UW-MADISON COLLEGE OF ENGINEERING DEPARTMENT OF BIOMEDICAL ENGINEERING A device for in vivo 2-photon imaging of synapses in mobile mice David Leinweber – Team Leader Jon Seaton – Communicator Mark Reagan – BSAC Jay Sekhon – BWIG Client: Giulio Tononi Ph.D./M.D. Advisor: Mitch Tyler 12/12/2008FINAL REPORT December 12, 2008 2 Table of Contents Background ............................................................................................... 3 Design Problem ........................................................................................... 4 Client Requirements.................................................................................... 4 Similar Devices ........................................................................................... 5 Phase 1 Design Alternatives........................................................................ 6 Phase 1 Design Matrix .............................................................................. 11 First Final Phase 1 Design......................................................................... 11 Phase 1 Testing.......................................................................................... 17 Second Final Phase 1 Design .................................................................... 18 Phase 2 Design Alternatives...................................................................... 21 Phase 2 Design Matrix .............................................................................. 24 Final Head Restraint Design ..................................................................... 24 Synergy of Ball and Head Restraint.......................................................... 27 Testing of Final Prototype......................................................................... 28 Future Work .............................................................................................. 29 Appendix A ............................................................................................... 30 Appendix B ............................................................................................... 35 Appendix C ............................................................................................... 38 Appendix D ............................................................................................... 41 Appendix E................................................................................................ 43 Appendix F................................................................................................ 45 Appendix G ............................................................................................... 47 References ................................................................................................. 49FINAL REPORT December 12, 2008 3 Background: The function of sleep remains one of the greatest unsolved mysteries in modern science. A multitude of theories have been proposed regarding the necessity of sleep, but none have been substantively established. Sleep is known to be an essential process of life, taking up almost 1/3 of the average human’s lifetime. Furthermore, it is such a necessity that deprivation has been known to cause health problems in rats, leading even to death (Everson 1995). Additionally, the effects of sleep have been studied on a variety of life factors. Retaining homeostasis is a commonly accepted view, since studies have shown that lack of sleep hampers healthy metabolic activity and immune system response (Zager, 2007). Furthermore, sleep has been linked to proper memory function—lack of sleep has been shown to correlate with cognitive impairment, decreasing by as much as 38% in comparison to a control (Turner, 2007). However, these functions all provide effects of sleep; they do not examine the purpose of it. A hypothesis has been proposed by Dr. Giulio Tononi that sleep is used for synaptic downsizing. Specifically, he states that “The synaptic homeostasis hypothesis claims that plastic processes during wakefulness result in a net increase in synaptic strength in many brain circuits; during sleep, synaptic strength is globally downscaled to a baseline level that is energetically sustainable and beneficial for memory and performance” (Tononi, 2005). An experiment to test this hypothesis has been proposed using 2-photon microscopy to image synaptic activity in the brain in awake and sleeping mice.FINAL REPORT December 12, 2008 4 Client Requirements: The clients, Dr. Giulio Tononi and Dr. Ugo Faraguna would like our team to develop and construct a device for use with a 2-photon microscope. The device is made up of two modules: a frame that holds the mouse’s skull in a fixed position for microscopy of the cranial window, and a “treadmill” that allows freedom of movement for the mouse. The client would like the treadmill to be done as quickly as possible for design purposes and for training the animals. Important considerations include the fact that the device must have no electrical components; the treadmill should ideally provide no movement restrictions; and that the device must fit between the lens of the microscope and the table on which the microscope rests. Design Problem: In order to support the hypothesis of synaptic downscaling as a neurological function of sleep, a device capable of holding a mouse’s head in a fixed position for 2-photon microscopy is necessary. The device should be broken into two parts: A) A stage upon which the mouse can have freedom of movement. (Phase 1) B) A stereotaxic frame for keeping the mouse’s head in a rigid position. (Phase 2)FINAL REPORT December 12, 2008 5 Similar Devices: Researchers at Princeton University have developed a similar frame for use with a 2-photon microscope (Fig. 1). (Image from Dombeck, 2007) The device is designed for use primarily with awake, mobile mice. Of particular note is the use of optical computer tracking mice for recording the specific movement and speed of the ball upon which the mouse rests. Additionally, the use of pressurized air to lift the ball and create an extremely low-friction environment allows for examination of brain activity solely for locomotion, avoiding potential issues with using the head restraint as something with which to exert leverage to move the ball.FINAL REPORT December 12, 2008 6 Product Uniqueness: Our design will be superficially similar to the
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