Supplementary Chapt. 2-optically imaging the brain(1)

Supplementary Chapt. 2-optically imaging the brain(1) - ...

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Unformatted text preview: Supplementary Chapter 2 Methods for Optically Imaging the Brain The major advances in our understanding of how the brain works relied on electrophysiological recordings from individual neurons. For example, we learned how action potentials are generated by recordings from the giant axons in squid, and how synaptic transmission works by recordings from both the giant stellate ganglion in squid and from the brilliant study of Bernard Katz and his students who used the neuromuscular junction as the model synapse. Moreover, we learned how neurons in the motor cortex initiate movements by recording from individual neurons in awake, behaving monkeys and how visual images are processed and represented in the visual portions of the brain from the studies of Hubel and Wiesel, who recorded from individual neurons in the visual cortex of cats and monkeys. Now scientists are able to observe the functional activity, not of single neurons, but rather of large regions in a living brain in response not only to sensory stimuli or motor movements, but also to far more complex conditions that involve cognitive functions. The there are two major types of methods that allow investigators to observe activity over large portions of the brain in a living animal. One way, and the first that we will discuss, is to apply a voltage sensitive dye (VSD) to the cortex. The dye fluoresces when illuminated with light, but the key feature is the intensity of the fluorescence changes with voltage. As will be explained below, this allows the changes in the electrical activity of an entire region of cortex to be optically monitored both before and while a stimulus is presented or while the animal is engaged in a behavior. The second way does not actually record the electrical activity of the brain, but rather it utilizes methods that detect changes in the blood flow to regions of the brain. It has been long known that increases in neuronal activity in the brain are reliably accompanied by changes in local blood flow together with a more pronounced conversion of oxyhemoglobin to hemoglobin as oxygen is delivered to the metabolically active neurons. The regional changes in blood flow coupled with the enhanced conversion of oxyhemoglobin to hemoglobin in neural regions with higher neuronal activity are detected either optically, with a camera, which then generates images of brain regions where regional blood flow is highest, or with highly specialized equipment that use extremely powerful magnets and detectors that create images which also show regions of greatest blood flow. Imaging reveals how functional systems are embedded in the physical structure of the brain. Optical Imaging of Neuronal Activity With Voltage Sensitive Dyes The use of voltage sensitive dyes is simple in principle. To optically image electrical activity, the voltage sensitive dye is first applied to the brain in an intact, living animal. The dye molecules applied to the brain then become embedded in the membranes of neurons. Each molecule of dye has a hydrophobic end, which is anchored in the membrane, and a hydrophilic side that points outwards to the extracellular space. Because the dye molecule is charged, it moves as the membrane voltage changes, where hyperpolarizations pull the dye molecules into the membrane and depolarizations push them further out into the extracellular space. The movements of the dye within the membrane affect their probability of absorbing light at the excitation wavelength and emitting light at the emission wavelength in a linear fashion. Thus hyperpolarization reduces the intensity of 1 the emitted fluorescence whereas depolarization increases the emitted fluorescence. The changes in fluorescence are monitored with light ­measuring devices. Since the changes in the emitted fluorescene of the stained neurons correlate linearly with their electrical activity, the changes in the intensity of the fluorescence provide an accurate indication of the changes in membrane potential. Moreover, the dye response is very fast and is in the microsecond range. With the use of an array of photodetectors positioned in the microscope image plane, the activity, i.e., the fluorescence, across the portion of the cortex being imaged is captured by the photodetectors. The fluorescence in each photodetector comprises a pixel, and thus the pattern of activity (fluorescence) across the cortex at any point in time is expressed in the relative fluorescence of all the pixels in the array of photodetectors. But what aspect of the electrical activity is captured in each pixel? During in vivo imaging of the cortex, a single pixel contains the blurred images of various neuronal parts— including the dendrites, axons and somata of a small population of neurons — rather than a single cell. The signal from the voltage sensitive dye is linearly related to the stained membrane area, and most of the dye signal originates from cortical dendrites rather than cell bodies, because their membrane areas are orders of magnitude larger than that of neuronal somata. Therefore, the voltage sensitive dye signal in vivo mainly reflects dendritic activity, rather than action potentials. Notice that in the absence of a stimulus, the record in each pixel tells you nothing about the electrical state of that portion of the cortex; the investigator has no idea whether any of the neurons in each pixel are slightly depolarized or slightly hyperpolarized or some combination of both. What is of interest in each pixel is the overall change in the excitability of a small number of neurons; whether they hyperpolarize or depolarize in response to a stimulus or an event. Thus it is the pattern of changes in excitability that are evoked in each region of the cortex in response to a stimulus that is recorded by the changes in fluoresence in each pixel and captured simultaneously for all pixels across the cortex in one image. Optical imaging of brain activity To a computer Video Camera Metal Visual stimulus Lunate Chamber sulcus Chamber V1 VSD apply VSD before experiment Figure 1. 2 An example of the experimental arrangement used with voltage sensitive dyes is shown in the Figs 1. The top left panel of Figure 1 on the previous page shows a monkey with a chamber over its visual cortex that had previously been surgically attached to its skull. A camera fits over the chamber and measures the changes in fluorescence over the cortical surface that occur while the monkey watches a video screen. The signals from the camera are sent to a computer. The computer breaks the signal up into a large number of small pixels and then analyzes the changes in amplitude of the fluorescent signals in each pixel as visual stimuli are shown. The top right panel shows a circular portion of the skull that has been removed to expose the primary visual cortex (V1). A metal chamber is permanently mounted on the skull over VI. The tough membrane that covers the brain, the dura, is surgically removed over V1. In the lower right panel, an artificial dura is placed over V1 to prevent the brain from drying out. The dura is attached to a plastic ring that has a tube attached. Before an experiment begins, the voltage sensitive dye is applied to the visual cortex by injecting it through the tube, as indicted. The lower left panel shows a cartoon of the membrane of a neuron upon which voltage sensitive dye is applied. Several molecules of voltage sensitive dye are inserted in the membrane to various depth, as well as a voltage gated potassium channel. see through left eye V1 activated by left eye see through right eye V1 no stimulation V1 activated by right eye regions that respond to right eye (black stripes) regions that respond to left eye (white stripes) Ocular Dominance right eye -left eye Fig. 2 Figure 2 shows the results of a study that used voltage sensitive dyes to map the arrangement of ocular dominance columns in V1. The left panel in Fig. 2 shows the cortex 3 through the artificial dura and illustrates the rich cortical vascular supply. The monkey is then first exposed to visual stimuli but its right eye is covered so only the left eye is able to view the visual signals presented on the video screen. The dark stripes on the cortex shown in the top right panel schematically show the changes in fluorescence that viewing signals though the left eye would evoke. The dark stripes are increases in fluoresence caused by cortical depolarization resulting from excitation of the left eye. The panel below shows the changes in fluorescence due to viewing the same scene with only the right eye, when the left eye was covered. At first glance, the stripped patterns evoked by each eye look as if they are the same. A closer inspection reveals that they actually alternate. This becomes obvious when the pattern evoked by the right eye is subtracted from the pattern evoked by the left eye. The subtraction renders the enhanced fluorescence evoked by the right eye dark (it is positive) and the enhanced fluorescence evoked by the left eye white (since the subtraction makes it negative). The subtracted traces are shown in the bottom right panel and illustrate the alternating strips of cortex that are dominated by the right and left eyes. Another feature that has been studied with voltage sensitive dyes is the arrangement of orientation columns in V1. In this study (Fig. 3), lines of a particular orientation were presented and the response recorded. The orthogonal orientation was then presented and its response obtained. The two responses were then subtracted. The idea is that a preferred orientation will produce a large response and its orthogonal will evoke no response, and hence there would be a large difference. A less than optimal orientation for that location will evoke only a small response, as would its orthogonal, and hence subtraction will result no difference or only a small difference. The subtraction was done for each of the orientations shown below. At each location, one orientation and its orthogonal produced the maximal difference, and the orientation that evoked the largest response was then taken as the preferred orientation for that location. The values where then color ­coded and the color ­coded map is shown in Fig. 3. Fig. 3. Map of orientation preferences in V1 obtained with voltage sensitive dyes. From: Blasdel, G. and Salama, G. (1986) Voltage ­sensitive dyes reveal a modular organization in monkey striate cortex. Nature, vol 321, pp 579 ­ 585. One of its real advantages is that animals prepared for experiments with voltage sensitive dyes can be used again and again for years. After an experiment is finished, the 4 camera can be removed and the metal well can be sealed with a sterile cap. The monkey can then returned to its cage and another experiment can be conducted at a later time. Even repeated dye applications have no deleterious effects on the cortex. For these reasons, each monkey can be studied in the same or different experiments over many months or years. Imaging Intrinsic Signals in the Brain The previous section showed that optical imaging of cortical activity offers several advantages over conventional electrophysiological techniques, in which electrodes are inserted into the brain and neurons are studied one at a time. With voltage sensitive dyes, for example, investigators can map a relatively large region of the cortex, obtain successive maps to different stimuli in the same cortical area and follow variations over time. But the use of voltage sensitive dyes is not the only way that optical imaging of the cortex can be achieved. Another way is to monitor the change in the reflectance of light due to activity ­induced changes in blood flow. As mentioned previously, brain tissue has a highly regulated vascular supply that continuously diverts blood from neural areas that are less active to neural areas that are most active. Active neurons have a higher metabolism than inactive neurons and thus require greater amounts of oxygen. Consequently, there is a more pronounced conversion from oxyhemoglobin to hemoglobin around neurons firing action potentials than around neurons that are not firing action potentials. The changes in the conversion from oxyhemoglobin to hemoglobin are especially important for imaging because the reflectance of light is reduced by the conversion, and thus active regions, with less oxyhemoglobin and more hemoglobin, reflect less light than less active or inactive areas. Intrinsic changes are monitored by illuminating the cortex while monitoring the light reflected in a cortical region with a camera attached to a microscope. The camera divides the cortex into a number of small pixels and measures the reflected light in each pixel before a stimulus is presented and during the presentation of the stimulus. This is basically the same procedure as described above for voltage sensitive dyes, except here it is the difference in the amount of reflected light that is measured rather than the difference in fluorescent intensity. The maps obtained with intrinsic imaging are very similar to those obtained with voltage sensitive dyes. This is illustrated by the maps of ocular dominance in the primary visual cortex obtained with both methods, as shown in Fig. 4 below. Ocular Dominance Columns Voltage sensitive dye Intrinsic Imaging Fig. 4. Maps of ocular dominance columns showing that the maps obtained with voltage sensitive dyes are similar to those obtained with intrinsic imaging. From Frostig et al., Proc. Natl. Acad. Sci. USA Vol. 87, pp. 6082 ­6086, August 5 1990 Advantages and disadvantages of voltage sensitive dyes and intrinsic imaging Imaging of intrinsic signals and imaging with voltage sensitive dyes each offer advantages but each method has its disadvantages as well. The principal advantage of intrinsic imaging is that nothing has to be applied or added to the brain to imagine intrinsic signals. Moreover, if the skull is thinned down, the images can be viewed through the thinned skull without cutting or removing the dura. This is in contrast to imaging with voltage sensitive dyes, which requires surgical implantation of an artificial dura and the application of dye to the cortex. The main disadvantage of imaging intrinsic signals is that the signals do not correspond to the electrical activity of the cortex and are more than an order of magnitude slower than the electrical events. The reason for the slow time course is that electrical activity is not being viewed directly but rather what is viewed are changes in hemodynamics, which are only indirectly related to electrical activity. Thus, the time scales of intrinsic imaging are on the order of seconds whereas the time scale of imaging voltage sensitive dyes is on the order of microseconds, the same time as the electrical events themselves. The events that the two methods measure, changes in membrane potential and changes in hemodynamics, are coupled, in that the normal neuronal activity requires an adequate blood supply and transfer of oxygen. Nevertheless, I still find it quite remarkable that the maps produced by the two methods are in such close agreement. Indeed, it is for these reasons that both techniques have found favor and are used to evaluate the functional organization of the cerebral cortex. 6 ...
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