such photosensitive proteins can be introduced into neurons by attaching the genes that code for them into the genetic materi al of harmless viruses. The viruses are then injected into the brain, where they infect neurons and being expressing the proteins, which are inserted into the cell membrane. The genes can be modified so that the proteins will be expressed only in particular types of neurons => the effects of turnin g on/off particular types of neurons in a specific region of the brain can be observed. Body and Behavior Page 5