Supplementary Chapt. 1-fMRI copy(1)

Supplementary Chapt. 1-fMRI copy(1) - Supplementary...

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Unformatted text preview: Supplementary Chapter 1 Imaging the Brain with Magnetic Resonance Imaging and Functional Magnetic Resonance Imaging Imaging functional variations in the living brain has become possible with the recent development of techniques for detecting small, localized changes in metabolism or cerebral blood flow. To conserve energy, the brain regulates its blood flow such that active neurons with relatively high metabolic demands receive more blood than relatively inactive neurons. Detecting and mapping these local changes in cerebral blood flow forms the basis for two widely used methods of imaging the regions of brain that are active during particular tasks; intrinsic signal imaging, which was discussed in a previous chapter, and functional magnetic resonance imaging (fMRI). fMRI is a special case of the more general method of magnetic resonance imaging (MRI). MRI is a method for obtaining a 3 ­dimensional image of the brain and body in a living person, without having to perform any surgery or inject any dye or radioactive material into the blood. The images, however, are static in that they do not show, or even given any indication of the brain areas that had greater or lesser activity during the MRI scan. In the sections below, we first consider how MRI machines work and how they create 3 ­D images of structures in the body and brain. We then turn to the more special case of fMRI, a technique now widely used to determine which brain areas are involved not only in specific sensory functions, such as seeing or hearing, but also which areas of the brain are activated during higher order cognitive functions as well. fMRI is an important method that is revealing the neural structures and systems involved in the most complex cognitive functions that humans perform. Magnetic Resonance Imaging (MRI) Patients being examined with MRI are placed inside the cylindrical MRI machine (Fig. 1). The MRI machine visualizes the body and brain using a combination of radio waves and a very powerful magnetic field. A typical research MRI scanner has a strength of about 3 ­4 teslas, a force about 50,000 times stronger than the Earth’s magnetic field. Figure 1. A: A cutaway A B view of an MRI scanner. B: A technician viewing a “slice” of the brain generated by MRI scanning. See text for a more explanation of a “slice” and how slices of the brain are generated by MRI. 1 In MRI, signals are produced by protons in brain tissue. The basic idea is that protons, which are the nuclei of hydrogen atoms, can be made to resonate when placed in a strong magnetic field and a radio frequency pulse is the applied to them (see Fig. 2 below). Different brain structures can be imaged because their protons have different properties. Thus proton resonance differs in fat and water, and depends upon whether the water is intracellular or extracellular, in the blood or in the cerebral spinal fluid. The influence of a magnetic field on protons is shown schematically in Fig. 2. When a patient is first placed in the MRI machine, and a magnetic field has not yet been turned on, each proton in his or her tissue rotates around its axis, acting as a small magnet with its own dipole. Normally, protons are randomly directed so the tissue essentially has no net dipole, as shown in Fig, 2 ­1. When a vertical magnetic field is applied to the tissue, as in Fig. 2 ­2, all the protons align with the field. Magnetic Resonance Imaging Fig. 2. Behavior of protons, the nuclei of p protons spin around their axes, creating individual magnetic fields with random directions p p p p p p p p p p p 1 1 1 p no external magnetic field no external magnetic field no external magnetic field p p vertical external magnetic field vertical external magnetic field vertical external magnetic field p p p p p p 2 2 2 Radio frequency p p p p p p p p p p pulse turned on Radio frequency pulse Radio frequency pulse protons align with vertical magnetic field but proton rotations are out of phase protons align with vertical magnetic field but proton rotations are out of phase protons align with vertical magnetic field but proton rotations are out of phase proton rotations are in phase and generate a large signal proton rotations are in phase and generate a large signal proton rotations are in phase and generate a large signal p p pp 3 3 3 p p p p p p p p p p rotating protons begin to “dephase” and signal strength is reduced rotating protons begin to “dephase” and signal strength is reduced p p p p p p p 4 4 p Radio frequency pulse turned off Radio frequency pulse turned off p p p p p p dephasing continues further reducing signal strength dephasing continues further reducing signal strength p p 5 5 p Radio frequency pulse turned off Radio frequency pulse turned off p p p Radio frequency pulse turned off Radio frequency Radio frequency pulse turned off pulse turned off 6 6 6 p p p p p p p p p p p p p p p protons realign with vertical magnetic field protons realign protons realign with vertical magnetic with vertical magnetic field field 2 hydrogen atoms, in the absence of a magnetic field (1) and when a strong vertical magnetic field is presented (2), as in an MRI machine. When a horizontal magnetic field is pulsed at a radio frequency (3), the proton rotations align and are in phase thereby generating a large signal that is detected by the MRI machine. When the radio frequency pulse is turned off (4, 5 and 6), protons begin to de ­phase and signal strength is reduced. The alignments create a net magnetic field that is also vertical, but the proton rotations are out of phase and thus create no net detectable signal. The key event occurs when second magnetic field formed by a radio frequency (RF) pulse (a high frequency sine wave) is applied, as in Fig. 2 ­3. The radio frequency pulse acts horizontally, or orthogonal to the vertical magnetic field. The horizontal radio frequency pulses “tip” the protons so they rotate in the horizontal plane synchronously, or in phase with one another, and the synchronous activity creates a large signal that is measured by MRI. When the RF pulse is turned off, the rotating protons begin to move out of phase with one another, i.e., they de ­phase. The de ­phasing leads to a loss of signal intensity (Figs. 2 ­4 to 6). Protons have different de ­ phasing rates depending upon whether they are embedded in fat, cerebrospinal fluid, myelin or the membranes of somata. By using detectors sensitive to the radio frequencies emitted by the oscillating protons and computational techniques of the processor in the MRI machine, images of the living human brain can be obtained with remarkable resolution, as shown in Fig. 3. MRI- Sagittal Scan Fig. 3. MRI sagittal scan of the brain. The various parietal lobe brain regions are labeled and can be clearly seen. frontal lobe corpus callosum midbrain occipital lobe pons cerebellum medulla MRI images are seen as “slices” The whole brain cannot be scanned at once but rather it is scanned in slices that are only a few millimeters thick. Think of an MRI slice in the same way you think of a slice of white bread, as part of the entire loaf. The loaf, the entire brain, is the sum of all the slices. Each MRI slice in turn is divided up into small square units called voxels. Typically in MRI, each voxel represents a small area of brain that is roughly 3 mm x 3 mm, but voxels can be made smaller for specialized purposes. Slices can be taken from any part of the brain and can be obtained at any desired angle. For example, it is possible with MRI to obtain images of the living brain in slices from any of the three cardinal planes, coronal, sagittal or horizontal, and at any level (Fig. 4). What this means is that the investigator can pick exactly which area he or she wants a picture of and obtain images in the form of slices that are each just a few mm thick. 3 MRI images can be obtained in the three cardinal planes horizontal coronal sagittal sagittal with slice Fig. 4. MRI images in the horizontal, coronal and sagittal planes are shown. The far right panel shows a sagittal MRI slice. The superimposed lines indicate how horizontal slices from the same image at different depths could be created. It is important to remember that the MRI brain images are static; they say nothing about the degree of activity occurring in various brain areas nor how activity shifts to different regions under different conditions. Rather the images are important for diagnostic purposes; to evaluate whether damage from a stroke (Fig. 5) or other injuries has occurred, or whether a tumor is present and so on. Horizontal Slices showing damage caused by a massive stroke on the right cerebral hemisphere Fig. 5. MRI slices from a patient who suffered a massive stroke to the right cerebral cortex that extended from the frontal into the parietal and temporal lobe (red area in brain on far right). The brain level at which each slice was obtained is shown in the brain drawing on the far right. Damage in each slice is outlined with red lines. Next we turn to a variant of MRI called functional resonance magnetic imaging (fRMI), which uses changes in blood flow to identify regions of the brain that are activated by specific stimuli or particular behaviors or cognitive processes. BOLD Signals Allow Activity in the Brain to be Mapped with Functional Magnetic Resonance imaging (fMRI) fMRI allows the differences in the functional activity in various parts of the brain to be imaged in a living person while he or she is performing a task of any complexity. Like MRI, fMRI does not require injections of dyes, radioactive substances or X ­rays. The reason is that fMRI measurements are sensitive to changes in the oxygenated level of blood. Blood rich in oxygen evokes a larger signal than deoxygenated blood, as explained in Fig. 6. In other words, the magnetic properties of deoxygenated iron in blood hemoglobin are such that a voxel becomes slightly brighter or darker in response to changes in the oxygenation or 4 deoxygenation of hemoglobin in that voxel. Recall that when a brain area is activated, it begins to use more oxygen and within seconds the brain microvasculature responds by increasing the flow of oxygen ­rich blood to the active area. Thus, localized changes in activation can be inferred from the changes in the brightness of voxels at each time point. This means that blood itself serves as a contrast agent. Indeed, fMRI signals are known as BOLD signals, BOLD referring to blood oxygenation level dependent changes in the magnetic resonance signals. Such fluctuations are detected using statistical imaging processing techniques to produce maps of brain regions that are active during various tasks. Color is then added to the image to create a color ­coded map of the most active areas. Fig. 6. In regions of heightened neuronal activity the supply of oxygenated blood is greater than its consumption, leading to a higher than normal ratio of oxygenated to deoxygenated blood. Because the two forms of hemoglobin have different effects on the de ­phasing of protons, they produce different magnetic resonant signals. A; In the unstimulated condition, there is little activation of neurons; blood flow is not increased and a relatively large proportion of the hemoglobin is in the deoxy form. Because deoxyhemoglobin promotes de ­phasing of the rotating protons, the proton rotations are not synchronized and the MRI signal is relatively weak. B: In the stimulated condition, the subject is exposed to a stimulus or is engaged in a task that requires activation of some portions of their brain. When neurons become active, blood flow increases and thus directs an excessive amount of oxygenated blood to the active area. Thus, the proportion of deoxyhemoglobin decreases. Thus de ­phasing of proton rotation is much slower, and thus protons remain synchronized for a longer time thereby generating a correspondingly stronger magnetic resonance signal. The stronger signal is processed as darker voxels in those areas where enhanced blood flow occurred. Functional Magnetic Resonance Imaging (fMRI) LGN LGN V5 V5 V1 V1 Occiptial lobes An example of a color ­coded map is shown in Fig. 7. The colored areas in Fig. 7 correspond to areas that were activated by a moving visual stimulus, compared with imaging obtained with the eyes closed. Visual areas are activated bilaterally, inclucing the primary visual cortex (V1) and the visual motion area (V5) in the occipital ­temporal region. The lateral geniculate nucleus (LGN) is also activated bilaterally. Fig. 7. BOLD signals in visual system. The BOLD (blood oxygen level dependent) signal is superimposed on a horizontal slice of the brain imaged by fMRI. Optogenetics together with fMRI show that BOLD signals are evoked 5 by excitation The assumption that BOLD signals are due to increased excitatory activity is central to the interpretation and the utility of fMRI studies in both basic and clinical research. The BOLD signals in fMRI, however, measure changes in blood flow (hemodynamics) rather than trains of action potentials generated by neurons. The hemodynamics reflect complex and incompletely understood changes in cerebral blood flow and cerebral metabolic rate of oxygen consumption following neuronal activity. It is therefore unclear as to exactly what aspect of neural activity generates BOLD. Candidate circuit elements for triggering various kinds of BOLD signals include the action potentials of excitatory neurons, subthreshold changes in the membrane potentials of neurons, the action potentials of axons passing through a region, or even the changes in membrane potentials of glial cells, the supporting cells in the brain. To put the interpretation of BOLD on a firmer footing, a method is needed that can evoke discharges confined to a localized region of the brain while the brain is simultaneously imaged with fMRI. What this combination of methodologies would have to show is that the BOLD signal not only is confined to the structures in which the excitatory activity occurred but also follows the neural excitation in time by a few seconds. These are exactly the features that were recently shown by Lee and his colleagues (Lee et al., Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature , 2010, Vol. 465 (10), pp 788 ­792). The way they did it was to use channelrhodopsin 2 (ChR2) to optogenetically drive neuronal activity while obtaining BOLD signals with fMRI. Recall from a previous chapter that ChR2 is a microbial derived ion channel that is activated by blue light. When the channel is activated, it is permeable to Na+ ions and thus depolarizes the cell. What these investigators did was to transfect cells in the motor cortex of rats with ChR2 via viral vectors. The viral vector had a fusion construct in front of the promotor for calcium activated calmodulin ­dependent protein kinase II (CaMKII). The construct was ChR2 fused with Yellow Fluorescent Protein (YFP). Thus, only the CaMKII cortical cells expressed ChR2 and YFP. YFP gene opsin gene Fig. 7 The fusion gene that was introduced into cells in the ChR2 motor cortex of rats with a viral vector. promotor CaMKII The significance of the co ­expression with CaMKII is that there are several types of cortical cells. Excitatory principal cells, which send their projections to lower regions, all express CaMKII. GABAergic cortical cells, whose axons are confined to the cortex, and other types of cortical cells, do not express CaMKII. Those cells are not transfected with ChR2 and thus cannot be excited by light. By making a restricted class of cells sensitive to light, i.e., excitatory principal cells expressing CaMKII, the investigators could selectively manipulate the activity of those cells while leaving other circuit elements unperturbed. Using rats with transfected cortical cells, the investigators demonstrate that stimulation 6 of the CaMKII ­expressing excitatory neurons elicited positive BOLD signals, but only in the region of the brain (the motor cortex) that was excited by light stimulated. To show these features they first show that light did indeed excite neurons in the motor cortex. To simultaneously stimulate and record from the cortical cells, a metal microelectorde was attached to an optical fiber, with the tip of the electrode deeper than the tip of the fiber to ensure illumination of the recorded neurons. They called this arrangement an “optrode”. A cannula was cemented onto the skull over the motor cortex, as shown in Fig. 9. An optrode was inserted in the cannula so that the motor cortex could be activated by blue light while action potentials were recorded by the microelectorde. As shown in Fig. 9, illumination of the motor cortex with blue light evoked firings from the transfected cortical cells. The firings were evoked almost immediately by the light activation with no apparent latency. optrode Fig. 9. A A: sagittal section of a transfected cortical neurons B rat brain showing the arrangement of the sagittal section of rat brain cannula (not labeled) and the optrode. The blue light shows illumination of transfected cells. B: Sections of the rat C brain showing the transfection of neurons in the motor Responses of cortical cortex. The neurons neurons to fluoresce because they light stimulation are transfected with YFP. C: recordings of neural activity from motor cortex with an optrode that records the action potentials from a few neurons. Notice the increase in firing when the blue light was applied to the motor cortex. After showing that light excited the transfected cells, they next recorded BOLD signals while illuminating the motor cortex and showed that activation of the motor cortex evoked a strong BOLD signal confined to the motor cortex (Fig. 10, left panel). Of importance is that there was a latency of about 4 ms between the onset of light, and hence the activation of the cortex, and the onset of the BOLD signal (Fig. 10, middle panel). This is important since it takes several ms for the vasculature to react to the enhanced neural excitation, which accounts for the several second latency between the light and BOLD signal. As a control, they also performed the exact same manipulation in rats in which the motor cortex was injected with a saline solution that did not contain the viral vector. As can be seen in the right panel of Fig. 10 (right panel,  ­opsin/+light), there was no BOLD signal in rats whose cortical cells were not transfected. 7 A B fMRI BOLD signal C Fig. 10. BOLD signal change (%) A: BOL 4s D sign als in slice light on - 20 sec s of brai n. Asterisk in slice shown in middle row on far left is location of optrode and thus locus of light source. BOLD signal was largely confined to motor cortex, at locations at and around optrode. B: Time course of BOLD signal in motor cortex. Notice the delay of ~4 ms between the onset of light and the initial BOLD signal. C: Light applied to motor cortex in rats that were not transfected with ChR2 did not evoke BOLD. BOLD signals can be evoked in nuclei that receive innervation from motor cortex The authors did not stop here, but rather extended their assessment by asking whether the targets of the axonal projections of the activated cortical cells could also evoke BOLD signals. In essence, what they are asking is whether this technique of optically activating specific cell groups could be used together with fRMI to map neural circuits; that is, can the cells in other regions of the brain that are connected with and thus activated by the group is optically stimulated be determined? As a first step the authors traced the one of the projections of the transfected cells to a nucleus in the thalamus. They could trace the projection because the transfection caused the ChR2 ­YFR to be inserted not only in the cell bodies, but throughout the entire neurons including their axons. Thus the projections could be seen from the YFP in sections of the brain taken after the animals were sacrificed (Fig. 12B, far right panel). When the motor cortex was stimulated with blue light, the optrode in the cortex and another electrode implanted in the thalamus both recorded action potentials (Fig. 11). The latencies of the thalamic action potentials, however, were longer than the latencies of the cortical action potentials. The longer latency of the thalamic action potentials was due to the conduction time from cortex to thalamus. Since light activation of the cortex evoked action potentials in cortex and the thalamus, it follows that light activation of the cortex should also evoke BOLD signals in both cortex and thalamus. As shown in Fig. 12, fMRI slices taken through the thalamus had prominent BOLD signals that were evoked while the cortex was stimulated with light. Moreover, the sections in Fig. 12B shows that the transfected cells in the cortex sent projections to the thalamus from which BOLD signals were recorded. The fibers of the cortical neurons, which had YFP, are shown in the right panel of Fig. 12B. These fibers from the motor cortex then excited the thalamic neurons, resulting in the BOLD signal. 8 A Fig. 11. A: Stimulus and B recording arrangement. Optrode in cortex delivered light and recorded light evoked activity. A second electrode implanted in thalamus records activation of thalamus. Light activation of motor C cortex (M1 in panel A) motor cortex recording evokes firing in both motor cortex (B, top panel) and in thalamus (B, lower panel). The difference in firing Thalamus recording latency between motor cortex and thalamus is shown in C. Thalamic discharges occurred about motor cortex optical stimulation (15 ms) 7.75 ms after cortical discharge, showing that the cortical cells drove the thalamic cells via their excitatory projections. Fig. 12. A: Section of rat brain showing optrode that provided light stimulation of cortex, and the region of the cortex that represent the slices from which BOLD signals were obtained upon stimulation of cortex. 1, 2 show the position along the rostro ­caudal (front to back) axis of the brain from which the two coronal sections in panel C were obtained. B: Green region shows that this region of the thalamus is innervated by cortical neurons transfected with the ChR2 ­YFP fusion gene. Left panel is low power view of thalamus while right panel is higher magnification that shows fibers in thalamus that are the axons of transfected cortical neurons. 9 Returning to the idea that “The Gain in Brain is Mainly in the Stain” In earlier portions of this course, we emphasized the importance of stains, since stains allow investigators to see the structure of neurons and allows investigators to trace their connections. Connections, in turn, are what determine functional systems and functional systems are the operational units upon which the brain works and it is the interactions among functional systems that endow the nervous system with its remarkable abilities. The studies cited above showed that BOLD signals are indeed due to the local changes in hemodynamics caused by local excitation, i.e., action potentials. But these studies also showed that fMRI within the living and intact mammalian brain reveals BOLD signals in targets distant from the neurons that were stimulated. What this suggests is that the method of optrode fMRI, i.e., transfecting a specified region of the brain to induce a particular neuronal population in that region to express ChR2 ­YFP coupled with fMRI, has an enormous potential for evaluating the connectivity of neural regions in a more detailed level and with greater specificity than had previously been possible. With this method, as with classical methods for tracing axonal connections, the only connections that are stained are those that the transfected cells make with their targets. Normally, those connections are the only ones that could be determined because they are the only connections that can be seen through the axons stained with a fluorescent protein, or with some other stain in classical tract tracing studies. But now comes the extraordinary potential advantages offered by optrode fMRI; the investigator may be able “see” the entire functional circuit engaged by the light activation of the specified cell group of transfected cells. They could see the full circuit, not with stained cells and stained axons, but with BOLD signals. Stated differently, it would allow a specified group of cells in a restricted region of the brain to be stimulated with light, and then would allow the functional connections of that cell group to be mapped with the BOLD signals evoked in slices that could be taken from all levels of the nervous system and at any plane, coronal, horizontal or sagittal. This would indeed be a revolutionary technology that has the potential to reveal the normal operations of functional circuitry and what changes to that circuitry occur in various pathologies or neurological disorders. 10 ...
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This note was uploaded on 09/19/2011 for the course BIO 365R taught by Professor Draper during the Spring '08 term at University of Texas.

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