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| About My Lab | |
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DR. TIMOTHY H. MURPHY
General areas of research:
Structure-function of brain circuits in relation to stroke. High resolution imaging CNS synaptic plasticity/physiology: in vivo imaging of synaptic interactions Imaging neurochemistry during stroke. Monitoring of oxidative stress New work: In vivo imaging of mitochondria suggests mediators of delayed cell death may be activated within 5 min of stroke onset. Liu R.R. and Murphy T.H. (2009) Reversible cyclosporine A sensitive mitochondrial depolarization occurs within minutes of stroke onset in mouse somatosensory cortex in vivo, a two-photon imaging study. EPub J. of Biol. Chem. 
 
New work: Imaging rapid redistribution of sensory-evoked depolarization through existing cortical pathways after targeted stroke in mice. Results of Voltage Sesitive Dye (VSD) imaging revealed that patterns
of sensory-evoked depolarization redistribute within hours after an ischemic stroke in the
forelimb region of the somatosensory cortex in mice. Published in
Proceedings of the
National Academy of Sciences of the United States of America 106 (2009): 11759-11764. see also: * Supporting Information (PDF).
New work: Stroke effects on brain circuit structure-function.
Publication:
In vivo voltage-sensitive dye imaging in adult mice reveals
that somatosensory maps lost to stroke are replaced over weeks
by new structural and functional circuits with prolonged modes
of activation within both the peri-infarct zone and distant sites.
Brown C.E., Aminoltejari K., Erb H., Winship I.R., and Murphy T.H.
J. Neurosci. 2009 29:1719-1734.
New work: In vivo synaptic physiology and optogenetic brain mapping.
Publication:
Ayling OG, Harrison TC, Boyd JD, Goroshkov A, Murphy TH. Automated light-based mapping of motor
cortex by photoactivation of channelrhodopsin-2 transgenic mice.
Nature Methods 6 (2009): 219-224.
Optical stimulation of motor cortex fixed brain example shown, left. Maps of mouse forelimb motor cortex derived from light-evoked muscle activity, middle raw EMG thumbnails, and right grayscale map of EMG amplitude. Stroke brain circuit structure-function research questions/plan:
Our aim is to help stroke victims regain brain function through understanding of how synaptic networks are damaged and recover from interruptions in brain blood flow. We hope that advances learned in our lab will translate into new treatments and hope. There are two major goals for our research: 1) Reveal the key chemical and electrical events that lead to early stroke-induced damage to synaptic networks in intact animals by employing high-resolution two-photon and other forms of in vivo imaging. The hope is that if we can better understand these processes we might be able to stop them. 2) Understand the structural and functional basis of adaptive changes to brain circuitry that accompany stroke recovery. A key component of stroke recovery is the re-mapping of function from damaged brain areas to surviving areas. Although new areas of activation occur, how information flows in and out reorganized cortical networks on the millisecond timescale and by what circuitry is unclear. Our goal is to understand these changes with the aim of using this knowledge to promote the recovery of human stroke patients.
Tools for mapping brain circuit function. 
Intrinsic optical signal imaging/mapping provides a means of assessing cortical circuits involved in processing touch and permits linking microscopic structure of vessels and synapses to function PLoS Biol (2007) 5:e119..
Brain mapping of forelimb somatosensory cortex using intrinsic signal imaging (IOS), darkened area indicates the response to contralateral forelimb stimulation. For free software and hardware description see: Harrison TC, Sigler A, and Murphy TH. (2009) Simple and cost-effective hardware and software for functional brain mapping using Intrinsic Optical Signal imaging as well as our software page. J Neurosci Methods 182:211-218. Tools for creating targeted ischemia (stroke).
Laser induced small artery clotting by photoactivation of rose bengal, process occurs over 2 min PLoS Biol (2007) 5:e119, permits targeted stroke in mouse somatosensory cortex (arteriole shown ~40 mm). See also Sigler A, Goroshkov A, and Murphy TH (2008) Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope. J Neurosci Methods 170:35-44. Stroke damage to synapses is apparent within minutes, but can be reversed with prompt reperfusion of blood.
Rapid and reversible dendritic damage. Reversible damage to dendrites during ischemia and reperfusion (images shown are from layer I cortex) J Neurosci (2008) 28:1756-1772.
Imaging waves of stroke damage. Changes in reflected light signal (ratio to preischemic condition, scaled between 95-105% of baseline reflectance) associated with ischemic depolarization over a 5 min period within a 3.1 mm wide brain window. The star on the upper right marks the time when ischemic depolarization was observed by monitoring the EEG . The time per frame is 1s and the movie starts 28s before the induction of ischemia as defined by EEG suppression. At the beginning of the movie the image is a uniform gray that corresponds to 100% of preischemic reflected light levels. Within 30s the surface vessels darken followed by the surrounding tissues indicating deoxygenation. ~170s after occlusion a wave of brightening (increased light scattering) begins to move across the cortex starting with the most anterior and lateral tissues. This wave leads to damaged synaptic networks J Neurosci (2008) 28:1756-1772. Changes to the structure and function of somatosensory circuits support stroke recovery weeks after ischemia. 
Synapse elimination in vivo (time in hours).
Synapse addition in vivo J Neurosci (2007) 27:4101-4109. Changes to the function of single neurons that support re-mapping of sensory function weeks after ischemia.  Layer 2 neurons that are normally responsive to only a single limb exhibit broader receptive fields and can respond to all 4 limbs as sensory responses are re-mapped. Winship IR and Murphy TH (2008) In vivo calcium imaging reveals functional rewiring of single somatosensory neurons after stroke. J. Neurosci. 28:6592-6606  Circuit and cellular level re-mapping model after stroke based on un-masking of sub-threshold connections between related regions of cortex. Winship IR and Murphy TH (2009) Re-mapping the somatosensory cortex brain after stroke: insight from imaging the synapse to network. The Neuroscientist in press(doi:10.1177/1073858409333076). Detailed Research Plans and Background
The stroke is the most common neurological disorder affecting our aged population currently. Stroke is defined as a sudden loss of blood flow to the brain making it a disorder of vascular plumbing. However, it is apparent that the stroke-plumbing problem evolves to affect the brain s electrical system through the ischemia-induced loss of synapses and circuits. It would be a great advantage to have approaches that can stop damage to neurons and brain synaptic networks from occurring during stroke. The first objective of this work is to uncover the key events that lead to stroke-induced damage of synaptic networks in intact animals by employing high-resolution two-photon and other forms of in vivo imaging. Originally developed by Winfred Denk and Watt Webb (1990), two-photon microscopy uses pulsed infrared light to excite fluorophores by the combined power of two long wavelength photons. One of the key advantages of this approach is that it allows one to image structures deep within thick biological specimens, whilst achieving micron level resolution. Combining this imaging technique with transgenic mice engineered to express fluorescent proteins within a subset of neurons (Feng et al., 2000) has enabled us and other investigators to track changes in neuronal structures in living animals over days, weeks and even months (Grutzendler et al., 2002; Trachtenberg et al., 2002). Here we describe recent results using this approach to examine the acute vulnerability of synaptic circuits to stroke, as well as the remarkable degree of plasticity (ability to change) in dendritic spines in regions adjacent to tissues lost to stroke damage (ie. the peri-infarct zone) (see Aim 2). Dendrites play a fundamental role in cell to cell communication in the brain, as they are the post-synaptic targets of most synapses in the brain. A large percentage of dendrites are decorated with tiny protrusions known as dendritic spines, which usually possess at least one excitatory synapse (Arellano et al., 2007). When dendrites become ischemic, they undergo a relatively stereotyped pattern of degeneration with the most salient morphological feature being the appearance of varicosities or beads along the shaft of the dendrite (Zhang et al. 2005; Hasbani et al., 2001; Park et al., 1996). We have recently shown the dendritic wiring of neurons can be degraded within 1-3 minutes following stroke. Surprisingly, the structure and function of the brain s synaptic wiring system was severely compromised only 1-3 minutes after stroke by an event termed ischemic depolarization. However, if blood flow was promptly restored, as can occur using stroke treatments with clot-busting drugs, the synapses bounced back from severe deformation (Murphy et al. 2008). These data suggest that ischemic depolarization is the major damaging event associated with stroke's effect on synapses, and suggest that strategies that control this process would be fruitful avenues for stroke prevention and treatment. A number of damaging events have already been identified in a variety of different systems that could underlie stroke damage and therapies for protecting brain circuitry and neurons are largely based on these studies. However, it is unclear what the exact timing and sequence of damaging events is after stroke? Currently we are producing an accurate timeline of potentially damaging events that occur in intact brain in response to stroke at the level of individual synapses and brain circuits. The two major classes of events that could damage brain neurons and synaptic circuitry are over-excitation of neurons (excitotoxicity), and a production of reactive oxygen metabolites produced by normal metabolism gone wrong (oxidative stress). In excitotoxicity messages which are normally involved in communication between neurons (and rely on the passage of ions such as calcium) are now increased in volume to the point where they damage neurons. In oxidative stress byproducts of normal cellular metabolism such as reactive oxygen species are now either over-produced or over-accumulate resulting in damage to brain proteins, membranes, and loss of cells. Although these two classes of mechanisms have been identified, if, when, and where they occur is not fully understood in intact animals and is the subject of our acute stroke imaging work. Our experiments will accurately describe the early events associated with stroke damage in an intact animal model. Although these studies themselves do not aim to treat stroke, we will identify potentially damaging events in intact animal models and will help to prioritize future treatment strategies and may be even suggest re-examining some current practices. By using a mouse stroke model we can also take advantage of mutant mice that can model aspects of human stroke and cardiovascular disease, allowing the experiments to be translated to those at risk for stroke through heritable disorders. Why is it important to describe the early events of stroke? We believe that an accurate description of the events that underlie stroke damage will be critical for stroke therapeutics. For example, if we find that the damaging effects of too much synaptic communication are largely over with within minutes following ischemia it may not be appropriate to design neuroprotective strategies that try to block these damaging events hours after ischemic onset. Furthermore, if we determine that reactive oxygen species only accumulate when blood flow is restored we may recommend that patients be treated with appropriate therapies only upon restoration of blood perfusion. Surrounding the death and destruction of the ischemic core, is a region of hypo-perfused tissue known as the peri-infarct zone (Hossmann, 2006). Currently, there is some debate as to what exactly constitutes the peri-infarct zone or how large this area extends. For the sake of simplicity we will define it as the region of surviving tissue immediately surrounding the core of the infarct, which in a rodent could be less than a millimeter whereas in humans, this region may be several orders of magnitude larger (Carmichael, 2005). In the days to weeks following an ischemic insult, accumulating data suggest that the peri-infarct region is a hot spot for neuronal plasticity that subserves functional recovery from stroke (Witte, 1998). In particular, peri-infarct zones undergo significant changes in the expression of growth promoting and inhibitory factors that are essential for neuronal re-wiring, angiogenesis and neurogenesis (Carmichael, 2006). Furthermore, anatomical studies have shown that for several weeks after focal stroke, peri-infarct regions show increased synaptogenesis, are enriched with histochemical markers of axonal sprouting such as GAP-43, and receive new intracortical projections (Dancause et al., 2005; Ito et al., 2006; Stroemer et al., 1995). Recovery from brain injury such as stroke is dependent upon how well surviving brain tissues assume the responsibilities of those lost to injury. Given the topographic organization of the cerebral cortex, functionally homologous areas situated in close proximity to the site of injury (i.e. peri-infarct regions) are most likely to adopt new functional roles after stroke. Despite considerable progress in our understanding of cortical plasticity after stroke, many fundamental questions remain. For instance, we do not know the spatiotemporal dynamics with which sensory information flows in and out of re-organized cortical networks or the precise structural changes that underlie these changes. We are currently combining network-imaging approaches with 2-photon imaging to address these questions. Imaging miniature synaptic activity at single synapses using calcium imaging. Mackenzie et al. J Neurosci (1999) 19:RC13
3D reconstructions of cortical synapses in culture. Shown below are a 3-D reconstruction of a rat cortical neuron spine from electron microscopic images. Analysis of spine function using calcium imaging suggests that larger more complex spines (having larger and multiple PSDs; indicated in dark color) have a greater quantal amplitude suggesting that structural changes to dendrites may be required for synaptic plasticity.
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