A Functional and Structural Analysis of Cells in Mice Visual Cortex

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A FUNCTIONAL AND STRUCTURAL ANALYSIS OF CELLS IN MICE VISUAL CORTEX BY JASMINE ZEKI A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr Alfred Beulig Sarasota, Florida March, 2012

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ii A cknowledgments I would like to thank my sponsor, Dr Alfred Beulig, for his continued support, patience, and advice throughout the writing process. I am very grateful for fi nding the email, which he had purposely left out, which asked for one of his students to travel to Houston for a thesis project. It was a very strange way to find an internship, but a perfect way at the same time! I would also like to thank Dr Leo Demski and Dr Gordon Bauer for joining my committee and for their support through my baccalaureate exam. Next I would like to thank Dr Jacob Reimer for accepting me as an intern in his laboratory. The experience at Baylor College of Medicine, working under a New College Alum, was a once in a lifetime experience. I cannot thank him enough for helping me grow both as a scientist and as a person. The entire Tolias laboratory has my eternal gratitude for what was truly a once in a lifetime experience. Many people say that they could not have gone through the thesis experience without the support of their family. This could not be more valid for me; my family is truly my rock and strongest support system. Their advice and love from the beginning of my inter nship until now had helped in more ways than I could ever hope to express in words. I love you all. Finally I would like to thank my friends both at New College and at home in Chicago. Their love and support has been crucial to my success this year.

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iii Table of Contents Chapter 1 Introduction ................................ ................................ ................................ .................. 1 Mice Visual Cortex: Justifying the project ................................ ................................ ....................... 1 Chronic Window Surgery ................................ ................................ ................................ ................. 5 Intrinsic Imaging ................................ ................................ ................................ ............................... 7 OGB staining and Calcium Imaging ................................ ................................ ................................ .. 8 Single Cell Electroporation ................................ ................................ ................................ ............... 9 Modified Rabies Virus ................................ ................................ ................................ .................... 10 Small scale summary ................................ ................................ ................................ ...................... 11 Chapter 2 Materials and Methods ................................ ................................ .............................. 13 Subjects ................................ ................................ ................................ ................................ .......... 13 Equipment and data collecting protocol ................................ ................................ ....................... 13 Two Photon Microscope and Manipulator ................................ ................................ ............ 13 Chronic Window Surgery ................................ ................................ ................................ ....... 15 Intrinsic Imaging ................................ ................................ ................................ ..................... 16 Calcium Imaging ................................ ................................ ................................ ..................... 17 Visual Stimulus and Tuning ................................ ................................ ................................ .... 18 Single Cell Electroporation ................................ ................................ ................................ ..... 1 9 Rabies Virus Injection ................................ ................................ ................................ ............ 20 Perfusion and making microscope slides ................................ ................................ ............... 20 Experimental Design ................................ ................................ ................................ ...................... 21 Intrinsic Imaging ................................ ................................ ................................ ..................... 21 Calcium Imaging ................................ ................................ ................................ ..................... 21 Single Cell Electroporation ................................ ................................ ................................ ..... 22 Rabies Injection ................................ ................................ ................................ ...................... 22 Chapter 3 Results ................................ ................................ ................................ ......................... 23 Intrinsic Imaging ................................ ................................ ................................ ............................. 2 3 Calcium Imaging ................................ ................................ ................................ ............................. 25 Single Cell Electr oporation ................................ ................................ ................................ ............. 33 Rabies Injection ................................ ................................ ................................ .............................. 33 Chapter 4 Discussion ................................ ................................ ................................ .................... 3 8

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iv Concluding the results of Calcium Imaging ................................ ................................ .................... 38 Concluding the results of Single Cell Electroporation ................................ ................................ .... 39 Concluding the results of Rabies Injection ................................ ................................ ..................... 39 Future Directions ................................ ................................ ................................ ........................... 40 Appendix Operational Procedures ................................ ................................ .............................. 42 Anesthesia ................................ ................................ ................................ ................................ ...... 42 Pipette Puller ................................ ................................ ................................ ................................ 42 Pipette Holder and Manipulator ................................ ................................ ................................ .... 43 Bibliography ................................ ................................ ................................ ................................ .. 45

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v List of Tables and Figures Figure 1 ................................ ................................ ................................ ................................ .......... 13 Figure 2 ................................ ................................ ................................ ................................ .......... 14 Figure 3 ................................ ................................ ................................ ................................ .......... 15 Figure 4 ................................ ................................ ................................ ................................ .......... 15 Figure 5 ................................ ................................ ................................ ................................ .......... 17 Figure 6 ................................ ................................ ................................ ................................ .......... 17 Figure 7 ................................ ................................ ................................ ................................ .......... 18 Figure 8 ................................ ................................ ................................ ................................ .......... 18 Figure 9 ................................ ................................ ................................ ................................ .......... 19 Figure 10 ................................ ................................ ................................ ................................ ........ 19 Figure 11 ................................ ................................ ................................ ................................ ........ 20 Table 1 ................................ ................................ ................................ ................................ ............ 23 Figure 12 ................................ ................................ ................................ ................................ ........ 23 Figure 13 ................................ ................................ ................................ ................................ ........ 24 Figure 14 ................................ ................................ ................................ ................................ ........ 25 Figure 15 ................................ ................................ ................................ ................................ ........ 25 Figure 16 ................................ ................................ ................................ ................................ ........ 26 Figure 17 ................................ ................................ ................................ ................................ ........ 26 Figure 18 ................................ ................................ ................................ ................................ ........ 27 Figure 19 ................................ ................................ ................................ ................................ ........ 27 Figure 20 ................................ ................................ ................................ ................................ ........ 28 Figure 21 ................................ ................................ ................................ ................................ ........ 28 Figure 22 ................................ ................................ ................................ ................................ ........ 29 Figure 23 ................................ ................................ ................................ ................................ ........ 29 Figure 24 ................................ ................................ ................................ ................................ ........ 30 Figure 25 ................................ ................................ ................................ ................................ ........ 31 Figure 26 ................................ ................................ ................................ ................................ ........ 32 Figure 27 ................................ ................................ ................................ ................................ ........ 32 Figure 28 ................................ ................................ ................................ ................................ ........ 33 Table 2 ................................ ................................ ................................ ................................ ............ 33

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vi Table 3 ................................ ................................ ................................ ................................ ............ 34 Table 4 ................................ ................................ ................................ ................................ ............ 35 Table 5 ................................ ................................ ................................ ................................ ............ 36 Table 6 ................................ ................................ ................................ ................................ ............ 37 Figure 29 ................................ ................................ ................................ ................................ ........ 37 Figure 30 ................................ ................................ ................................ ................................ ........ 37 Figure 31 ................................ ................................ ................................ ................................ ........ 40 Figure A1 ................................ ................................ ................................ ................................ ........ 42 Figure A2 ................................ ................................ ................................ ................................ ........ 43 Figure A3 ................................ ................................ ................................ ................................ ........ 43 Figure A4 ................................ ................................ ................................ ................................ ........ 44

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vii A bstract This thesis examines new procedures used to study the functional and structural properties in the primary visual cortex (V1) in mice. Chronic window surgery was used to create an opening in the mice skulls that can easily be continually accessed and image d. Two Photon microscopy was used to achieve a deeper view into a live mouse than would be available in other form s of microscopy. Intrinsic Imaging function ed to establish the area viewed under the Two Photon microscope as V1 by showing blood flow with two orientations of a moving bar stimulus. Calcium Imaging wa s used to show cell activity when a mouse is presented with two different moving bar stimuli which have varying m oving bar orientations. Cells we re found to be tuned or not and then compared to other cells in the same area to examine the effect of proximity of cells on creating functional relationship s between them Single cell electroporation wa s used to introduce a rabies plasmid to a single cell while keeping it alive. Once a successful ele ctroporation occurred, the cell received an injection of a modified rabies virus. If this step was successful, the pre synaptic connections to a single post synaptic cell would be demonstrated. Overall, the results showed that the procedures used to ob tain data were very successful. Intrinsic Imaging showed blood flow orthogonal to each other when presented with two different orientations of moving bars. Calcium Imaging showed changing results from groups of cells that were similarly tuned to areas of cells that were all differently tuned. T his raised the question of the e ffect of the two different moving bar stimuli on the results obtained. Single cell electroporation proved to be a difficult but good method of giving a single cell the plasmid required for later rabies infection. The rabies infection was successful for those cells

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viii which were successfully electroporated. Slices of the successfully infected brains were mounted on micr oscope slides so that pre synaptic cells could be counted. In conclusion, the methods used in this study were a successful way of gathering functional and structural data of mice visual cortex separately. The next step is to obtain method to connect th ese two sets of data to find a connection between functional and structural data. Dr Alfred Beulig

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1 Chapter 1: Introduction Mice Visu al Cortex: Justifying the project The visual pathway from the eye to the visual cortex is one that is well understood. Before an in depth discussion of the visual cortex begins it is essential to understand where the information comes from: the retina of the eye. The retina acts as a starting place for neural visual processing. It has five layers, two of which are composed of neural processes and three which are composed of cell bodies. The three layers of cell bodies are termed the outer nuclear layer inner nuclear layer, and the ganglion cell layer (Squire et al 2008). The outer nuclear layer is farthest from the center of the eye and contains photoreceptors called rods and cones. These rods and cones function under low (rods) and high (cones) amou nts of light to produce graded potentials. The outer plexiform layer is the ne x t layer and contains interneurons, the horizontal cells The next layer is celluar, comprised of b ipolar cells also producing graded action potentials from reflected light levels. Fa r ther to the interior of the eyeball is the inner horizontal layer with h orizontal and amacrine cells These function to create an antagonistic center surround relationship in receptive field s of t he bipolar cells. The ganglion cell layer contains cell bodies of retinal neurons whose axons go on to form the optic nerve, which is the pathway from the retina to the further visual processing stations. The two neural processing layers are called the i nner and outer plexiform layers in which interactions between retinal neurons occurs (Squire et al 2008). Continuing from the retina, ganglion cells project to the optic chiasm and cross where axons from the medial and lateral areas of the retina cross and join, but only half of the total axons in the retina cross. From here the retinal axons from both eyes travel down the optic

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2 tract to the lateral geniculate nucleus (LGN) (Squire et al 2008). The LGN has six layers, which each receive inputs from on e eye. From the LGN the axons travel to the striate cortex, or the primary visual cortex. Therefore, visual information from each eye can be separated through the lateral geniculate nucleus all the way to the cortical neurons. This direct pathway is sti ll under research, which is part of the research to be investigated in the current project. The main focus of physiological studies of the primary visual cortex has been directed towards behaviors of individual neurons. It has been well established tha t cells which respond et al 2008). Ocular columns have been found compromising cells that are either dominated by one eye or prefer similar orientations. Similarly, one finds that orientation preferences of cells tend to continue in rows across the cortex. Most of this research has been done in cats and monkeys (Squire et al 2008). To relate these occurrences to retinal inpu ts a retinotopic map can be produced by positioning a stimulus to a certain point in the retina and marking the area that responds on the visual cortex. Mice have not always been known to have a similar cortex organization to other higher mammals (i.e cats and monkeys). mammals which have more sophisticated visual systems than mice, such as cats and monkeys. Using mice as a study subject has several benefits. First, very simply put, they are more r eadily available and easy to take care of than other higher mammals. Second, recently transgenic mice have become available to be used in studies examining in vivo circuit labeling and manipulation, like in this experiment (Huberman 2011). A negative th ought in using mice is a small size limitation, but this can be beneficial. In mice, it is significantly easier to visualize the entire visual cortex at one time. In terms of smaller details of the visual system, mice also present a

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3 few key benefits. Th e retina is predominantly made of rods, which are used in low light vision, meaning their visual system can still function well with minimal lights, which is needed when using microscopes. The first to recognize that mice were a valuable subject to study cortex structure was Drager in 1975. She suggested that mice responded to specifically orientated stimuli, just as overall tuning seems to be less sharp in mice than it is in cats and monkeys 2003). This is because there are significantly fewer photoreceptors, specifically in the mouse retina than in the monkey or cat eye (Hubuner 2011). Hubener conclude d that homogeneous tuning did not occur across the entire visual cortex, rather in subgroups of homogeneous tuning. Homogeneous tuning is groups of cells which respond to the same moving bar orientation Heterogeneous tuning is groups of cells which are t uned for different moving bar orientations. This study looks to answer the question of whether the mice visual cortex is in fact homogeneously or heterogeneously tuned. There are six layers present in the visual cortex: layer 1, layer 2, layer 3, layer 4, layer 5, and layer 6; though layer 2 and layer 3 are typically referred to as one layer. Also, layer 4 is subdivided into layers A, B, and C. This study examines neurons in layer 2/3, which have different properties than those in layers 4 and 5. Ori ented cells are commonly found in layer 2/3, while fields of commonly functioning cells are found in layer 4, and layer 5 contains cells that are not oriented and have large visual receptive fields (Mangini 1980). Whether the cells in layer2/3 are homog eneously or heterogeneously tuned continues to still be a debate with suggestions varying from completely heterogeneously tuned, to patches of cells that are either homogeneously or heterogeneously tuned, to completely homogeneously tuned. Through

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4 methods later discu ssed, the project looks to demonstrate that layer 2/3 cells are heterogeneously tuned. In a study by Jia et al in 2010 dendritic spines were shown not be to tuned to a specific orie ntation preference. They show that, as consistent with the salt and pepper mouse brain model the same orientation preference did not meet at one dendrite, but instead they were widely dispersed over the whole dendritic tree. It was shown that neurons in layer 2/3 of the brain are hotspots for specific sensory fe atures. The dendrites studied are identified as able to receive heterogeneous signals that code for many different orientations. Even if a neuron was highly tuned for a specific output signal it was able to receive input signals for many different orient ation pre ferences. From their data the group concluded that models that suggest convergent inputs to single dendrites are incorrect, and instead the correct model is a summation of widely distributed inputs. However, they do suggest that in other areas of the brain it may be possible that there are clustered sensory inputs, which is the argument that Ko et al (2011) discussed. Ko s (2011) findings were almost exactly the opposite of what Jia et al found. Using both in vitro and in vivo imaging, t hey found that orientation tuning preference was directly related to dendrite connection probability. Neurons with the same preferred orient ed stimuli were shown to be connected at twice the rate as those with right angle stimuli preference. Similarly, n eurons were connected that responded to naturalistic stimuli more than those which responded with unrelated responses. A naturalistic stimuli is one that mimics the orientations of objects that occur naturally, i.e, not a moving bar. Neuronal pairs were also studied; the pairs were shown to be more likely to be connected when they responded similarly to stimuli. This research suggests that there is in fact a correlation between function and connectivity of

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5 neurons on a small scale. The argument between these two res earch studies was investigated in the current project. Chronic Window Surgery In mouse brain research there are two common techniques used to view the brain under a two photon microscope: bone thinning and cranial window. Using bone thinning allows the experimenter to cause little to no disturbance in the brain, which is beneficial because it is believed that once a bone piece is removed neural circuits will modify themselve s or die. The most efficient use of this techni que assembled and modified throughout life and how glial and other cells function in the living 2010). Yang argues that there is significant damage caused by the inflammatory reaction of the brain when a piece of the skull is removed; damage that may change structures within the brain. It is also argued that there is a one to two week opaque period after the bone removing surgery in which the area remains opaque, but that immediately aft er thinning the bone the area can be imaged. Finally it is noted tha t bone thinned mice tend to live longer and are able to be imaged longer (over two years) than those with an open skull (several weeks to months). However there are li mitations to bone thinning having to do with skull thickness, deterioration, and area size. Thickness of the bone can be an issue for a few reasons relating to the thickness being optimal for a quality image to be produced. If the bone is not thinned enough or unevenly t hen the fluorescent structures within the cortex can be abnormal. To avoid this, Yang suggests that the bone thickness should be between 15 and 25 m to ensure quality images of synapses without causing damage to the cortex from pushing the skull down.

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6 De terioration can also occur if the same animal is imaged more than five times in a row, and over time the bone will continually thin with each optical section, which can cause different images to be produced between sessions (Holtmatt 2009). This does not occur in the chronic window since there is no bone left to thin. Finally, in a thinned bone preparation only a small area (about 0.2mm) can be studied, while in bone removal the area can be much larger (2 5mm). Because of this Yang concludes that bone t hinning should be used to study the connections in the live cortex while the chronic window technique should be used to image a large portion of the cortex or in drug studies (to cover the area of cortex with a drug). The chronic window surgery is used in the current experiment for reasons best described by Holtmatt et al (2009) ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The enti (2009). One of the main goals of this project is to establish connectivity of neurons and to reconstruct major axonal arbors with numerous pre synaptic cells. Another is to obtain as many optical imaging sessions as possible at one time, which is possible with the chronic window because there is no damage that a deteriorating skull could cause to the brain. Holtmatt et al (2009) even suggests that the window can be combined with in trinsic imaging and injection of viral vectors, w hich are both essential parts of this project. To counter the argument that chronic window surgery can change structure of the neural circuitry, Holtmatt et al (2009) ent changes in the expression of glial complexities of dendritic and axonal arbors a

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7 et al 2009). For these reasons, this project concluded that a chronic window was the better suited technique for this project. Intrinsic Imaging The goal of int rinsic imaging in relation to this project was to create a retinotopic map to ensure that the area being examined was the primary visual cortex (V1). There are two main types of imaging that create maps like this: those that study intrinsic signals and th ose that study voltage sensitive dyes. For the purposes of this study, imaging focusing on intrinsic signals, i.e. hemoglobin movement, will be discussed. As stated by Grinvald (2001) dimensional functional organization of a given cortical area is a key step towards revealing the mechanisms of Currently the simplest and most efficient way to do this is to map slow intrinsic changes in the brain, such as the absorption of cytochromes or hemoglobi n. This experiment mapped the presence of oxygenated versus deoxygenated hemoglobin in relation to the light reflected back because of the changes. These intrinsic changes are essentially activity dependent vascular responses (Cannestra 1996). Hemoglobin intrinsic et al made it possible to map out these occurrences (Grinva l d 2001). As previously stated the intrinsic changes are activity dependent, t his activity is a response to stimuli presented to the subject. There are two types of stimuli presented: episodic and continuous periodic. In episodic presentation the responses to different stimuli are averaged to a blank stimulus without a moving obj ect, in order to create a map. This blank stimulus creates data of blood flow normally, which is then compared to blood flow with a

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8 stimulus. The comparison of the two creates a map. Continu ous periodic stimulation generates data continuously of slow he modynamic responses in a certain space which is used to calculate neuronal responses (Kalatsky 2003). In this experiment a continuous/period stimulus is presented: there is a moving bar presented followed by a short static image, and then the process rep eats with the moving bar at a different orientation. The two orientations used for this experiment were 0 and 90 or horizontal and vertical. The hemoglobin changes are color coded for stimulus orientation and non active regions do not show a color, which creates a retinotopic map (Scheutt 2002). A retinotopic map is the desired product from intrinsic imaging in this project. OGB staining and Calcium Imaging Calcium imaging of neural networks is a very good technique used to observe neuronal functi on in vivo By injecting fluorescent indicator dyes, such as OGB (Oregon Green 488 BAP TA 1 acetoxymentyl ester), into the brain w ith targeted pressure, areas of dense neurons can be imaged in a live subject up to 500 m below the surface (Garaschuk et al 2006). When coupled with two photon imaging and stimuli presentation intracellular calcium signals can be monitored in anesthetized mice. Using this technique, individual neurons can be studied and analyzed in real time, with the ability to study multi ple single neurons at a time (Stosiek et al 2003). The injection of the dye is accomplished using a technique called multicell bolus loading (MCBL). The dye is hydrolyzed by inter cellular esterases into the cells of interest after injection. This all ows for the calcium transient s, which are activity dependent to be monitored by two photon movie imaging (Garaschuk et al 2006). The targeting involved in this technique allows

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9 for less diffusion of the dyes over time, making it easier to image a specif ic part of the brain. It can be used to study the activity of whole populations of neurons at a time (Stosiek et al 2003). The technique is particularly beneficial because it can be used at any stage of development, even in fully developed adult mice. S ince the technique is using a target area and specific dyes it can be used in chronic experiments, or even experiments that require multiple injections. However, there is a limitation to this technique: it cannot be used to study subcellular structures. This is due to the concentration of the dye commonly used in the process as well as a reduction in image contrast because of the background being stained by the injection (Garaschuk et al 2006). Single Cell Electroporation As neurobiologic al research interest steers more towards single cell properties and functions it is increasingly necessary to be able to target single cells or networks. Judkewitz et al (2009) presents a procedure that makes this possible: single cell electroporation. In this technique, two photon imaging is used to visualize the cell within a target area and a pipette is used to deliver a charge which opens the cell membrane, allowing any plasmid or vir us to be inserted into the cell. The approach offers many benefits those pertinent to this study are that single cells can be studied, a number of cells can be tightly controlled and studied, and the arrangement of cells can be specifically defined. Genetic properties such as gene products, of a single cell can be clo sely monitored and even modified, allowing a better understanding of the relationship between specific genes and beha vior. Previously, single cells could not be targeted as easily because they could not be visualized well; this is where two photon microsc opy comes into the technique. The use of a two photon microscope allows for the experimenter to clearly

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10 monitor where a cell is in relation to the pipette used. With the use of a two photon microscope cells can be selected based on their anatomy and pr eviously known function, or their placement in the studied area (Judkewitz et al 2009). With more precision the dendrites or axons of a neuron could also be targeted. Because of the precision available, single cell electroporation was used in this exper iment to deliver the rabies plasmid to a target cell. Modified rabies virus The ability to mark cellular connections in the visual system was made available by a technique involving a modified rabies virus. The need to show connections of single neuron s and specific cell types became clear. Before transsynaptic tracers were used, but they only marked the synaptic connection, not the entir e connecting cell, so the number of synapses crossed and connecting cells was very unclear (Wickersham et al 2007) The idea of viral vectors sprung from this problem: they have the ability to cross directly connected synapses from a starting cell. Wickersham et al (2007) produced a modified rabies virus which could spread from a single cell to only one synaptic con nection because the genes required for infection are not p resent in secondary connections. They show the rabies virus over other virus (such as the herpes virus) because it has a higher infection ability and it has lower cytopathicity. Rabies has the abi lity to spread to only specifically connected neurons, not surrounding non connected neurons. The virus required modification because otherwise it would spread across many synapses. The first step in modification was to stop the spread of the virus acr oss many synapses. This was achieved by deleting the gene that produced the infectious particles, which, in rabies, is the glycoprotein gene. The glycoprotein is required for trans synaptic spread, but not for

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11 transcription or replication of the virus. In place of the glycoprotein a green fluorescent protein (GFP) was placed in the coding sequence so that the connected cells would become marked (Wickersham et al 2007). When the glycoprotein is deleted, its ability to replicate the virus is still presen t, which allows the virus to replicate and express GFP in infected cells. Second, in order to target a single neural population the use of an avian sarcoma virus was required because it has very specific receptor interactions. The receptor for this vir us (TVA) is detected by the envelope protein for the avian sarcoma virus (EnvA) which causes only the cells that express TVA to be infected with the virus. By combining the rabies virus with EnvA, only a specific neural network that expressed TVA is infec ted by the rabies virus. Before rabies infection the cells must be initially infected with the glycoprotein gene (Wickersham et al 2007). In this experiment, this occurs in the electroporation step, which will be explained in further detail in the method s section. The glycoprotein gene is only present in a single initial cell, which allowed for the rabies virus to spread across one synapse because the connected cells do not have the glycoprotein gene, therefore stopping the spread of the virus (Wickersha m et al 2007). The ability of the virus to only cross one synapse shows its precision in unambiguously marking presynaptic neurons to the original cell. This technique is very important because there has never before been a technique which can so preci sely mark single synapse connections. Small scale summary We used intrinsic imaging with calcium imaging to show the functional connections of the neurons present in V1. The intrinsic imaging was used to show the area of the brain that is activated wh en the mouse is presented with a visual stimulation from a computer. The neurons are monitored for calcium transients over a long set of visual stimulations and mapped out,

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12 which will show which cells are related functionally. Then, cells were electropo rated with a rabies plasmid, and then receive d a rabies injection which show ed the pre synaptic connections from a single cell. Overall, the g oal of the following experiment wa s to combine all of the above techniques into one smooth flowing project. We aimed to show that each of these techniques can benefit the others and can be used simultaneously. In combining techniques, we hope d to reach a better understanding of how structure and function are related on the cellular level in mice primary visual cor tex.

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13 Chapter 2: Materials and Methods Subjects Specific transgenic Cre reporter mice were used for this project, the strain used was C57BL6/J. They were twenty eight all black, adult, male mice used in total. The mice were kept in cages with beddin g, food, water, and cotton to bury under. Equipment and data collecting protocol Two Photon Microscope and Manipulator Essentially, two photon microscopy is used to see a more precisely focused image at a location deeper than the surface. Two photon microscopes function to minimize the signal to noise ratio by restricting photon excitation in space which then targets the signal source and collects the photons to form the final image (Scanziani 2009). They are a form of non linear optical microscopy which means that they create contrast by using multiple photons in higher order light interactions. Because thi s study is looking at the deeper layers of the visual cortex, two photon microscopy was required to view the field of study. Figure 1: A photo of the entire two photon set up including the preparation mounting and pipette holder

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14 The following explanation of the theory of two photon microscopy is relatively basic because t wo photon microscopy is not the main topic of the project. A laser emits a beam of light between 760 command, which goes through an attenuator that deflects light, concentrating the beam. The beam then continues thro ugh the objective and to the preparation, whose fluorophores reflect the light back up. From there, the beam hits a dichroic beam splitter, which passes a very specific length of light and reflects the rest. The beam then hits a seconda ry dichroic beam splitter, which passes red light and reflects green light. These separated beams go into their own separate photo multiplier tubes (PMT). From there the beams are reflected back to the preparation and an image is created in both the gree n and red channels which are separate as a result of the dichroic beam splitters On a computer the researcher can manipulate what wavelengths and power the laser emits. They can also change the frequencies of viewing the images and the size of the ar ea being viewed. These controls are easily found in the computer program for both the laser and the two photon microscope. The position of the objective is changed with a manipulator (seen in Figure 2). The three knobs change the position in the x, y, a nd z planes. The position relative to a set zero is shown on the machine which controls the manipulator. Figure 2: Objective manipulator

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15 Chronic Window Surgery The first step of the surgery was to put the mouse under isoflurane anesthesia, place it in a stereotax, and place it on a heating pad set to 37 degrees Celsius. The set up i s shown in shav ed and sterilized then the tools were sterilized using the glass bead sterilizer. A section of the skin was cut off and the fascia underneath the skull was scraped away until the area wa s dry. The mar gins were sealed with superglue, then the margins and left hemisphere were covered with bone cement and a headbar was placed onto the area. A mouse in the headbar set up is shown in Figure 4. Once dry, a cir cular hole the size of a three millimeter cove rslip was drilled into the skull and the bone was removed Onc e the bleeding had stopped a three millimeter coverslip with a 0.66mm hole in it was placed over an area that was relatively free of blood vessels and secured with super glue. The brain was always kept wet. Figure 3: Stereotax, lights, and glass bead sterilizer Figure 4: A mouse under anesthesia in a headbar with a heating pad

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16 Some problems may occur from the three main steps in this procedure: surgery to make a chronic window may damage the brain or cortex, the desired area of study (V1) could not be found, a visual map may not be obtained, or the OGB injection may not cover a large enough area. However, there are ways to fix or avoid each of these issues. The chronic window surgery need ed to be done w ith extra care, and if issues did occur bleeding was irrigated properly with cortex buffer and gelfoam to be stopped, and the dura was sometimes removed in order to get a better field of view. In order to ensure that V1 is reached we measure d exactly 2.7 millimeters away from the meet ing point of the lam b doid and midsaggital sutures or move d the chronic window as needed. Intrinsic Imaging Intrinsic imaging was used to obtain a map of a visually responsive area of the cortex, which in this experiment is V1. First, the mouse was placed in the two photon set up with proper anesthesia. The computer monitor was placed on the left side of the mouse then the enti re preparation was covered. It was ensured that there was no light from the monitor escaping to the outside of the covered preparation. The microscope was switched to the camera mode and the lights were turned off. A bar stimulus was run first at zero d egrees, then at ninety degrees. If the visual maps obtained were orthogonal to each other then the researcher established that the area viewed was V1. If a visual map wa s not obtained the status of the mouse was checked to ensure that it wa s breathing no rmally and that its left eye was open so that it wa s able to see the stimulus.

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17 Calcium Imaging The pipettes were made using the pipette puller as directed in Appendix A, a nd the tips were broken to between 0.5 3 microns in diameter T he OGB solu tion was made using OGB, 4l of P luronic, 36l S ulfr a hodamine, and 0.5l A lexa ; then injected into the pipettes. The mouse was placed under the two photon microscope set up and the objective and pipette manipulator were lined up for an i njection. T he pipette was moved s lightly under the sur face of the brain to an area that wa s both clear of blood vessels and highly populated with cells at a depth of about 250 300 m below the dura T he OGB solution was injected without pun cturing through a cell, while the solution spre ad throughout the viewing area. If the injected area was not large enough the second pipette was used to make another injection. Figure 6: An area of cells under the OGB which had been taken up by the cells, and are now green under the Two Photon Microscope Figure 5: An area of cells immediately after an OGB injection, the lighter area shows as red under the Two Photon Microscope

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18 The da ta gathered from this was video of calcium transients, which show the activity of the group of cells o bserved. The resulting graph ic s show how bright a cell fluoresces as a function of time at different orientations. Visual Stimulus and Tuning Two different stimuli were used in this experiment for the calcium imaging section. First, a stimulus developed by Ko et al is a square grati ng stimulus (Shown in Figure 7.) This uses well defined bars crossing through the screen at different directions and orientations. Between each direction and orientation there is a pause of a blank screen. The results from this stimulus presentatio n suggeste d that there were no groups of similarly tuned cells in the visual cortex of a mouse. However, several researchers in the laboratory performing this experiment believed the contrary. They developed a reverse correlation sinusoidal stimu lus (Shown in Figure 8) that used the same principles as Ko et al bars were not sharply defined and there was no pause in between bars of different orientations or those moving in di fferent directions. The results of these differences in stimuli will be reviewed in the discussion section. Figure 7: Ko et al Figure 8:

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19 Single Cell Electroporation This step used the chronic windo w previously explained. T he solution for this in jection was a combination of 15l of three single plasmids (TVA, g protein, and tdToma to), 15l internal solution, and 0.5l Alexa. T he same pipette program was used as the c alcium injecti on procedure, and the pipettes were filled with the solution. The mouse was placed under the two photon microscope set up and the ob jective and pipette manipulator were lined up for an injection. The manipulator was used to find the same area o f cells that were stu died with the calcium imaging. T he pipette was brought close to a target cell and then close to its membrane when a sound was used to know when the pipette had touched the cell. A set voltage (between 10 and 14 volts) was used to e lectroporate the cell with the solution inside the pipette. If a cell was properly filled it turned red. This was repeated for as many cells as desired. The window was closed and the researcher wait ed three days to know if the procedure was successful. Figure 9: A pipette coming close to a target cell Figure 10: A cell being electroporated

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20 Rabies Virus Injection Three days after the electroporation the covering was removed and the mouse was placed under the two photon set up. The electroporated cells were now expressing td Tomato If so, the same pipettes as previously used were made and filled with a combination of rabies virus and alexa The objective and the pipette manipulator were lined up and the electroporated cells were found with the two photon microscope and the pipette was positioned close to the target cells. A n injection of the rabies solution was made within 50 100 microns of the electroporated cells Then the researcher waited five to seven days for a primary infectio n, and then eight to ten days for a secondary infection After eight to ten days the mouse was brought back to the two photon set up and check ed for a secondary infection, which was seen as many green cells. If there wa s an infection the mouse was perfus ed, as explained in the next section, after taking scans and stack images of the cells. If there wa s no infection then the procedure was repeated until there wa s an infection. Perfusion and making microscope slides Onc e a secondary rabies infection was f ound, the mouse was perfused. First, the mouse was euthanize d with isoflourane, and the heart was exposed T he left ventricle of the heart was punctured and then phosphate buffer solution ( PBS ) was run through the mouse until the right ventricle and then t he right atrium expanded Once the liver had c ompletely lost color, or turned a more pale red, then the line was switched to run 4% paraformaldehy d e solution through the mouse. This was c ontinue d for about five Figure 11: A microtome

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21 minutes, or until the mou se wa s completely stiff. The brain was dissected out and placed in 4% paraformaldehyde overnight. A 3% agarose solution was made then the brain was placed in it right before the agarose turn ed solid. The agarose was trimmed down then mounted on the micr otome (shown in Figure 11). Using the microtome, 100 micrometer slices of the brain were made. Slices were stored bri efly in PBS and then mounted on Thermo Scientific Superfrost Plus Gold slides. Four to five brain slices were placed on a single microsco pe slide A good seal was made, then the slides were left to dry over night. The cells present on each slice were counted and recorded the next day. Experimental design previous studies that are commonly agreed upon. Intrinsic Imaging The independent variable in this section is the orientation of the moving bar stimulus. The two orien tations used were zero and ninety degrees. The dependent variable is the blood flow which is shown in a visual map. There will not be any statistical analysis done for the results of this section, only the visual maps obtained. Calcium Imaging As in the previous experiment, orientation and direction of the moving bar stimulus was the independent variable. The stimulus orientations vary from zero to one hundred and eighty degrees while the direction can be sideways, up and down, or diagonally. Calcium tr ansients

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22 observed during the stimulation period are the dependent variable. The data obtained from this section will be shown in the form of graphs of the calcium transient graphs and tuning curve graphs. Single cell electroporation The independent var iable in this section is the solution of the three plasmids electroporated into the cells. The dependent variable in this section is if the cells live and express the plasmids. A cell is determined to be expressing the plasmids if they appear red since o ne of the plasmids is td Tomato which appears red. The data obtained will be pictures of the expressing cells td Tomato. Rabies Injection The rabies injection itself is the independent variable. The dependent variable is if the pre synaptic cells express the rabies virus. Cells are determined to express the rabies virus by appearing green. The data collected from this secti on were counts of the amount of pre synaptic cells expressing the rabies virus. Means, medians, and standard deviations will be done with each mouse then compared among each other.

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23 Chapter 3: Results Intrinsic Imaging Table 1: A table of the procedures different subjects underwent. Cells highlighted in purple indicate procedures performed by the author on her own. Blue cells indicate procedures that occu rred before the author arrived to the lab. Figure 12: A visual map after a stimulus was presented at which the bars were oriented to 0. The clear lines of color indicate a visually responsive area The axes are the location of the map in reference to the cortex. The numbers are the pixels in the picture.

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24 The purpose of this procedure is simply to verify that the area being viewed under the Two Photon microscope is in fact V1, the area of interest for this study. As seen in Figures 12 and 13, stimuli that vary direction by 90 should be orthogonal to each other. Also, there should be clear lines of colors, not colors mixed among each other without a pattern. If the clear lines of colors occur the researcher can assume that the area under the microscope is in fact V1. Errors may occur within this larger area when the wrong subregion of the brain is reached, which can be determined when there are no clear lines of color in a visual map This can be as simple to fix as moving the prepara tion under the microscope However, it is also possible that the researcher would have to take the mouse back to the chronic window surgery to make viewing area larger. Figure 13: A visual map after a stimulus was presented at which the bars were oriented to 90. The clear lines of color are orthogonal to those shown above in Figure 12 which established that V1 is the area exposed.

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25 Calcium Imaging The cells chosen to examine from Figure 14 were: Cell 1: x axis= 50 65, y axis=160 145 Cell 2: x axis= 85 105, y axis= 130 115 Cell 3: x axis= 85 105, y axis= 10 25 Cell 4: x axis= 120 135, y axis= 160 175 Cell 5: x axis= 105 125, y axis= 50 65 The following figures are specific to different cells. Differences in the fluorescence level graphs are subtle, but the high spikes are different for each cell. Each cell studied showed a different tuning preference, which explains the urve. Cell 1: 400 0 50 100 150 200 250 300 350 Fluorescence Frame Number Fluorescence Levels over time Figure 14: A m ap extracted from a video of cells undergoing calcium imaging. Five cells were picked that seemed to be the brightest yellow or green, indicating that they were active at the time the picture was captured. The data is from mouse 271. Figure 15: A graph representing how the fluorescence levels fluctuate over time. The high spikes are calcium events.

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26 Cell 2: 640 660 680 700 720 740 760 0 5 10 15 Mean Fluorescence Orientation Tuning Curve 400 900 0 50 100 150 200 250 300 350 Fluorescence Frame Number Fluorescence Levels over time Figure 16: A Tuning Curve. The strength of the calcium events at each orientation is calculated and averaged across all trials. Then, the averages of each orientation are graphed. In this case, the cell was most active when the bar stimulus was at the f ourteenth orientation. Figure 17: A graph representing how the fluorescence levels fluctuate over time. The high spikes are calcium events.

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27 Cell 3: 700 720 740 760 780 800 820 840 0 5 10 15 Mean Fluorescence Orientation Tuning Curve 400 0 50 100 150 200 250 300 350 Fluorescence Frame Number Fluorescence Levels over time Figure 18: A Tuning Curve. The strength of the calcium events at each orientation is calculated and averaged across all trials. In this case, the cell was most active when the bar stimulus was at the thirteenth orientation. Figure 19: A graph representing how the fluorescence levels fluctuate over time. The high spikes are calcium events.

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28 Cell 4: 640 650 660 670 680 690 700 710 720 0 5 10 15 Mean Fluorescence Orientations Tuning Curve 400 0 50 100 150 200 250 300 350 Fluorescence Frame Number Fluorescence Level over time Figure 20: A Tuning Curve. The strength of the calcium events at each orientation is calculated and averaged across all trials. In this case, the cell did not have a specific orientation at which it was most active and is the refore said to not be tuned. Figure 21: A graph representing how the fluorescence levels fluctuate over time. The high spikes are calcium events.

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29 Cell 5: 720 730 740 750 760 770 780 790 0 5 10 15 Mean Fluorescence Orientations Tuning Curve 400 900 0 50 100 150 200 250 300 350 Fluorescence Frame Number Fluorescence Levels over time Figure 22: A Tuning Curve. The strength of the calcium events at each orientation is calculated and averaged across all trials. In this case, the cell was about equally active at orientation five and six, but this is still not considered a well tuned cell because one orientation is not clearly preferred. Figure 23: A graph representing how the fluorescence levels fluctuate over time. The high spikes are calcium events.

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30 The results of this section explored t uning of five cells from a video taken of mouse 264. The stimulus used in this analysis was the Baylor developed stimulus. Figures 15, 17, 19, 21, and 23 all showed how fluorescence levels changed and fluctuated over time (frame numbe rs increased over time). This fluctuation occurs as a result of the cell responding differently to the varying ori entations of the moving bar stimulus. The higher spikes in the area show strong calcium events, which are later processed by strength to determine which orientation caused that spike. If these spikes occur more than once for the same orientation the cell is said to prefer that orientation, or to be tuned for that orientation. Figures 16, 18, 20, 22, and 24 are Tuning Curves of the five cells chosen to be studied. Tuning curves are calculated by taking the averages of the fluorescence levels found or a g iven orientation over the course of the stimulus presentation. This means that there is one final value of fluorescence for each orientation. This is then graphed and the highest peak is 595 600 605 610 615 620 0 5 10 15 20 Mean Fluorescence Orientations Tuning Curve Figure 24: A Tuning Curve. The strength of the calcium events at each orientation is calculated and averaged across all trials. In this case, the cell did not have a specific orientation at which it was most active and is therefore said to not be tuned.

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31 determined to be the preferred orientation. In Figures 16 and 18 a clear high peak is shown at 14 in Figure 16 and at 13 for Figure 18. This means that those cells are tuned to their respective orientations. Figure 22 shows a high peak, but it occurs between two orientations, 5 and 6. This means that the cell is not t uned for a specific orientation, but it is an active cell. Figures 20 and 24 show no high peaks, meaning that the cells are not tuned to any specific orientations. Figure 25: An orientation selectivity map made from an imaging session on mouse 12709 when the Baylor stimulus was used. The areas of pink are all cells that have the same orientation preference.

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32 Figure 26: An orientation selectivity map made fr om an imaging session on mouse 12709 when the Ko et al stimulus was used. The different colors indicate different orientation preferences. Figure 27: An orientation selectivity map of mouse 271 that clearly shows the salt and pepper model of orientation preference organization. It can be clearly seen that there is a great amount of variance in the orientation preferences of the cells in this area.

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33 Single Cell Electroporation Rabies Injection Four mice expressed rabies infected post synaptic cells but mouse 12814 showed pre synaptic cells without an infected post synaptic cell It is not included in the data. Mouse 12651 Slide Number Slice Number Cells Notes Left Right 5 1 0 0 2 0 0 3 0 0 4 0 1 bottom 5 0 0 6 1 0 0 2 0 1 bottom w/ dendrite 3 0 1 bottom 4 0 0 5 0 0 7 1 0 0 2 0 0 3 0 1 middle bottom 4 0 0 5 0 0 8 1 0 0 Figure 28: Four cells expressing tD Tomato after being electroporated three days prior. This indicated that the cells were ready to receive a rabies injection. Axons and dendrites are also present in this f igure. Table 2: Raw data of the counted cells in mouse 12651. Data from slides 1 4 and 16 18 were not included because no cells were present.

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34 2 0 0 3 0 0 4 0 1 bottom 5 0 0 9 1 0 0 right dendrite 2 0 0 3 0 0 4 0 1 top 10 1 0 0 2 0 0 3 0 0 4 0 1 top 11 1 0 3 top 2 0 2 top 3 0 4 top 4 0 3 top 12 1 0 4 top 2 0 13 top 3 0 22 top w/ dendrites 4 0 25 top 13 1 0 44 top 2 0 85 top 3 0 91 top & electroporated cell 4 0 47 top 14 1 0 20 top 2 0 6 top 3 0 5 top 4 0 2 top 5 0 1 top 15 1 0 0 2 0 1 top 3 0 1 middle top 4 0 0 5 0 2 top outside Total Cells =388 Mouse 264 Slide Number Slice Number Cells Notes Left Right 5 1 0 0 2 1 0 bottom 3 1 0 bottom 4 0 0 5 0 0 6 1 0 0 2 0 0 3 0 0 dendrites 4 1 0 bottom 7 1 0 0 dendrites 2 0 1 3 0 0 4 0 1 9 1 1 0 very close to center 2 0 0 3 0 0 4 0 0 13 1 0 0 2 3 0 3 4 0 Table 3: Raw data of the counted cells in mouse 264. Data from slides 1 4, 8, 10 12 and 17 18 were not included because no cells were present.

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35 4 11 0 14 1 28 0 one separate from cluster 2 51 1 3 57 0 4 32 0 15 1 19 0 2 5 0 3 2 0 4 0 0 16 1 1 2 0 0 3 0 0 4 0 0 Total = 220 Mouse 222 Slide Number Slice Number Cells Notes Left Right 5 1 0 0 2 0 0 3 0 1 top 4 0 0 5 0 0 10 1 0 0 2 0 1 top 3 0 0 4 0 1 top 11 1 0 3 top 2 0 2 top 3 0 8 top with other dendrites 4 0 0 chunk of brain gone 12 1 0 25 2 0 21 one big cluster of ten 3 0 4 chunk missing 4 0 9 top 13 1 0 2 more to middle 2 0 0 3 0 0 4 0 0 5 0 0 super thick slice 14 1 0 0 2 0 0 3 0 0 super thick slice 4 0 0 5 0 0 15 1 0 0 missing chunk of brain 2 0 0 missing chunk of brain 3 0 0 missing chunk of brain 4 0 0 missing chunk of brain 5 0 0 missing chunk of brain 6 0 0 missing chunk of brain Total Cells= 77 Table 4: Raw data of the counted cells in mouse 222. Data from slides 1 4 and 6 9 were not included because no cells were present.

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36 Mo use 254 Slide Number Slice Number Cells Notes 3 1 0 2 0 3 0 4 0 5 0 6 1 4 1 1 2 0 3 0 4 1 5 0 6 0 6 1 0 2 1 Dendrites 3 0 4 1 5 1 8 1 0 2 0 3 0 4 2 5 1 9 1 1 2 0 3 0 Dendrites 4 7 10 1 4 2 4 3 3 Next to each other 11 1 6 2 5 3 5 12 1 6 2 14 3 17 13 1 28 2 47 3 41 14 1 44 2 44 3 26 15 1 15 2 11 3 4 16 1 1 2 2 3 0 Total= 388 Table 5: Raw data of the counted cells in mouse 254. Data from slides 1 2, 5, and 7 were not included because no cells were present.

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37 Mouse Number Cell Count 12651 388 264 220 222 77 254 344 Total Cells 1029 Mean 257.25 Median 282 Standard Deviation 139.6409085 Table 6: Simple statistical analysis performed on the mice used as data in this section. electroporated cell which shows glows red. synaptic cells to the electroporated cell in Figure 26.

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38 Chapter 4: Discussion Concluding the results of Calcium Imaging When examining the area as a whole, the researcher must consider if the cells studied showed the same preferences to the same orientations. In this area of cells none of the cells were tuned for the same orientation. Ko et al found that none of t he cells in a given area were tuned to the same orientation, that cells in a given area show a salt and pepper arrangement (as shown in Figures 26 and 27) meaning that no groups of similar orientations were found. They found so using their own moving bar stimul us which presents clear bars without any pauses in lines are not clear, and the stimulus also includes breaks in between presentations of bars. This specific area of cells showed no similar tuning using the Baylor stimulus, but other areas showed obvious groupings of similarly tuned cells as shown in Figure 25 This raised the question of if the style of stimuli reports different results. The lab continue d to examine this relationship between stimuli and tuning groupings of cells. This yielded the conclusion that the duration of the stimuli was causing the confusing results: Ko et al t was found that the shorter stimuli gave misleading results; due to the shorter duration a stronger signal was created in the neuropil than in the cell. The neuropil is all tuned the same, so the g to the incorrect conclusion that all the cells were tuned similarly.

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39 Concluding the results of Single Cell Electroporation The results from this section are relatively straight forward. First, it can be determined that a cell was successfully electr oporated if it glows red after the pipette moves away from it. This establishes that the plasmid entered the cell without killing it. Second, if the cell is glowing red three days after being electroporated it is determined to be expressing tD Tomato, me aning that the plasmid was successfully taken up by the cell. In Figure 25 four cells were successfully electroporated, which is a very high number. Typically only one or two cells would be successfully electroporated and expressing tD Tomato after three days. Problems with this section occurred mostly in the technique of electroporating. Cells are very delicate and can easily die from the process. Most often the pipette was held to the cell for too long of a period of time. The only way to fix this s eemed to be practice, once the researcher had a better idea o f the timing of the process few e r cells died. Sometimes the pipette would travel through a cell which would cause the pipette to become clogged. In this case the pipette would have to be removed from the brain and replaced. Concluding the results of Rabies Injection The results from this section are based on cell counts taken of slices of mice brain on microscope slides. Tables 2, 3, 4, and 5 show raw data of the cell counts. Table 6 shows simple statistics performed based on the raw data. The mean was 257.25 cells per brain with a standard devation of 139.64 cells. Needless to say this is a rather large standard deviation and suggests a great area for improvement. The data from mouse 222 most likely caused this great error. The small number of cells counted in mouse 222 was caus ed by poor slicing of the brain with the microtome. Had the brain been better sliced it can be assumed that the cell count

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40 would have been more similar to the other mice. This animal was included in the data because even though the slicing was not as wel l done as the other mice, the brain still had many cells and connections visible, and therefore provided valuable data. The large numbers of cells count ed in the other three mice showed the importance of examining the cellular connections. In these thr ee mice there were only one or two post synaptic cells. The large number of pre synaptic suggests a great deal of complex networks that are interconnected. This network is not very well understood, from mammals such as mice to humans. This project offers a stepping stone in understanding how the cells in a brain are connected and what networks these connections make. Future Directions Structure and function were separately studied in this study. It is important to have the procedures to collect th ese data well established and understood before moving forward towards further study. This goal was achieved throughout the course of this study but raised the question of connecting the two forms of data. Do structure and function have a relationship of sig nificance? Ko et al (2011) found that cells which were connected to each other had dendrites that were similarly tuned. Our research looks to examine if cells which are connected to each other share common tuning as well. In order to do this a common structural background must be established. This can be done using DAPI, an antibody that dyes all cells yellow in a Figure 31: An area of cells dyed with the DAPI antibody.

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41 given area. An example of an area stained with DAPI is shown in Figure 28. DAPI is added to a slice of the brain once it has been mounted on a microscope slide. After the DAPI has dyed the cells a stack image can be taken of the slide and compared stacks taken of slides with rabies infected pre synaptic cells. Overlapping these data is fairly simple, a common structural point is found and compared among the sta ck images. The next step is to compare stack images from the functional calcium imaging to the structural results with the rabies and DAPI cells connected. There are two ways to approach this problem: manually overlapping the data or creating an equation or computer program to overlap the data. Manually overlapping would not only be tedious, but also very difficult. Structure is not consider e d in the depth required for overlapping by the researcher. While it is possible to overlap the data a fa ster solution would be easier and more accurate. Writing a computer program to recognize cells based on their position in the brain would solve this. A computer program to recognize cells already exists. The next step would be to fine tune this program to be able to relate the cell to a structure that the computer can recognize in both a functional set of data and a nother computer program. If this task is able to be accomplished a new window for relating structure to function of cells across the brain w ould be opened, which could be taken into many directions.

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42 Appendix Operational Procedures Anesthesia Open two oxygen tanks and the oxygen line connected to the Isoflurane Anesthesia machine (shown in Fi gure A1). Turn the oxygen flow on the machine to three and turn the isoflurane flow rate to three. Make sure the oxygen and isoflurane flow tubes are connected to the induction chamber, place the mouse inside, and close the chamber. Once the mouse is as leep, which typically takes two to three minutes, move it to the appropriate set up (varies depending on the step of the procedure), connect both flow tubes to the set up, and finally turn the flow rate on the machine down to one and a half. Pipette Puller For each injection pipettes must be made with a specific tip width. Since the widths are very small, a pipette puller (shown in Figure A2) is used to make the pipettes. Each pipette size will be mentioned by section, the following is general instructions on how to work the machine. Turn the machine on and open the top. With gloves on, insert a glass tube into the machine and feed it through so that both ends can be secured. Type in the program number, which is different for each ste p, close the stop, and click enter to begin the pulling. Once the program has run, remove both pipettes created. If necessary, break the tip to make the end Figure A1: Isoflurane Anesthesia Machine

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43 width bigger. This is done under the microscope with a filament that is brought up to the pipette tip. Make sure that the pipette is clean and continue to inject whichever solution necessary. Pipette Holder and Manipulator Place the pipette into the pipette holder (shown in Figure A3) and screw the top on to secure it. Especially for the electroporation section, make sure the metal filament is properly in the pipette to ensure the proper circuit is made Using the pipette manipulator (shown in Figure A4) and the microscope to follow the tip, brain. The buttons on the top right side control the speed at which the pipette is Figure A3: Pipette Holder and Microscope Figure A2: Pipette Puller and Microscope to break pipettes

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44 moved. The buttons in the bottom center as well as the turn dials can be used to move the pipette once the speed is selected. Great attention to the speed at which the pipette moves is required because if it is moved too quick ly the pipette can easily be broken. Figure A4: Pipette Manipulator

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45 Bibliography Cannestra, a F., Blood, a J., Black, K. L., & Toga, a W. (1996). The evolution of optical signals in human and rodent cortex. NeuroImage 3 (3 Pt 1), 202 8. doi: 10.1006/nimg.1996.0022. Drager, U. (1975). Receptive fields of single cells and topography in mouse visual c ortex. Journal o f Computational Neuroscience 160 269 290. Garaschuk, O., Milo, R., & Konnerth, A. (2006). Targeted bulk loading of fluorescent indicators for two photon b rain imaging in vivo. Nature Protocols,1(1), 380 386. Grinvald, A. et al (2001). In vivo optical imaging of cortical architecture and dynamics (Technical Report GC AG/99 6). The Grodetsky Center for Res earch of Higher Brain Functions: Modern Techniques in Neuroscience Research. Holtmaat, A., Bonhoeffer, T., Chow, D. K., Chuckowree, J., De Paola, V., Hofer, S. B., et al. (2009). Long term, high resolution imaging in the mouse neocortex through a chronic cranial window. Nature protocols 4 (8), 1128 44. Hube ner, M. (2003). Mouse visual cortex. Current Opinion in Neurobiology 13 413 420. Huberman, A. D., & Niell, C. M. (2011). What can mice tell us about how vision works? Trends in neurosciences 34 (9), 464 473. Elsevier Ltd. doi: 10.1016/j.tins.2011.07.002 Jia, H., Rochefort, N., Chen, X., & Konnerth, A. (2010). Dendritic organization of sensory input to cortical neurons in vivo. Nature,464, 1307 1312.

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46 Judkewitz, B., Rizzi, M., Kitamura, K., & Hausser, M. (2009). Targeted single cell electroporation of mam malian neurons in vivo. Nature Protocols,4(6), 862 869. Kalatsky, V. a, & Stryker, M. P. (2003). New paradigm for optical imaging: temporally encoded maps of intrinsic signal. Neuron 38 (4), 529 45. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1276560 6. Ko, H., Hofer, S. B., Pichler, B., Buchanan, K. a, Sjstrm, P. J., & Mrsic Flogel, Thomas D. (2011). Functional specificity of local synaptic connections in neocortical networks. Nature 1 7. Mangini, N. J., & Pearlman, A. L. (1980). Laminar distribut ion of receptive field properties in the primary visual cortex of the m ouse. The Journal of Comparative Neurologyy 193 203 222. Schuett, S., Bonhoeffer T., & Hu, M. (2002). Mapping retinotopic structure in mouse visual cortex with o pti cal i maging. Structure 22 (15), 6549 6559. Squire, L., Berg, D., Bloom, F., du Lac, S., Ghosh, A., & Spitzer, N. (2008). Fundamental neuroscience. (3rd ed., pp. 639 642, 649 654). Amsterdam: Elsevier. Stosiek, C., Garaschuk, O., Holthoff, K., & Konnerth, A. (2003). In vivo two photon calcium imaging of neuronal networks. Proceedings of the National Academy of Science Neuroscience, 100(12), 7319 7324. W ickersham, I. R., Lyon, D. C., Barnard, R. J. O., Mori, T., Finke, S., Conzelmann, K. K., et al. (2007). Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53 (5), 639 47. doi: 10.1016/j.neuron.2007.01.033.

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47 Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., & Gan, W. biao. (2010). Thinned skull cranial windo w technique for long term imaging of the cortex in live mice. Nature Protocols 5 (2), 213 220.