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Spatially resolving acute functional changes in descending cortical motor output after targeted stroke Anenberg, Eitan 2015

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SPATIALLY RESOLVING ACUTE FUNCTIONAL CHANGES IN DESCENDING CORTICAL MOTOR OUTPUT AFTER TARGETED STROKE  by  Eitan Anenberg B.Sc (Hons.) The University of Western Ontario, 2011  THESIS SUBMITTED IN PARTIAL FULFILLEMENT FOR THE REQUIREMENTS OF   MASTER OF SCIENCE  in  The Faculty of Graduate and Postdoctoral Studies  (Neuroscience)  The University of British Columbia (Vancouver) April 2015 © Eitan Anenberg 2015  ii	  Abstract   We evaluated the effects of mini-strokes targeted to individual pial arterioles on motor and cortical function within the first hours after ischemia.  This was done in Thy-1 line 18 channelrhodopsin-2 (ChR2) transgenic mice. Using optogenetics, we directly assessed both the excitability and motor output of cortical neurons in a rapid, repeated, and relatively non-invasive manner independent of behavioral state or training. Occlusion of individual arterioles within the motor cortex led to a ministroke that was verified using laser speckle contrast imaging. Surprisingly, ministrokes targeted to a relatively small region of the forelimb motor map, with an ischemic core of 0.07 ± 0.03 mm2, impaired motor responses evoked from points across widespread areas of motor cortex even 1.5 mm away. Contrasting averaged ChR2-evoked electroencephalographic, spinal (ChR2 evoked potential), and electromyographic responses revealed a mismatch between measures of cortical excitability and motor output within 60 min after stroke. This mismatch suggests that apparently excitable cortical neurons (even >1 mm into peri-infarct areas, away from the infarct core) were impaired in their capacity to generate spinal potentials leading to even more severe deficits in motor output at muscles. We suggest that ischemia, targeted to a subset of motor cortex, leads to relatively small reductions in excitability within motor cortex, and cumulative depression of both descending spinal circuits and motor output in response to the activation of widespread cortical territories even outside of the area directly affected by the ischemia.    	   	   iii	   Preface   This thesis is based on  work previously published - Anenberg E, Arstikaitis, P., Niitsu, Y., Harrison, T.C., Boyd, J.D., Hilton, B.J., Tetzlaff, W., Murphy T.H. (2014). Ministrokes in Channelrhodopsin-2 Transgenic Mice Reveal Widespread Deficits in Motor Output Despite Maintenance of cortical Neuronal Excitability. Journal of Neuroscience.  Pilot work for this project was conducted by Yoichi Niitsu and Pamela Arstikaitis.  The software used to operate previously designed hardware (Ayling, Harrison, Boyd, Goroshkov, & Murphy, 2009) was developed by  Dr. Jamie Boyd. Brett Hilton trained me to perform laminectomies. I conducted the majority of experimental procedures and all data analyses.  Dr. Timothy H. Murphy supervised the project, worked with me to write the published manuscript, and he provided financial support.   Approval for animal experiments was issued by the Animal Care Committee of the University of British Columbia (Protocols A09-0665 and A10-0140).      	  	  	  iv	  	  Table of Contents 	  Abstract.......................................................................................................................................ii	  Preface....................................................................................................................................... iii	  Table	  of	  Contents ....................................................................................................................iv	  List	  of	  Figures............................................................................................................................ v	  List	  of	  Acknowledgements.............................................................................................................. vii	  Dedication .............................................................................................................................. viii	  Introduction ..............................................................................................................................1	  Overview .............................................................................................................................................................1	  The	  etiology	  of	  stroke....................................................................................................................................2	  The	  pathophysiology	  of	  ischemic	  stoke.................................................................................................2	  The	  evolving	  ischemic	  penumbra ............................................................................................................3	  Photothombosis,	  a	  model	  for	  targeted	  focal	  cortical	  stroke.........................................................4	  Optogenetics,	  light	  activated	  channels,	  non-­‐invasive	  cell	  type	  specific	  stimulation .........4	  Anatomy,	  organization	  and	  efferent	  projections	  of	  motor	  cortex .............................................6	  Automated	  light-­‐based	  mapping	  of	  the	  mouse	  motor	  cortex ......................................................7	  Immediately	  after	  stroke	  electrically	  silent	  but	  viable	  neurons	  exist ......................................9	  Light	  based	  probing	  to	  spatially	  resolve	  immediate	  changes	  in	  neuronal	  excitability.. 10	  Objective	  and	  hypothesis ................................................................................................... 13	  Materials	  and	  methods ....................................................................................................... 14	  Animals	  and	  surgery ................................................................................................................................... 14	  Optogenetic	  motor	  mapping	  and	  assessment	  of	  neuronal	  excitability ................................ 15	  Laser	  speckle	  contrast	  imaging.............................................................................................................. 17	  Targeted	  photothrombosis ...................................................................................................................... 17	  Statistics ........................................................................................................................................................... 18	  Results ...................................................................................................................................... 19	  Light-­‐based	  motor	  mapping .................................................................................................................... 19	  Evolution	  of	  the	  targeted	  stroke............................................................................................................ 22	  Differential	  effect	  of	  stroke	  on	  neuronal	  excitability,	  spinal	  potentials,	  and	  motor	  maps.............................................................................................................................................................................. 25	  Discussion ............................................................................................................................... 36	  Mapping	  neuronal	  excitability	  with	  channelrhodopsin-­‐2 .......................................................... 36	  The	  nature	  of	  the	  ChR2	  electrophysiological	  signal...................................................................... 37	  Neuronal	  excitability	  is	  less	  affected	  than	  spinal	  potentials	  and	  motor	  output	  after	  stroke................................................................................................................................................................. 39	  Implications	  and	  future	  directions	  (conclusions).......................................................................... 43	  References............................................................................................................................... 45	  	   	   v	   List of Figures   Fig 1.  Experimental arrangement for ChR2 cortical and motor mapping……...….20 Fig 2.  Evolution of the targeted stroke.  ……………………………………………...24 Fig 3.  Assessment of motor output after stroke. …………………………………..…26 Fig 4. Assessment of motor output and neuronal excitability after stroke……..…...29 Fig 5. The spatial relationship between changes in neuronal excitability, motor output, and the ministroke for group data……………………………………………30 Fig 6. The effect of stroke on ChR2-mediated and cortically evoked dorsal spinal potentials………………………………………………………………………………...34 Fig 7. Summary diagram showing selective breakdown of post stroke motor cortex output………………………………………………………………………………...….42            	  vi	   List of Abbreviations 	  	  	  	  CDP	  –	  (spinal)	  Cord	  dorsum	  potential	  	  ChR2	  –	  Channelrhodopsin-­‐2	  	  EEG	  –	  Electroencephalography	  	  EMG	  –	  Electromyography	  	  IOS	  –	  Intrinsic	  Signal	  Imaging	  	  PET	  –	  Positron	  Emission	  Tomography	  	  	   	   vii	  	  Acknowledgements 	   Thank you, to my supervisor Timothy Murphy, and my colleagues, for the knowledge and experience I gained from the unwritten failures and published successes.  The environment fostered by Tim, of constant building, rebuilding and refining both ideas and instruments was invaluable to me.  Thank you to my committee, Brian Macvicar, Jeremy Seamans, and Matthew Ramer for your guidance and continued interest in my work.  Jeffrey LeDue and Jamie Boyd for training me to converse with both hardware and data. And lastly, thank you to all my friends for their support, especially fellow graduate students, for this shared experience.  viii	   Dedication 	  	     погибший старой ведьмы и старения потерянный колдун 	   	   1	  Introduction  Overview  Motor representations were first mapped onto the cortex by associating muscle twitches with stimulated regions (Penfield & Boldrey, 1937).  With this procedure, of simultaneous cortical stimulation and observation of movement, a motor organization dubbed the ‘homunculus’ was revealed in the form of a cortical map. These motor cortical representations are dynamic; they are altered by injury and have the capacity reorganize during recovery (R J Nudo, Milliken, Jenkins, & Merzenich, 1996; R J Nudo, Wise, SiFuentes, & Milliken, 1996). Here we aim to briefly describe the structural and functional anatomy that underlies these motor maps. Next, we will present technological advancements in motor mapping, specifically how the activation of light gated ion channels in transgenic mice, optogenetics, has facilitated experiments that aim to spatially resolve acute changes in the topography of motor maps (motor output) after targeted cortical stroke. These experiments demonstrated that the post stroke brain was optogenetically excitable, yet there was a serial and graded breakdown in output detected in the spinal cord and then the musculature.  Finally, we will present our current understanding of the nature of these light evoked electrophysiological responses and discuss how these data contribute to our understanding of how ischemic lesions affect physiological processes required for motor output.     2	  The etiology of stroke  Stroke is a result of a rupture or blockage in a blood vessel. Interruption of blood flow in the brain, an ischemic stroke, leads to a cascade of biochemical and electrical events that resolve in tissue damage.  This insult can manifest itself as potentially devastating sensorimotor, cognitive, and ultimately behavioral deficit (Lo, 2008; Mergenthaler, Dirnagl, & Meisel, 2004). Different types of stroke cause variable patterns and progressions of damage. Functional outcome after stroke is largely dependent on the size and location of the insult (Richard Green, Odergren, & Ashwood, 2003). The origin and types of stroke are diverse. With progressive culmination of smaller lesions possibly leading to deficits which are often associated with vascular dementia and Alzheimer’s disease (Okamoto et al., 2009; Wang et al., 2012). Our interest is to study the effects of a single focal stroke targeted to motor cortex.  The pathophysiology of ischemic stoke  The metabolic cost to support brain function is extremely high.  This organ occupies only 2% of the total body mass but accounts for the use of 25% of the glucose and 20% of body’s oxygen budget (Attwell & Laughlin, 2001; Hofmeijer & van Putten, 2012). The majority of metabolic support in the brain is provided by oxidative metabolism. Consequently, the brain is very sensitive to disruption in its oxygen and glucose supply, making an ischemic stroke devastating to its function.  Synaptic function in the brain is metabolically demanding; in addition to the synthesis of neurotransmitters being biochemically linked to oxidative metabolism (Waagepetersen, Sonnewald, Larsson, & Schousboe, 1999) rectification of ion flux resultant from events such as 	   	   3	  synaptic transmission is in part dependent on energy demanding active membrane bound transporters (Attwell & Laughlin, 2001).  Membrane failure introduces ions and neurotransmitters to the extracellular environment; This disrupts neuronal communication, signals apoptosis (Lipton & Nicotera, February; Orrenius, Zhivotovsky, & Nicotera, 2003) and can lead to necrosis (Mergenthaler et al., 2004).   The evolving ischemic penumbra  Many human strokes are local (near the middle cerebral artery) and result in a focal lesion with a surrounding area with reduced blood flow which is at risk to develop into an infarct. Damage at the core of the lesion is considered unsalvageable (Lo, 2008); however, nearby regions with reduced blood flow and partial function (Zhang & Murphy, 2007) may be the target of neuroprotective strategies. The penumbra is defined as tissue at risk due to a partial reduction in blood flow. Whether tissue survives or progresses into infarction is directly related to oxygen availability via local blood flow. Over time after ischemic stroke, secondary clotting and damage to penumbral vasculature occurs as perfusion to the vessels themselves is compromised. In most models of stroke, inflammation and blood extravasation leads to an expansion of the penumbra (Dirnagl, Niwa, Sixt, & Villringer, 1994). In related work, using models of focal ischemia, it was observed that the size of the occluded or poorly perfused area increased spontaneously over time. This observation is in accordance with PET imaging (Baron, 1999) and human perfusion/diffusion mismatch data from MR imaging data (Neumann-Haefelin et al., 1999).  We can monitor/image cortical blood flow to observe evolution of the penumbra and relate alterations in blood flow to the integrity of evoked responses. Below we discuss the animal model used in these experiments to make a ‘mini’ ischemic stroke. 4	  Photothombosis, a model for targeted focal cortical stroke   Photothrombosis is a relatively non-invasive method for targeting focal ischemic lesions of controlled size.  First, rosebengal, a light sensitive dye, is injected intravenously. Next, vessels or regions of interest are targeted irradiated with green light (exciting the dye).  The resultant photochemical reaction produces radicals, irritating the vasculature, leading to an aggregation of red blood cells and ischemia (Watson, Dietrich, Busto, Wachtel, & Ginsberg, 1985).  This method is not limited to but is most practical for application in optically accessible tissue.  This makes it common to application in the cortex.  Alternative methods for making ischemic strokes are ligation of larger vessels, such as the middle cerebral artery.  However, these model can be variable (Tudela et al., 2014) perhaps due to the intrinsic differences in the anatomy of the vasculature in the cortex.  These procedures are also surgically invasive, arguable introducing confounds associated with surgical procedures. We chose to use photothrombosis to reliably target a limited region of the cortex.   Optogenetics, light activated channels, non-invasive cell type specific stimulation  Slightly over a decade ago, it became possible to activate neurons with light rather than electrical stimulation. The light activated ion channel, Channelrhodopsin-2 (ChR2), isolated form Chlamydomonas reinhardtii, can be expressed in the membrane of cells of interest, namely those which are excitable. This relatively non-selective ion/cation channel has a reversal potential of near 0 mV, when photoactivated it opens and allows the flux of ions largely according to the electrochemical gradient (although the channel has some intrinsic properties that make it bias to passing certain elements).  This can 	   	   5	  bring a neuron past threshold for firing, leading to action potentials.  This can be done reliably at stimulation rates up to 30 Hz (Boyden, Zhang, Bamberg, Nagel, & Deisseroth, 2005).  Peak excitation of this variant of ChR2 is 470nm (Nagel et al., 2003). Red shifted variants of channelrhodopsins have been engineered to facilitate imaging reflectance or florescence at short wavelengths and also to decrease light scattering and increasing the depth of light penetration into the brain for stimulation (Lin, Knutsen, Muller, Kleinfeld, & Tsien, 2013).  Here we will concentrate on the variant sensitive to blue light.   It was used as it can be stimulated without spectral overlap from/into imaging of blood flow (red/infrared) and the induction of stroke through rose bengal photoactivation (532nm). The combination of genetic engineering and optical stimulation allows for non-invasive cell type specific excitation with high temporal and spatial resolution. Although optogenetic stimulation is cell type specific, one must consider that polysynaptic responses originating form light activated cells can recruit networks of functionally connected neurons (Lim et al., 2012; Mohajerani et al., 2013) of various identities. Here we use transgenic mice (Arenkiel et al., 2007) which within the cortex, express ChR2 predominantly in layer 5 pyramidal neurons (Lim et al., 2012).  These neurons, when stimulated, lead to motor output (Hira et al., 2009; Kiritani, Wickersham, Seung, & Shepherd, 2012).  Here we will stimulate an array of sites across the cortex while recording in the contralateral forelimb to generate a cortical representation of the limb (Ayling et al., 2009) and explore how topography of these wide scale maps are altered by local targeted ischemia.   6	  Anatomy, organization and efferent projections of motor cortex  The ultimate output of the brain is typically realized in the form of movement.  This is mediated in part by the motor cortex.  This frontal cortical region is classically categorized into 6 layers of cells.  In the mouse, output is largely mediated by large excitatory pyramidal neurons (Betz cells), which are found in the fifth layer of the cortex.  The motor region is characterized by its agranular nature (lacks the dense amount of inhibitory neurons which can be found in layer 4 of sensory cortex).  Mouse motor cortex and is situated approximately 2 mm lateral of Bregma (Fig. 1).  The ultimate/terminal output of these Betz cells is the muscle (here the forelimb).  This contact is achieved via lower motor neurons in the spinal cord which are activated through the large number synapses from local interneurons contacted from the cortex (corticospinal tract), or through the striatum (corticostriatal tract) (Anderson, Sheets, Kiritani, & Shepherd, 2010). We acknowledge that stimulation of these Betz cells can lead to cortical responses from all efferent pathways, such as the corticobulbar tract, or the corticoreticular tract and rubrospinal tracts, which is particularly important for movement in rodents. Any tract connected to these stimulated output neurons could be activated. In the following experiments we focus on the corticospinal tract as it is the predominant projection from layer5 cortical neurons (Lemon, 2008; Lemon & Griffiths, 2005). Individual neurons in motor cortex can code for limb position, velocity (Paninski, Fellows, Hatsopoulos, & Donoghue, 2004), direction (Georgopoulos, Kalaska, Caminiti, & Massey, 1982), and force (Kalaska, Cohen, Hyde, & Prud’homme, 1989).  These neurons are not organized in a clear ‘map’ but are overlapping in their distribution 	   	   7	  (Harrison & Murphy, 2012).  This makes it apparent that the motor cortex is organized in a fashion far more complex than revealed by early mapping experiments. The ‘salt and pepper’ arrangement of these scattered neurons where some preferentially respond to different kinematic or kinetic stimuli may contribute to overlapping somatotopic representations. Despite this, certain regions of the brain, when directly stimulated just above excitation threshold for the musculature, reveals a ‘muscle map’(Asanuma & Rosén, 1972). It is noteworthy that stimulation above this threshold yields movement from multiple muscles (Tennant et al., 2011).  This can be due to any and all of the following reasons: overlapping cortical somatopy, upper motor neurons having the ability to communicate with more than one lower motor neuron (through spinal circuitry), and technical limitations associated with uncertainty as to the volume of tissue stimulated.  The presence of spinal interneurons as relays between upper and lower motor neurons is a characteristic of rodents.  In primates upper motor neurons can synapse directly to lower ones in the spinal cord (Lemon & Griffiths, 2005).  Despite the limitation of uncertainty as to what information a motor map may encode, the integrity of muscle maps, is related to motor function, dysfunction and recovery.  Focal injury can reduce the area of a motor map, this is reflected as a deficit in the behaviour associated with the region ablated in the experimental animals.  Recovery coincides with a reorganization and recovery of map topography (Dancause et al., 2005; R J Nudo, Wise, et al., 1996).  Automated light-based mapping of the mouse motor cortex   The refinement of this technique has been focused on three fronts.  Firstly one can improve spatial resolution. This can be done by reducing the area stimulated by piercing high resistance microelectrodes for stimulation into the brain (ICMS) (Donoghue & 8	  Sanes, 1987; Tennant et al., 2012) while observing motor responses.  This can be done repeatedly at multiple locations to generate a map. Alternatively one can insert an array of stimulation electrodes (Schneider, Devanne, Lavoie, & Capaday, 2002) into the brain.  These techniques are arguably very damaging to the brain; consequently, some have focused on the application of non-invasive stimulation techniques such as transcranial magnetic stimulation. Despite being able to stimulate through the intact skull; TMS activates a far larger area than ICMS. Lastly, one wishes to specify which cell type is being stimulated in order to better isolate from where the measured responses originate. This has been made possible with the advent of optogenetic stimulation techniques. Light stimulation of Channelrhodopsin-2 (ChR2) leads to opening of cation channels; when expressed in neurons the resulting current leads to firing of action potentials (Boyden et al., 2005; Nagel et al., 2003). Expression of the opsin can be targeted to cells of different genetic identity, and even to specific areas with local injections (Madisen et al., 2012).  In transgenic mice used in experiments discussed here, B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J strain, cortical expression of ChR2 is predominantly in  layer 5 pyramidal neurons (Ayling et al., 2009). Recording in the musculature while photo stimulating the cortex of a mouse expressing ChR2 in neurons responsible for motor output with a 100µm diameter laser facilitates repeated, relatively non-invasive, cell type specific generation of relatively high-resolution motor cortical representations of limbs on the cortex.  Here we can apply use this technique to investigate how motor representations are affected at time points earlier after stroke than previously investigated.  We are able to do this without the use of invasive electrode stimulation and with the added advantage of offering cell type specific stimulation.  	   	   9	  Immediately after stroke electrically silent but viable neurons exist  The preservation of neuronal excitability, maintenance their electrical potential and capacity to fire, is vital for stroke recovery. Neurons that surround the irreversibly damaged infarct core (Lo, 2008) can maintain their excitability (Symon et al., 1977; Astrup et al., 1981; Hossmann, 1994) and are the medium of functional remapping (Murphy and Corbett, 2009). Neurons within hypoperfused tissue can have intact membrane potentials while they are unable to fire sensation-evoked action potentials. (K.-A. Hossmann, 1971; K. A. Hossmann, 1994).  This prolonged electrical depression after stroke has been described as a “stunned brain” (Carmichael, 2012), a dysfunctional state which hinders functional recovery (Clarkson, Huang, Macisaac, Mody, & Carmichael, 2010).  Whether this prolonged depression of activity is related to a lack of membrane hyperpolarization (Xu & Pulsinelli, 1994), deficits in signal initiation/propagation (Schafer et al., 2009), or due to a defect that selectively impairs synaptic transmission (Gao, Howard, & Xu, 1998) is unclear.  This is due to ischemia affecting upstream presynaptic pathways (Li, Lei, & Xu, 2009; Pang, Deng, Ruan, & Xu, 2002), and potentially enhancing some form of local inhibition (Clarkson et al., 2010). The classic concept of viability thresholds for ischemia differentiates between two critical flow rates; the threshold of electrical and membrane failure. Optogenetics allows for us to probe responses from synaptically silent cells above membrane failure.     10	  Light based probing to spatially resolve immediate changes in neuronal excitability   Recovery of motor function after stroke is dependent on the capacity of the brain to reorganize surviving circuitry (Randolph J Nudo, 2006; R J Nudo, Plautz, & Frost, 2001).  Motor maps exhibit plasticity (Kerr, Cheng, & Jones, 2011) and alterations during training (Kleim, Barbay, & Nudo, 1998; R J Nudo, Milliken, et al., 1996; Tennant et al., 2012) and their remapping over days to weeks after stroke is related to the recovery of motor behavior (Castro-Alamancos & Borrel, 1995; R J Nudo, Wise, et al., 1996)(Castro-Alamancos and Borrel, 1995; Nudo et al., 1996b). However, assessment of motor function can be challenging in experimental animals given that stroke often produces a period of prolonged synaptic depression (Astrup et al., 1981; Astrup, Symon, Branston, & Lassen, 1977; Heiss, 2000; K.-A. Hossmann, 1971; K. A. Hossmann, 1994). Furthermore, to adequately assess  motor function sophisticated behavioral tests that can be difficult to implement and interpret in mice are required, particularly at early time points after ischemia (Alaverdashvili & Whishaw, 2010; Levin, Kleim, & Wolf, 2009; Moon, Alaverdashvili, Cross, & Whishaw, 2009) In vivo synaptic function is typically assayed via cortical recordings of sensory evoked responses  (Zhang and Murphy 2007). These types of experiments established that in the immediate hours after ischemia, intact and electrically viable neurons exist but they are unable to fire. Deficits within sensory responses are likely due to depression of local synaptic transmission (Hershkowitz, et al., 1993; Shih et al. 2013) potentially overshadowing other deficits such as impaired axonal conduction (Dunn, Bolay, Moskowitz, & Boas, 2001)which is pertinent to efferent motor processes. This ‘synaptically silent’ brain state has made assaying efferent function challenging. 	   	   11	  Additionally, results from the aforementioned studies are only attributable to cells that respond to ascending sensory signals leaving the function of motor neurons unresolved. As much of the optogenetic stimulation induced extracellular response is dependent on target cells having a hyperpolarized membrane potential rather than an ability to produce action potentials (Chen, Mohajerani, Xie, & Murphy, 2012). Light stimulation can overcome this electrical silence and evoke a presumably largely photocurrent based response during ischemia.  Recently, the light-activated ion channel channelrhodopsin-2 (ChR2) (Boyden et al., 2005; Nagel et al., 2003) has been used as a means of assessing the excitability  (Xiong & Jin, 2012) of neurons in somatosensory (Chen et al., 2012) and motor cortex (Xie, Chen, Anenberg, & Murphy, 2013)within the first minutes to hours following global ischemia. This approach revealed neurons that were silent with respect to sensory processing, but were nonetheless excitable after reperfusion. These excitable neurons may be available to mediate circuit-level plasticity that could contribute to recovery of function. However, this previous work was limited to global ischemia and did not evaluate focal ischemia models where an affected core and a penumbra with a partial reduction in blood flow was present. Here, we extended this previous work to focal ischemia and have directly evaluated motor function using ChR2 stimulation and electromyographic recordings. Given that ischemia impairs initiation (Schafer et al., 2009) and conduction (H Bolay & Dalkara, 1998) of neuronal signals, and can also alter the excitation-inhibition balance of cortex (Buchkremer-Ratzmann, August, Hagemann, & Witte, 1996; Bütefisch, Netz, Weßling, Seitz, & Hömberg, 2003; Carmichael, 2012; Schmidt, Bruehl, Frahm, Redecker, & Witte, 2012; J. H. Wang, 2003), optogenetics can 12	  potentially differentiate between effects on general neuronal excitability and selective depression of motor output. In the case of motor function we have applied optogenetics to automate the mapping of motor cortex (Ayling et al., 2009; Hira et al., 2009) to better resolve effects within both the ischemic core and penumbra by examining changes in neuronal excitability and the topography of motor maps within the initial 2.5 h after stroke. We have focused on this time interval because this is when treatments, such as thrombolysis and other interventions, are performed (Cook, Teves, & Tymianski, 2012; Hill et al., 2012). We report that a ministroke directed to a subset of the forelimb motor map has a significantly larger effect on motor output than that expected from its action on local cortical neuronal excitability.  	   	   13	  Objective and hypothesis  The objective of this work was to use recently developed optogenetic methods for mapping output from motor cortex to assess and spatially resolve the immediate effects of a local ischemic lesion.  The working hypothesis was that optogenetic stimulation could overcome the electrical silence induced by some aspects of ischemia, thereby allowing us to investigate the immediate functional consequences of stroke on cortical, spinal, and muscular responses  Aim 1: Assess changes in local blood flow in the ischemic penumbra using laser speckle contrast imaging (the evolution of the infarct).  Aim 2: Map the neuronal and associated muscular excitability of the ischemic penumbra in excitatory neurons using optogenetic methods during the first 2.5h of focal stroke.  Aim 3: Assess the serial transmission motor output after stroke (by monitoring cortical, spinal, and muscular ChR2 evoked extracellular fields)      14	  Materials and methods  Animals and surgery  All animal protocols were approved by The University of British Columbia Animal Care Committee.  Male and Female Jackson Labs Line-18 stock 007612, strain B6Cg-Tg (Thy1-COP4/EyFP)18Gfng/J Channelrhodopsin-2 transgenic mice  from an established breeding colony aged 2-10 months weighing 20-35g were used.  Mice were anesthetized using 2% isoflurane in air which was reduced to 1-1.5% during surgery. Body temperature was maintained at 37±0.50C using a heating pad and feedback from a rectal probe. The mouse was glued in a custom made head hold (Harrison et al., 2009) and a craniectomy was made over the right motor cortex without removing the dura.  Agarose dissolved in HEPES buffered ACSF (pH 7.3) to 1.5% was placed over the surface of the cortex at 37°C.  The surface of the cortex was then covered with a No.1 130-160 µm thick glass cover slip.   A laminectomy was performed over a region corresponding to the fourth cervical vertebrae.  After an incision was made to expose the spinal cord, the bone was removed with rongeurs.  The dura was left intact.   A silver ball electrode was inserted superiorly to the exposed area over the dorsal column the spinal cord and stabilized in agarose. Teflon coated silver wires were surgically implanted into the left extensor carpi ulnaris for electromyography (EMG) and placed on the medial edge of the craniectomy on the surface of cortex for electroencephalography (EEG). Ground electrodes were placed subcutaneously in the forepaw, hindlimb, and above the nose. The skin of the forelimb was sealed with cyanoacrylate. Isoflorane is a potent vasodilator and does not 	   	   15	  allow the animal to maintain muscle tone when anesthetized. Consequently, before light based mapping or assessing hemodynamic responses, anesthesia was switched to a ketamine xylazine combination (100/10 mg/kg) and supplemented as required (Ayling et al., 2009; Harrison, Ayling, & Murphy, 2012; Scott & Murphy, 2012).  Optogenetic motor mapping and assessment of neuronal excitability  The methodology for light-based motor mapping has been described in detail in other work (Ayling et al., 2009). Custom software written in Igor pro (v6.2 Wavemetrics) moved a scanning stage (ASI MS-2000) with the mouse beneath a fixed 473nm laser focused to a diameter of ~100 µm.  Laser pulses were 10 ms in duration.  Sites of stimulation were 300µm apart and typically arranged in a 12X14 grid of sites that were stimulated in semi-random order, with the requirement that sites had to be further than 750µm each other to be stimulated sequentially. Stimulation of the grid was repeated three times and the resultant recordings from each independent site of cortical activation and muscular activity in the periphery were averaged in order to generate a motor and neuronal excitability map. ChR2 evoked Electromyographic (EMG) electroencephalographic (EEG) (Xie et al., 2013), and  dorsal spinal potential recordings were amplified 1000 times (Model 1700, A-M Systems), digitized (National Instruments NIDAQ), and then recorded and monitored online in Igor pro.  The amplitude of EMG signals were quantified as the root mean square of the signal and the amplitude of the EEG was the maximum of the rectified integrated signal over 50 ms after stimulus onset.  Laser intensity ranged from 1-5 mW and was adjusted to the lowest power settings capable of generating stable motor maps. Only responses with an amplitude which exceeded three times the standard deviation of the 500 ms pre-stimulus period were 16	  included in mapping data.  If the EEG electrode encroached within the mapping grid a distinct light artifact in a minimal number of EEG map pixels from the laser striking the electrode was evident (Fig 4C), and was therefore removed from the data set. To aid analysis of sparsely sampled maps EEG and EMG response maps were upsampled, leading to a pixel size of 100x100µm, normalized to their respective means, aligned relative to the stroke location and averaged.  A potential concern is that the laser-induced photoactivation of ChR2 may spread to regions of cortex not affected by the ministroke since light scattering through tissue and other factors increases the apparent area of photoactivation.  The greatest responses are at the targeted photo activation site; however, intrinsic optical signal (IOS) imaging revealed that responses are detectable at a distance of 600-900 mm (Ayling et al., 2009).  The relatively sharp boundaries of motor maps (presumably due to a threshold) suggest that the area of activation associated with motor responses may be smaller than areas activated and recorded with IOS and here with EEG.  In mice in which recordings from the spinal cord were employed, stimulation was limited to 2 sites, either at the location of the vessel targeted for occlusion, or in a peri-infarct area within motor cortex.  These two stimulation sites were at an average distance of 1.3±0.2 mm of each other.  Recording ChR2-evoked EEG potentials in the spinal cord, and EMG responses from 200-300 repetitions of stimulation at of these two sites was performed before and after stroke.  Quantification of potentials recorded from the spinal cord was performed by measuring the peak  deflection in the signal.     	   	   17	  Laser speckle contrast imaging  The surface of the cortex was illuminated with a 784-nm 32 mW StockerYale SNF-XXX_885s-35 laser (Stocker & Yale, Salem NH) at a 30-degree angle with a beam expander and light intensity controlled by a polarizer. The flow of blood cells and resultant blurring in the interference patterns was detected by a CCD camera (Dalsa 1M60). Image acquisition was performed using EPIX XCAP software (v2.2).  To assess the ischemic penumbra 50 frames were acquired at 10Hz with a 10 ms exposure time.  The 1024x1024 image stack was variance filtered with a kernel size of 3x3 pixels and averaged into a single 32-bit image. Regions of interest selected for assessment of blood flow over time were 20x20 pixels (76x76µm) and centered over the vessel targeted for occlusion. Blood flow measurements were estimated as the inverse square of speckle contrast values and scaled between minimum and maximum flow rate (Ohtsubo & Asakura, 1976; Dunn et al., 2001; Cheng et al., 2003). To determine regional changes in blood flow images were Gaussian filtered, typically with a pixel radius of 15, subtracted from baseline images, and thresholded to contrast levels equivalent to 20, 40 and 60% reductions relative to mean image contrast. Maximal speckle contrast values were empirically determined from dead animals and minimum values were those associated with pre-stroke image contrast levels.  Targeted photothrombosis    The animals were placed under an upright microscope (Olympus BX51W1) and the target vessel was located through a 40x0.8 NA water immersion objective.  The animal was injected intravenously in the tail with 0.01-0.03mg/g Rose Bengal dye (Sigler 18	  et al., 2008; Mohajerani et al., 2011; Murphy, 2011; Risher et al., 2012) diluted in 0.02 ml HEPES buffered artificial CSF. Light from a 532 nm laser (Beta Electronics MgM-20) was directed at the target vessel through the epiflorescence path light path of the microscope.  Photoactivation of the dye was maintained until the clot formed; this was first confirmed visually and then with laser speckle imaging similar to previous studies  (Sigler et al., 2008; Sigler et al., 2009; Mohajerani et al., 2011) Statistics  EMG and EEG data were normalized to the average of values acquired in the baseline mapping session for each mouse. Wilcoxon signed rank tests were used to compare EEG, EMG, and blood flow measures to hypothetical baselines. Comparisons between stroke (n = 8) and sham (n = 7) EEG and EMG signals (with some maps missing at random due to the requirement of maintenance of the proper anesthetic plane for mapping) were performed with Mann–Whitney tests. Ischemic territories from these mice were compared with Kruskal–Wallis and Dunn's post hoc statistical tests. Linear regression analysis was applied to quantify the relationship between EEG and EMG signals as a function of distance from the infarct core. In the cohort of mice in which spinal potentials were recorded comparison of EEG, spinal potentials, and EMG signals after stroke were performed with one-way ANOVA followed by Bonferoni within group post hoc tests (n = 4). The same statistical testing was applied to the surface area of ischemic territories. Comparison of EEG, spinal potentials, EMG, and speckle values after stroke to those measured at baseline were performed with paired t tests. Statistical tests were performed using GraphPad Prism (version 5.0).   	   	   19	  Results  Light-based motor mapping  Line-18 transgenic mice express ChR-2 via the Thy-1 promoter largely in layer 5 pyramidal neurons (Arenkiel et al., 2007). Automated optogenetic motor mapping (Ayling et al., 2009; Hira et al., 2009) allowed for repeated fast acquisition of a cortical motor representation of the forelimb and averaged EEG responses that reflect aspects of cortical neuronal excitability. EMG recordings in the contralateral forelimb, recordings from the dorsal column of the spinal cord, and ChR2-evoked cortical EEG recordings provide simultaneous assessment of cortical neuronal and spinal excitability while evaluating motor output and regional blood flow (Fig. 1A–C) in response to cortical ChR2 activation. As each of these electrophysiological measurements is based on different but dependent physiological processes, this approach serves to assay selective vulnerabilities within the motor system. Unlike traditional intracortical microstimulation mapping methods which aim to determine the dominant response of a particular area (Tennant et al., 2011), we quantitatively map ChR2-evoked cortical and forelimb EMG responses across the entirety of motor cortex to assay the regional capacity of cells to drive motor output.  20	   Fig 1.  Experimental arrangement for ChR2 cortical and motor mapping. A, Green light image of cortex overlaid with a 12 × 14 grid of stimulation sites each separated by 300 µm (Ai). Traces of EMG (Aii) and EEG (Aiii) responses evoked from light stimulation directed at each site on cortex. Aiv, Quantification of the ChR2 evoked EEG response; the raw, rectified and integrated signal. Bi, Processed laser-speckle image displaying blood flow of surface vasculature, with higher velocity blood flow appearing as a darker tone. The arrow indicates the arteriole targeted for occlusion, selected for its 	   	   21	  location branching from the middle cerebral artery over a restricted region of the motor map. ChR2 evoked EMG (Bii) and EEG (Biii) responses from three repetitions averaged and scaled between minimal and maximal values represented as pixels ranged from black to white reveal a ChR2-evoked motor and neuronal excitability (EEG) map. Tracings of vasculature superimposed on the motor map, with the vessel targeted for occlusion colored in blue. Biv, Outline of the motor map after thresholding-excluding responses <25% of maximum. Location of vessels targeted for occlusion relative to center of motor maps (n = 8). Outline of thresholded motor map relative to forelimb and hindlimb cortical sensory representations and bregma based on previous observations (Ayling et al., 2009). C, Timeline of the experiment. Baseline maps and laser speckle imaging was performed. Photothrombosis was achieved by irradiating the target vessel after injection of rose bengal. Following induction of stroke, changes in blood flow were measured with laser speckle imaging before ChR2 mapping ensured. This procedure was repeated for 2.5 h.  22	   Evolution of the targeted stroke  Stroke was induced with a modified version of the rose bengal method (Watson et al., 1985) targeting individual surface pial arterioles (Sigler et al., 2008; Fig. 2A). Laser-speckle imaging and laser-optogenetic stimulation did not commence until 5–10 min after the occlusion was induced to avoid additional off-target photoactivation of the dye. Imaging after stroke revealed that within a 20 × 20 pixel (76 × 76 µm) area surrounding the targeted vessel, blood flow was reduced to 27.1 ± 3.9% of baseline (p < 0.05 n = 8 mice; Fig. 2B). Pial arterioles were targeted for occlusion were 46.5 ± 5.7 µm in diameter (n = 8). Within 40–50 min, blood flow decreased to <20% of baseline and continued to decline gradually throughout the experiment. The ischemic territory was calculated by subtracting baseline laser-speckle contrast images from those acquired after induction of stroke. Thresholding images at contrast values equivalent to 20, 40, and 60% of mean baseline blood flow values confirmed that the infarcted area was restricted to a subset of motor cortex (Fig. 2C,D). The targeted vessels were located on average 1.66 ± 0.43 laterally and ±0.21 mm caudally from the center of motor maps (Fig. 1B). The initial 0.07 ± 0.03 mm2 ischemic core defined by 20% blood flow expanded to 0.45 ± 0.27 mm2 within 130–140 min after irradiation. Similarly, hypoperfused areas corresponding to reductions in blood flow of 40% and 60% were initially restricted to 0.8 ± 0.3 mm2 and 1.6 ± 0.5 mm2 regions, but they expanded to 1.2 ± 0.02 and 2.3 ± 0.7 mm2 by 130–140 min after stroke, (Fig. 2C,D). As photoactivation and subsequent occlusion in this model of stroke is localized to surface arterioles, damage to deeper cortical layers occurs more 	   	   23	  gradually (Enright, Zhang, & Murphy, 2007) and is consistent with the findings where surface vascular networks can supply deeper cortical areas (Blinder et al., 2013; Schafer et al., 2009; Shih et al., 2013) 24	    Fig 2.  Evolution of the targeted stroke.  A, Laser-speckle image displaying blood flow of surface vasculature with a black outline of the corresponding motor map and the area with <20% blood mean blood flow after stroke in white. B, Percentage change in blood flow at the 76 × 76 µm region of interest at the vessel targeted for occlusion. C, The surface area of cortex with area corresponding to various thresholds of blood flow. D, Areas corresponding to 20, 40, and 60% blood flow superimposed onto laser speckle images at 70–80 min poststroke with the most ischemic area (<20% blood flow) outlined. Error bars in all graphs are SEM. Significant differences relative to baseline #p < 0.05, ##p < 0.01 and between groups *p < 0.05, **p < 0.01.  	   	   25	  Differential effect of stroke on neuronal excitability, spinal potentials, and motor maps After stroke, motor output (measured using EMGs) was reduced at all stimulated points within the motor map (Figs. 3A–C, 5A). Deficits in motor maps were more widespread and severe with motor output at the stroke core depressed to 28.8%±13.0 of baseline (see Fig. 5D; group data). Motor output at 1 and 1.5 mm from the stroke core was 58.1%±6.2 and 68.8%±4.6 of baseline, respectively, increasing as a function of distance (Fig. 5D; r2 = 0.92, p < 0.001, group data). There was no significant change in the number of responsive points in motor maps (Fig. 3C). Notably, the majority of the stimulated locations (for EMGs) were not directly encroached upon by the stroke core as defined by laser-speckle imaging (Fig. 2A). This is comparable to ministroke-induced remapping in sensory systems, where responses at the stroke core were attenuated, but cortical regions farther from the infarct were preserved (Sigler et al., 2008). Immediately after stroke, the average of all responses within motor maps were depressed to 59.1 ± 13.6% (p = 0.063) of baseline, whereas sham animals maintained motor output at 93.4 ± 34.0% of baseline (p = 0.344; Fig. 3B; based on EMG amplitude). The decrease in motor output continued after stroke, reaching 40.2 ± 12.6% (p < 0.001) of baseline by the last mapping session (130–160 min). Motor output in sham animals (n = 7) increased to 184.7 ± 34.7% (p < 0.05) of baseline at the corresponding time point (between groups comparison p < 0.001). Repetitive optogenetic stimulation significantly increased motor output in a time dependent manner in sham animals comparable with previous work (Ayling et al., 2009).     26	   Fig 3.  Assessment of motor output after stroke.  A, Motor maps generated from repeated mapping of a (Ai) sham animal and (Aii) one with a targeted stroke. B, The percentage change in the mean of ChR2 evoked EMG responses in the map over time. C, Change in motor map area based on the number of stimulation sites that evoked a motor response (EMG). Rescaled image from 130 to 160 min time point more clearly demonstrating spatial the extent of cortical stimulation regions that yielded motor responses. Error bars in all graphs are SEM. Significant 	   	   27	  differences #p < 0.05 with respect to baseline. Significant differences *p < 0.05, **p < 0.01, ***p < 0.001 between groups.  28	   In contrast to the deficit observed in motor maps, neuronal excitability as defined by ChR2-evoked EEG signal was relatively preserved across cortex (Fig. 4A,B). Signal amplitude, measured as the peak of the rectified integrated signal over a 50 ms period, was used to generate EEG maps to reflect baseline light-evoked neuronal excitability across cortex. Average neuronal excitability (EEG) values from across cortex within 40 min after stroke onset were 96.6 ± 2.1% of baseline (n = 6). These values were not significantly different from sham animals 90.1 ± 2.3% (n = 7). In maps collected during the final mapping session EEG maps collected from stimulation across the entire 3 × 4 mm cranial window were 73.3 ± 6.0% of baseline in the stroke group and 79.3 ± 2.6% of baseline in sham controls (Fig. 4B; based on EEG amplitude). Presumably, the 20% run-down in map amplitude in the sham control animals was due to time-dependent changes in this acute preparation. Although neuronal excitability was typically lower in stroke animals, no significant differences in the amplitude of the EEG responses were observed between stroke and sham control animals at any time point. Significant differences in neuronal excitability after stroke relative to baseline were reached in maps collected at 70–100 min poststroke (p < 0.05). In maps generated 10–40 min after stroke, a local depression to 80.4 ± 5.0% of baseline was detectable poststroke. This depression in ChR2-evoked EEG was local, as within 400µm of the occlusion site values of excitability were 95.9 ± 6.8% relative to the baseline map (Fig. 5A–D). As ministroke-induced depression of neuronal excitability was local it did not display a linear correspondence between distance and changes in excitability over millimeter scales (r2 = 0.30, p = 0.0651; Fig. 5D,E). 	   	   29	   Fig 4. Assessment of motor output and neuronal excitability after stroke.  A, ChR2-evoked EEG maps generated from repeated mapping of a (Ai) sham animal and (Aii) one with a targeted stroke. Gray values of pixels represent the amplitude of responses from stimulation of that specific location. Saturated pixels from light striking the EEG electrode, marked with a black circle, were not analyzed. B, The percentage change in the mean of EEG responses in the map over time. C, EEG traces from a single animal displaying the initial and delayed persistent response to ChR2 cortical stimulation in (Ci) sham and (Cii) stroke animals. D, The relative change in persistent activity after irradiation and ischemia. Error bars in all graphs are SEM. Significant differences #p < 0.05 with respect to baseline. Significant differences *p < 0.05, **p < 0.01, ***p < 0.001 between groups.  30	    Fig 5. The spatial relationship between changes in neuronal excitability, motor output, and the ministroke for group data. A, Average upsampled ChR-2 stimulated motor maps, (100 × 100 µm pixels) normalized to their respective mean, aligned based the location of the infarct and averaged together. Motor maps (average) at (Ai) baseline and (Aii) motor maps immediately after stroke (10–40 min; n = 6). B, Average upsampled neuronal excitability maps, (100 × 100 µm pixels) normalized to their respective mean, aligned based the location of the infarct and 	   	   31	  averaged together. ChR-2 stimulated EEG map at (Bi) baseline and (Bii) immediately after stroke (n = 6). C, Schematic of how plots in D were derived, with the amplitude of each map pixel being binned according to its distance to the infarct regardless of direction. D, Motor output (r2 = 0.92; p < 0.001) and neuronal excitability (r2 = 0.30; p = 0.0651) immediately after stroke relative to baseline maps as a function of distance from the occluded vessel. E, Schematic of the spatial changes in neuronal excitability and motor output imparted by targeted ischemia.  32	   After averaging ChR2-evoked EEG responses, we observed activity after the initial 50 ms evoked response that typically persisted for 500 ms (Fig. 4C). This activity may represent a cortical response dependent on widespread cortical activity (Lim et al., 2012). We do not discount that this could be generated through subcortical circuits mentioned previously. Persistent activity decreased to 81.1 ± 5.3% of baseline (p < 0.01) 10–40 min after stroke (n = 6), whereas activity at the same time point in sham animals was maintained at 106.3 ± 8% (n = 7; p < 0.05). Within 130–160 min after stroke, this activity was reduced to 70.5 ± 12.8%, whereas it was preserved at 99.1 ± 15.2% in sham animals (Fig. 4D).  In the cohort of mice in which spinal cord recordings were performed (Fig. 6A–F) blood flow at the target vessel was reduced to 24.7 ± 9.0% (p < 0.05) immediately after stroke (within 10 min), remaining stable (25.8 ± 10.4%) at 40–50 min poststroke (n = 4; Fig. 6C). Vessels targeted for occlusion were 58.3 ± 11.6 µm in diameter (n = 4). Within groups comparison of the surface area of ischemic territories after repeated-measures ANOVA (p < 0.001) demonstrated significant differences between groups (Fig. 6B). Descending spinal potentials evoked from light stimulation at the site of the occluded vessel within cortex were recorded within 10–40 min after stroke (Fig. 6A,D). We observed that cortically evoked (ChR2-mediated) spinal potentials were reduced to 42.3 ± 5.4% of baseline for sites in the stroke core and declined to 56.5 ± 11.6% (n = 4; p < 0.05) when evoked at cortical peri-infarct sites (1.3 ± 0.2 mm away; Fig. 6D,E). The latency of the peak of spinal potentials recorded at the peri-infart site increased to 36.3 ± 1.0 ms from 32.3 ± 2.9 ms (n = 4; p = 0.11). The increase in latency at the stroke core 	   	   33	  was not significant, 36.1 ± 0.8 ms from a baseline of 29.9 ± 2.23 ms (n = 4; p = 0.059; Fig. 6F). Simultaneous cortical EEG and EMG recordings acquired immediately after stroke confirmed that neuronal cortical excitability was maintained (90.5 ± 12.8%), whereas motor output by EMG was impaired (12.4 ± 1.5%) at the site of the infarct (p < 0.05) and in the stimulated peri-infarct area (83.7 ± 1.0%; 24.7 ± 9.8% p < 0.05; n = 4; Fig. 6E). These finding suggest a progressive loss of excitability as ChR2-evoked activity enters the spinal cord that may account for the relatively larger effect on motor output than direct cortical excitability.  34	     Fig 6. The effect of stroke on ChR2-mediated and cortically evoked dorsal spinal potentials.  A, Distance of the two sites optogentically stimulated, one being the stroke core and the other a hypoperfused peri-infarct region within motor cortex. B, The surface area of cortex with area corresponding to various thresholds of blood flow immediately after stroke (0–10 min; n = 4). Significant differences *p < 0.05, **p < 0.01, ***p < 0.001 for 	   	   35	  multiple-comparisons between these measures via Bonferroni Post Hoc testing after one-way ANOVA. C, Blood flow at the 76 × 76 µm region of interest at the vessel targeted for occlusion (n = 4). Significant differences # p < 0.05, ##p < 0.01 after t test. D, ChR2-evoked spinal cord potentials from stimulation of motor cortex at the site of the stroke (Di) and within a peri-infarct area (Dii) recorded along the dorsal column superior to the level of the fourth cervical vertebrae. E, ChR2-evoked cortical (EEG), spinal cord dorsum potential (CDP), and muscular (EMG) potentials recorded 10–40 min after stroke demonstrate the progressive attenuation of the motor output after stroke (n = 4). F, Latency to peak spinal response from ChR2 cortical stimulation from each stimulation site; at the stroke core and at the peri-infarct site.  36	  Discussion  Mapping neuronal excitability with channelrhodopsin-2  The canonical view of brain injury is that the magnitude of the deficit is proportional to infarct size (Lashley, 1931). In the immediate hours after focal ischemia it is unclear if this holds, as variable degrees of structural damage are possible depending on local perfusion levels (Zhang et al., 2005). Additionally,  there is an inherent nonlinearity in ischemic thresholds relative to cellular dysfunction (Baron, 2001; Baron & Marchal, 1999; K. A. Hossmann, 1994).  Ischemia imparts differential consequences on axonal (Enright et al., 2007), dendritic (Johansen et al., 1984), and post synaptic (Horner et al., 1998) neuronal structures. Remote deficits in connected regions (Mohajerani et al.; 2011; Carter et al., 2012) and selective vulnerability of different cell types (Khazipov et al., 1995) and areas of the brain (Zhao & Flavin, 2000) adds further complexity to deciphering the effects of focal lesions. Here, we characterized functional changes in optogenetically activated layer 5 neurons, a cell population responsible for motor output from cortex (Anderson et al., 2010). We addressed the differential effects of ischemia on cortical and muscular excitability with the spatial resolution to identify regional changes. The ischemic core, defined as the area with an ჼ80% reduction blood flow, was restricted to a portion of forelimb motor cortex; however, the functional deficit was apparent at all stimulated sites within motor maps. The infarct core was surrounded by an evolving and substantial amount of hypoperfused tissue. Over time, this developing penumbra expanded across motor cortex. Although the pyramidal tract is unexcitable during proximal middle cerebral occlusion (Hayrunnisa Bolay, Gürsoy-Özdemir, Ünal, & 	   	   37	  Dalkara, 2000), a smaller ischemic region was sufficient to cause a deficit in motor output. The pervasive effect of a small ischemic insult, notably also influencing remote areas of motor cortex during early stages of stroke, is suggestive of a unique vulnerability in the motor system. 	  The nature of the ChR2 electrophysiological signal  B6.Cg-Tg (Thy1-COP4/EYFP)18Gfng/J mice express ChR2 predominantly in layer5 pyramidal neurons (Ayling et al., 2009). Photoactivation of ChR2 leads to opening of cation channels; the resulting current leads to firing of action potentials (Boyden et al., 2005; Nagel et al., 2003). ChR2-evoked EEG responses may in part reflect action potentials; being depressed after application of the sodium channel blocker TTX while being insensitive to glutamate receptor antagonists (Chen et al. 2012). We treat the initial ChR2-evoked EEG response as a measure of neuronal excitability that reflects a combination of action potentials, photocurrent, from the initial 600-900µm area of activation (Ayling et al., 2009) and potentially widespread synaptic activity evoked by local stimulation (Lim et al., 2012). Maintenance of this photocurrent infers that cells have a viable membrane potential. Within layer 5, there is a high density of intracortical projection axons; these connections are likely involved in mediating complex movements by recruiting multiple areas of the motor cortex (Capaday et al., 2009; Schneider et al., 2002).  Photostimulation of layer 5 neurons can evoke motor responses (Ayling et al., 2009; Hira et al., 2009) and blocking intracortical glutamatergic synaptic transmission does not significantly affect gross motor output (Ayling et al., 2009).  This suggests that within the scope of our stimulation parameters, motor output is likely largely independent of local recurrent synaptic connections. Spinal and muscular responses measured during 38	  light based motor mapping are likely contributed to predominantly by activity of the corticospinal tract. This is due to the large number of corticospinal axons compared to other tracts (Lemon, 2008; Lemon & Griffiths, 2005).  Additionally, due to the short latency of ChR2 evoked spinal responses (Fig 5), it is unlikely that complex circuits, for example ones which operate under di-synaptic mechanisms that include inhibition/disinhibition and are integral in modulation of movements, such as the basal ganglia, are recruited by direct excitation of Betz cells. Similar logic can be applied to other long range circuits, such as those relayed from the cortex via the pons.	  Automated optogenetic motor mapping (Ayling et al., 2009; Hira et al., 2009) allows for repeated fast acquisition of a cortical motor representation of the forelimb and averaged evoked EEG responses that reflect aspects of cortical neuronal excitability. Electromyographic (EMG) recordings in the contralateral forelimb, recordings from the dorsal column of the spinal cord, and ChR2-evoked cortical electroencephalographic (EEG) recordings provide simultaneous assessment of cortical neuronal and spinal excitability while evaluating motor output in response to cortical ChR2 activation. As each of these electrophysiological measurements is based on different but dependent physiological processes, this approach serves to assay selective vulnerabilities within the motor system. We addressed the differential effects of ischemia on cortical and muscular excitability with the spatial resolution to identify regional changes in these measures. Contrasting measures of neuronal, spinal, and muscular activity facilitates study of the effect of stroke on the electrical viability of excitatory cortical neurons, their effectiveness in generating and disseminating efferent signals, and their transmission to target muscles (Anenberg et al., 2014).  	   	   39	   Neuronal excitability is less affected than spinal potentials and motor output after stroke  Preservation of neuronal excitability is vital for stroke recovery (Carmichael, 2012; Clarkson & Carmichael, 2009). Neurons that surround the irreversibly damaged infarct core (Lo, 2008) can maintain their excitability (K.-A. Hossmann, 1971; K. A. Hossmann, 1994; Symon et al., 1977)  are the medium of functional remapping (Murphy & Corbett, 2009). Neurons within hypoperfused tissue can have intact membrane potentials while being unable to fire sensation-evoked action potentials. By directly depolarizing neurons with photoactivation of ChR2, we revealed that neurons in motor areas subjected to ministrokes are still excitable by averaged EEG yet fail to produce muscle excitation. Our results suggest that cortical excitability may not always be a good predictor of the effects detected within target muscles.  Previous work mapping the spatial relationship between ministroke induced changes in cortical microcirculation and deficits in sensation-evoked responses highlighted the vulnerability of cortical processing (Zhang & Murphy, 2007). Deficits within sensory responses are likely due to depression of local synaptic transmission (Hershkowitz et al., 1993; Rosen & Morris, 1993; Shih et al., 2013), potentially overshadowing other deficits such as impaired axonal conduction (H Bolay & Dalkara, 1998) which is pertinent to efferent motor processes. By directly depolarizing neurons through photoactivation of ChR2, we revealed that synaptically silent neurons in sensory-motor areas are excitable, but display a deficit in transmitting motor output to muscles. We also demonstrate a deficit in ChR2-evoked delayed cortical responses (500 ms after photostimulation) as observed with EEG (Fig. 4C). This may represent a deficit in 40	  cortical processing dependent on synaptic transmission. Although ischemia induced deficits in synaptic transmission are considered to be detrimental (Hofmeijer & van Putten, 2012), the synaptic terminals required to make motor maps (Ayling et al., 2009) are remote from the sites of cortical ischemia (Lemon, 2008). This suggests vulnerability of processes in addition to cortical synaptic transmission may be responsible for the observed depression of motor maps (Fig. 3) which previous work suggests are evoked largely independently from local synaptic transmission (Ayling et al., 2009). Preservation of ChR2-evoked EEG cortical responses after stroke suggests that cortical neurons have a hyperpolarized membrane potential (Chen et al., 2012). Attenuation of cortically evoked spinal responses was observed (Fig. 6D,E) despite the maintenance of cortical excitability. The profound suppression of EMGs in response to ministrokes may be the result of signal propagation failure within projection axons that may be vulnerable to ischemia (Enright et al., 2007; Nukada & Dyck, 1987; Schafer et al., 2009). This supports the notion that deficits in transmission upstream from synaptic terminals (Hofmeijer and Van Putten, 2012) may be key contributors to forms of ischemic damage (Hinman, Rasband, & Carmichael, 2013). Perhaps experiments using imaging of spiking dependent florescence (Gcamp) from retrograde stimulation could resolve whether this mechanism is indictable for the observed effect. Only relatively minor deficits in ChR2-evoked EEG responses were detected in this ministroke model (Fig. 5B). We caution that the observation of maintained cortical excitability (evoked by photostimulation of ChR2) may not directly reflect action potential propagation in layer 5 neurons. Our recordings within brain and spinal cord are consistent with maintenance of cortical excitability and progressive failure within 	   	   41	  descending spinal circuits; however, this approach does not cellular resolution to directly assess this. A reduction in basal excitatory synaptic transmission onto inhibitory cells can indirectly increase neuronal excitability (Khazipov et al., 1995). Alternatively, increased inhibition after stroke may be contributing to deficits in motor output (Clarkson et al., 2010), perhaps being responsible for altered motor excitation thresholds (Hayrunnisa Bolay et al., 2000). These scenarios can lead to selective deficits in motor function, but may not necessarily be reflected in overall excitability (Capaday, 2004; Clarkson et al., 2010). It is likely that several factors contributed to the observed functional deficit. This may reflect a stroke-induced “stunned brain state” (Hinman et al., 2013) where inhibitory and excitatory connections undergo different levels of dysfunction (J. H. Wang, 2003).  Following focal ministroke, we use ChR2 to directly stimulate cortex and observe preserved cortical EEG activity, but significantly greater depression of motor output. These findings suggest caution when using measures of cortical activity as predictors of functional outcome following stroke.  The integrity of ChR2-evoked EEG responses in the presence of a significant deficit in spinal, and muscular responses demonstrates a deficit in serial processing that occurs downstream of the induced photocurrent. There is a nonlinear relationship between intensity of excitation delivered to motor cortex and muscle activation; thus, small changes in cortical excitability can have large effects on motor output when gated by spinal circuitry. Although this may be beneficial for motor control, here we report the severe consequence of a minor alteration in cortical excitability on descending spinal potentials and motor output (Fig. 7).   42	   	   Fig 7. Summary diagram showing selective breakdown of poststroke motor cortex output.  A, Neurons within motor cortex project along the dorsal column of the spinal cord and synapse ventrally eventually reaching a lower motor neuron and the musculature. B, Effects of ministroke on motor output. The stroke core is represented with black shading with hypoperfused tissue in gray. These neurons are still excitable (when measured with the ChR2-evoked averaged EEG response); however, there is a cumulative depression in the motor signal, with an attenuated signal in the spinal cord and a severe depression in motor output as detected in the muscle. These motor deficits were detectable when stimulation was targeted to cortical areas over a millimeter away from the stroke core, whereas depressed neuronal excitability was only detected at the stroke core.  	   	   43	   	  Implications and future directions (conclusions) 	   Focal stroke imparts remote and differential effects on neuronal, spinal, and muscular excitability within the initial hours after stroke. How the integrity of these electrophysiological responses evolves as cell death and cortical rewiring progresses over weeks following stroke is an active area of research (Benowitz & Carmichael, 2010; Dancause & Nudo, 2011; Kleim et al., 2002; Teskey, Flynn, Goertzen, Monfils, & Young, 2003). Motor function can recover over 6–8 weeks from ischemia targeted to areas of similar or larger size as studied here in mouse motor cortex (Harrison et al., 2012).  Part of this resilience may be due to effects outlined here, where cortical excitability is relatively preserved, facilitating plasticity (Murphy & Corbett, 2009). With bilateral optogenetic motor mapping techniques (Silasi, Boyd, LeDue, & Murphy, 2013) the effect of this stroke on the opposing hemisphere could be explored, perhaps contributing to our current understanding of the role of interhemispheric inhibition in affecting stroke induced alterations in both neuronal excitability and motor output.  The observed electrophysiological deficits highlight the importance of addressing white matter integrity in addition to ischemia induced deficits in synaptic transmission. Non-invasive and repeatable optogenetic motor mapping can be used as a platform to test the effect of different stroke models on neuronal excitability and motor output; additionally, the efficacy of therapeutics on preserving or recovery of these measures could be assayed. This technique also has applications beyond the scope of stroke research. The clear connection between cortical stimulation and spinal recordings inspired ongoing 44	  collaborative work where motor representations were mapped after spinal injuries (Hilton et al. 2014).  The finding that cortical excitability (measured here as the ChR2 evoked EEG response) is maintained during ischemia, paired with knowledge that local glutamatergic synaptic transmission is not required for light induced neurovascular coupling (Scott & Murphy, 2012) may be relevant for dissecting mechanisms for  acute stimulation based stroke interventions (Burnett et al., 2006) which can lead to full recovery (Davis, Lay, Chen-Bee, & Frostig, 2011; Lay, Davis, Chen-Bee, & Frostig, 2010, 2011).  We expect that optogenetic stimulation and blood flow imaging tools will be instrumental in the mechanistic study of neurovascular coupling during ischemia.                  	   	   45	  References   Alaverdashvili,	  M.,	  &	  Whishaw,	  I.	  Q.	  (2010).	  Compensation	  aids	  skilled	  reaching	  in	  aging	  and	  in	  recovery	  from	  forelimb	  motor	  cortex	  stroke	  in	  the	  rat.	  Neuroscience,	  167(1),	  21–30.	  doi:10.1016/j.neuroscience.2010.02.001	  Anderson,	  C.	  T.,	  Sheets,	  P.	  L.,	  Kiritani,	  T.,	  &	  Shepherd,	  G.	  M.	  G.	  (2010).	  Sublayer-­‐specific	  microcircuits	  of	  corticospinal	  and	  corticostriatal	  neurons	  in	  motor	  cortex.	  Nature	  Neuroscience,	  13(6),	  739–744.	  doi:10.1038/nn.2538	  Anenberg,	  E.,	  Arstikaitis,	  P.,	  Niitsu,	  Y.,	  Harrison,	  T.	  C.,	  Boyd,	  J.	  D.,	  Hilton,	  B.	  J.,	  …	  Murphy,	  T.	  H.	  (2014).	  Ministrokes	  in	  channelrhodopsin-­‐2	  transgenic	  mice	  reveal	  widespread	  deficits	  in	  motor	  output	  despite	  maintenance	  of	  cortical	  neuronal	  excitability.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  34(4),	  1094–1104.	  doi:10.1523/JNEUROSCI.1442-­‐13.2014	  Arenkiel,	  B.	  R.,	  Peca,	  J.,	  Davison,	  I.	  G.,	  Feliciano,	  C.,	  Deisseroth,	  K.,	  Augustine,	  G.	  J.,	  …	  Feng,	  G.	  (2007).	  In	  vivo	  light-­‐induced	  activation	  of	  neural	  circuitry	  in	  transgenic	  mice	  expressing	  channelrhodopsin-­‐2.	  Neuron,	  54(2),	  205–218.	  doi:10.1016/j.neuron.2007.03.005	  Asanuma,	  H.,	  &	  Rosén,	  I.	  (1972).	  Topographical	  organization	  of	  cortical	  efferent	  zones	  projecting	  to	  distal	  forelimb	  muscles	  in	  the	  monkey.	  Experimental	  Brain	  Research,	  14(3),	  243–256.	  Astrup,	  J.,	  Siesjö,	  B.	  K.,	  &	  Symon,	  L.	  (1981).	  Thresholds	  in	  cerebral	  ischemia	  -­‐	  the	  ischemic	  penumbra.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  12(6),	  723–725.	  46	  Astrup,	  J.,	  Symon,	  L.,	  Branston,	  N.	  M.,	  &	  Lassen,	  N.	  A.	  (1977).	  Cortical	  evoked	  potential	  and	  extracellular	  K+	  and	  H+	  at	  critical	  levels	  of	  brain	  ischemia.	  Stroke,	  8(1),	  51–57.	  doi:10.1161/01.STR.8.1.51	  Attwell,	  D.,	  &	  Laughlin,	  S.	  B.	  (2001).	  An	  energy	  budget	  for	  signaling	  in	  the	  grey	  matter	  of	  the	  brain.	  Journal	  of	  Cerebral	  Blood	  Flow	  and	  Metabolism:	  Official	  Journal	  of	  the	  International	  Society	  of	  Cerebral	  Blood	  Flow	  and	  Metabolism,	  21(10),	  1133–1145.	  doi:10.1097/00004647-­‐200110000-­‐00001	  Ayling,	  O.	  G.	  S.,	  Harrison,	  T.	  C.,	  Boyd,	  J.	  D.,	  Goroshkov,	  A.,	  &	  Murphy,	  T.	  H.	  (2009).	  Automated	  light-­‐based	  mapping	  of	  motor	  cortex	  by	  photoactivation	  of	  channelrhodopsin-­‐2	  transgenic	  mice.	  Nature	  Methods,	  6(3),	  219–224.	  doi:10.1038/nmeth.1303	  Baron,	  J.	  C.	  (1999).	  Mapping	  the	  ischaemic	  penumbra	  with	  PET:	  implications	  for	  acute	  stroke	  treatment.	  Cerebrovascular	  Diseases	  (Basel,	  Switzerland),	  9(4),	  193–201.	  doi:15955	  Baron,	  J.	  C.	  (2001).	  Perfusion	  thresholds	  in	  human	  cerebral	  ischemia:	  historical	  perspective	  and	  therapeutic	  implications.	  Cerebrovascular	  Diseases	  (Basel,	  Switzerland),	  11	  Suppl	  1,	  2–8.	  doi:49119	  Baron,	  J.	  C.,	  &	  Marchal,	  G.	  (1999).	  Ischemic	  core	  and	  penumbra	  in	  human	  stroke.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  30(5),	  1150–1153.	  Benowitz,	  L.	  I.,	  &	  Carmichael,	  S.	  T.	  (2010).	  Promoting	  axonal	  rewiring	  to	  improve	  outcome	  after	  stroke.	  Neurobiology	  of	  Disease,	  37(2),	  259–266.	  doi:10.1016/j.nbd.2009.11.009	  	   	   47	  Blinder,	  P.,	  Tsai,	  P.	  S.,	  Kaufhold,	  J.	  P.,	  Knutsen,	  P.	  M.,	  Suhl,	  H.,	  &	  Kleinfeld,	  D.	  (2013).	  The	  cortical	  angiome:	  an	  interconnected	  vascular	  network	  with	  noncolumnar	  patterns	  of	  blood	  flow.	  Nature	  Neuroscience,	  16(7),	  889–897.	  doi:10.1038/nn.3426	  Bolay,	  H.,	  &	  Dalkara,	  T.	  (1998).	  Mechanisms	  of	  motor	  dysfunction	  after	  transient	  MCA	  occlusion:	  persistent	  transmission	  failure	  in	  cortical	  synapses	  is	  a	  major	  determinant.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  29(9),	  1988–1993;	  discussion	  1994.	  Bolay,	  H.,	  Gürsoy-­‐Özdemir,	  Y.,	  Ünal,	  I.,	  &	  Dalkara,	  T.	  (2000).	  Altered	  mechanisms	  of	  motor-­‐evoked	  potential	  generation	  after	  transient	  focal	  cerebral	  ischemia	  in	  the	  rat:	  implications	  for	  transcranial	  magnetic	  stimulation.	  Brain	  Research,	  873(1),	  26–33.	  doi:10.1016/S0006-­‐8993(00)02466-­‐5	  Boyden,	  E.	  S.,	  Zhang,	  F.,	  Bamberg,	  E.,	  Nagel,	  G.,	  &	  Deisseroth,	  K.	  (2005).	  Millisecond-­‐timescale,	  genetically	  targeted	  optical	  control	  of	  neural	  activity.	  Nature	  Neuroscience,	  8(9),	  1263–1268.	  doi:10.1038/nn1525	  Buchkremer-­‐Ratzmann,	  I.,	  August,	  M.,	  Hagemann,	  G.,	  &	  Witte,	  O.	  W.	  (1996).	  Electrophysiological	  Transcortical	  Diaschisis	  After	  Cortical	  Photothrombosis	  in	  Rat	  Brain.	  Stroke,	  27(6),	  1105–1111.	  doi:10.1161/01.STR.27.6.1105	  Burnett,	  M.	  G.,	  Shimazu,	  T.,	  Szabados,	  T.,	  Muramatsu,	  H.,	  Detre,	  J.	  A.,	  &	  Greenberg,	  J.	  H.	  (2006).	  Electrical	  forepaw	  stimulation	  during	  reversible	  forebrain	  ischemia	  decreases	  infarct	  volume.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  37(5),	  1327–1331.	  doi:10.1161/01.STR.0000217305.82123.d8	  48	  Bütefisch,	  C.	  M.,	  Netz,	  J.,	  Weßling,	  M.,	  Seitz,	  R.	  J.,	  &	  Hömberg,	  V.	  (2003).	  Remote	  changes	  in	  cortical	  excitability	  after	  stroke.	  Brain,	  126(2),	  470–481.	  doi:10.1093/brain/awg044	  Capaday,	  C.	  (2004).	  The	  Integrated	  Nature	  of	  Motor	  Cortical	  Function.	  The	  Neuroscientist,	  10(3),	  207–220.	  doi:10.1177/107385403262109	  Capaday,	  C.,	  Ethier,	  C.,	  Brizzi,	  L.,	  Sik,	  A.,	  van	  Vreeswijk,	  C.,	  &	  Gingras,	  D.	  (2009).	  On	  the	  nature	  of	  the	  intrinsic	  connectivity	  of	  the	  cat	  motor	  cortex:	  evidence	  for	  a	  recurrent	  neural	  network	  topology.	  Journal	  of	  Neurophysiology,	  102(4),	  2131–2141.	  doi:10.1152/jn.91319.2008	  Carmichael,	  S.	  T.	  (2012).	  Brain	  excitability	  in	  stroke:	  the	  yin	  and	  yang	  of	  stroke	  progression.	  Archives	  of	  Neurology,	  69(2),	  161–167.	  doi:10.1001/archneurol.2011.1175	  Carter,	  A.	  R.,	  Shulman,	  G.	  L.,	  &	  Corbetta,	  M.	  (2012).	  Why	  use	  a	  connectivity-­‐based	  approach	  to	  study	  stroke	  and	  recovery	  of	  function?	  NeuroImage,	  62(4),	  2271–2280.	  doi:10.1016/j.neuroimage.2012.02.070	  Castro-­‐Alamancos,	  M.	  A.,	  &	  Borrel,	  J.	  (1995).	  Functional	  recovery	  of	  forelimb	  response	  capacity	  after	  forelimb	  primary	  motor	  cortex	  damage	  in	  the	  rat	  is	  due	  to	  the	  reorganization	  of	  adjacent	  areas	  of	  cortex.	  Neuroscience,	  68(3),	  793–805.	  Cheng,	  H.,	  Luo,	  Q.,	  Wang,	  Z.,	  Gong,	  H.,	  Chen,	  S.,	  Liang,	  W.,	  &	  Zeng,	  S.	  (2003).	  Efficient	  Characterization	  of	  Regional	  Mesenteric	  Blood	  Flow	  by	  Use	  of	  Laser	  Speckle	  Imaging.	  Applied	  Optics,	  42(28),	  5759–5764.	  doi:10.1364/AO.42.005759	  	   	   49	  Chen,	  S.,	  Mohajerani,	  M.	  H.,	  Xie,	  Y.,	  &	  Murphy,	  T.	  H.	  (2012).	  Optogenetic	  analysis	  of	  neuronal	  excitability	  during	  global	  ischemia	  reveals	  selective	  deficits	  in	  sensory	  processing	  following	  reperfusion	  in	  mouse	  cortex.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  32(39),	  13510–13519.	  doi:10.1523/JNEUROSCI.1439-­‐12.2012	  Clarkson,	  A.	  N.,	  &	  Carmichael,	  S.	  T.	  (2009).	  Cortical	  excitability	  and	  post-­‐stroke	  recovery.	  Biochemical	  Society	  Transactions,	  37(Pt	  6),	  1412–1414.	  doi:10.1042/BST0371412	  Clarkson,	  A.	  N.,	  Huang,	  B.	  S.,	  Macisaac,	  S.	  E.,	  Mody,	  I.,	  &	  Carmichael,	  S.	  T.	  (2010).	  Reducing	  excessive	  GABA-­‐mediated	  tonic	  inhibition	  promotes	  functional	  recovery	  after	  stroke.	  Nature,	  468(7321),	  305–309.	  doi:10.1038/nature09511	  Cook,	  D.	  J.,	  Teves,	  L.,	  &	  Tymianski,	  M.	  (2012).	  Treatment	  of	  stroke	  with	  a	  PSD-­‐95	  inhibitor	  in	  the	  gyrencephalic	  primate	  brain.	  Nature,	  483(7388),	  213–217.	  doi:10.1038/nature10841	  Cramer,	  S.	  C.,	  Shah,	  R.,	  Juranek,	  J.,	  Crafton,	  K.	  R.,	  &	  Le,	  V.	  (2006).	  Activity	  in	  the	  peri-­‐infarct	  rim	  in	  relation	  to	  recovery	  from	  stroke.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  37(1),	  111–115.	  doi:10.1161/01.STR.0000195135.70379.1f	  Dancause,	  N.,	  Barbay,	  S.,	  Frost,	  S.	  B.,	  Plautz,	  E.	  J.,	  Chen,	  D.,	  Zoubina,	  E.	  V.,	  …	  Nudo,	  R.	  J.	  (2005).	  Extensive	  cortical	  rewiring	  after	  brain	  injury.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  25(44),	  10167–10179.	  doi:10.1523/JNEUROSCI.3256-­‐05.2005	  50	  Dancause,	  N.,	  &	  Nudo,	  R.	  J.	  (2011).	  Shaping	  plasticity	  to	  enhance	  recovery	  after	  injury.	  Progress	  in	  Brain	  Research,	  192,	  273–295.	  doi:10.1016/B978-­‐0-­‐444-­‐53355-­‐5.00015-­‐4	  Davis,	  M.	  F.,	  Lay,	  C.	  C.,	  Chen-­‐Bee,	  C.	  H.,	  &	  Frostig,	  R.	  D.	  (2011).	  Amount	  but	  not	  pattern	  of	  protective	  sensory	  stimulation	  alters	  recovery	  after	  permanent	  middle	  cerebral	  artery	  occlusion.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  42(3),	  792–798.	  doi:10.1161/STROKEAHA.110.607135	  Dirnagl,	  U.,	  Niwa,	  K.,	  Sixt,	  G.,	  &	  Villringer,	  A.	  (1994).	  Cortical	  hypoperfusion	  after	  global	  forebrain	  ischemia	  in	  rats	  is	  not	  caused	  by	  microvascular	  leukocyte	  plugging.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  25(5),	  1028–1038.	  Donoghue,	  J.	  P.,	  &	  Sanes,	  J.	  N.	  (1987).	  Peripheral	  nerve	  injury	  in	  developing	  rats	  reorganizes	  representation	  pattern	  in	  motor	  cortex.	  Proceedings	  of	  the	  National	  Academy	  of	  Sciences,	  84(4),	  1123–1126.	  Dunn,	  A.	  K.,	  Bolay,	  H.,	  Moskowitz,	  M.	  A.,	  &	  Boas,	  D.	  A.	  (2001).	  Dynamic	  Imaging	  of	  Cerebral	  Blood	  Flow	  Using	  Laser	  Speckle.	  Journal	  of	  Cerebral	  Blood	  Flow	  &	  Metabolism,	  21(3),	  195–201.	  doi:10.1097/00004647-­‐200103000-­‐00002	  Enright,	  L.	  E.,	  Zhang,	  S.,	  &	  Murphy,	  T.	  H.	  (2007).	  Fine	  mapping	  of	  the	  spatial	  relationship	  between	  acute	  ischemia	  and	  dendritic	  structure	  indicates	  selective	  vulnerability	  of	  layer	  V	  neuron	  dendritic	  tufts	  within	  single	  neurons	  in	  vivo.	  Journal	  of	  Cerebral	  Blood	  Flow	  and	  Metabolism:	  Official	  Journal	  of	  the	  International	  Society	  of	  Cerebral	  Blood	  Flow	  and	  Metabolism,	  27(6),	  1185–1200.	  doi:10.1038/sj.jcbfm.9600428	  	   	   51	  Frostig,	  R.	  D.,	  Lay,	  C.	  C.,	  &	  Davis,	  M.	  F.	  (2013).	  A	  rat’s	  whiskers	  point	  the	  way	  toward	  a	  novel	  stimulus-­‐dependent,	  protective	  stroke	  therapy.	  The	  Neuroscientist:	  A	  Review	  Journal	  Bringing	  Neurobiology,	  Neurology	  and	  Psychiatry,	  19(3),	  313–328.	  doi:10.1177/1073858412462607	  Gao,	  T.	  M.,	  Howard,	  E.	  M.,	  &	  Xu,	  Z.	  C.	  (1998).	  Transient	  neurophysiological	  changes	  in	  CA3	  neurons	  and	  dentate	  granule	  cells	  after	  severe	  forebrain	  ischemia	  in	  vivo.	  Journal	  of	  Neurophysiology,	  80(6),	  2860–2869.	  Georgopoulos,	  A.	  P.,	  Kalaska,	  J.	  F.,	  Caminiti,	  R.,	  &	  Massey,	  J.	  T.	  (1982).	  On	  the	  relations	  between	  the	  direction	  of	  two-­‐dimensional	  arm	  movements	  and	  cell	  discharge	  in	  primate	  motor	  cortex.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  2(11),	  1527–1537.	  Harrison,	  T.	  C.,	  Ayling,	  O.	  G.	  S.,	  &	  Murphy,	  T.	  H.	  (2012).	  Distinct	  cortical	  circuit	  mechanisms	  for	  complex	  forelimb	  movement	  and	  motor	  map	  topography.	  Neuron,	  74(2),	  397–409.	  doi:10.1016/j.neuron.2012.02.028	  Harrison,	  T.	  C.,	  &	  Murphy,	  T.	  H.	  (2012).	  Towards	  a	  circuit	  mechanism	  for	  movement	  tuning	  in	  motor	  cortex.	  Frontiers	  in	  Neural	  Circuits,	  6,	  127.	  doi:10.3389/fncir.2012.00127	  Harrison,	  T.	  C.,	  Sigler,	  A.,	  &	  Murphy,	  T.	  H.	  (2009).	  Simple	  and	  cost-­‐effective	  hardware	  and	  software	  for	  functional	  brain	  mapping	  using	  intrinsic	  optical	  signal	  imaging.	  Journal	  of	  Neuroscience	  Methods,	  182(2),	  211–218.	  doi:10.1016/j.jneumeth.2009.06.021	  52	  Heiss,	  W.-­‐D.	  (2000).	  Ischemic	  Penumbra:	  Evidence	  From	  Functional	  Imaging	  in	  Man.	  Journal	  of	  Cerebral	  Blood	  Flow	  &	  Metabolism,	  20(9),	  1276–1293.	  doi:10.1097/00004647-­‐200009000-­‐00002	  Hershkowitz,	  N.,	  Katchman,	  A.	  N.,	  &	  Veregge,	  S.	  (1993).	  Site	  of	  synaptic	  depression	  during	  hypoxia:	  a	  patch-­‐clamp	  analysis.	  Journal	  of	  Neurophysiology,	  69(2),	  432–441.	  Hill,	  M.	  D.,	  Martin,	  R.	  H.,	  Mikulis,	  D.,	  Wong,	  J.	  H.,	  Silver,	  F.	  L.,	  Terbrugge,	  K.	  G.,	  …	  Tymianski,	  M.	  (2012).	  Safety	  and	  efficacy	  of	  NA-­‐1	  in	  patients	  with	  iatrogenic	  stroke	  after	  endovascular	  aneurysm	  repair	  (ENACT):	  a	  phase	  2,	  randomised,	  double-­‐blind,	  placebo-­‐controlled	  trial.	  Lancet	  Neurology,	  11(11),	  942–950.	  doi:10.1016/S1474-­‐4422(12)70225-­‐9	  B.J.	  Hilton,	  E.	  Anenberg,	  T.C.	  Harrison,	  J.D.	  Boyd,	  T.H.	  Murphy,	  W.	  Tetzlaff	  (2014).	  Motor	  cortical	  plasticity	  following	  spinal	  cord	  injury	  revealed	  optogenetically.	  ICORD	  Annual	  Meeting,	  Vancouver,	  BC,	  CANADA.	  	  	  Hinman,	  J.	  D.,	  Rasband,	  M.	  N.,	  &	  Carmichael,	  S.	  T.	  (2013).	  Remodeling	  of	  the	  axon	  initial	  segment	  after	  focal	  cortical	  and	  white	  matter	  stroke.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  44(1),	  182–189.	  doi:10.1161/STROKEAHA.112.668749	  Hira,	  R.,	  Honkura,	  N.,	  Noguchi,	  J.,	  Maruyama,	  Y.,	  Augustine,	  G.	  J.,	  Kasai,	  H.,	  &	  Matsuzaki,	  M.	  (2009).	  Transcranial	  optogenetic	  stimulation	  for	  functional	  mapping	  of	  the	  motor	  cortex.	  Journal	  of	  Neuroscience	  Methods,	  179(2),	  258–263.	  doi:10.1016/j.jneumeth.2009.02.001	  	   	   53	  Hofmeijer,	  J.,	  &	  van	  Putten,	  M.	  J.	  A.	  M.	  (2012).	  Ischemic	  cerebral	  damage:	  an	  appraisal	  of	  synaptic	  failure.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  43(2),	  607–615.	  doi:10.1161/STROKEAHA.111.632943	  Horner,	  C.	  H.,	  Davies,	  H.	  A.,	  &	  Stewart,	  M.	  G.	  (1998).	  Hippocampal	  synaptic	  density	  and	  glutamate	  immunoreactivity	  following	  transient	  cerebral	  ischaemia	  in	  the	  chick.	  European	  Journal	  of	  Neuroscience,	  10(12),	  3913–3917.	  doi:10.1046/j.1460-­‐9568.1998.00435.x	  Hossmann,	  K.-­‐A.	  (1971).	  Cortical	  steady	  potential,	  impedance	  and	  excitability	  changes	  during	  and	  after	  total	  ischemia	  of	  cat	  brain.	  Experimental	  Neurology,	  32(2),	  163–175.	  doi:10.1016/0014-­‐4886(71)90060-­‐4	  Hossmann,	  K.	  A.	  (1994).	  Viability	  thresholds	  and	  the	  penumbra	  of	  focal	  ischemia.	  Annals	  of	  Neurology,	  36(4),	  557–565.	  doi:10.1002/ana.410360404	  Johansen,	  F.	  F.,	  Balslev	  Jørgensen,	  M.,	  Ekström	  Von	  Lubitz,	  D.	  K.	  J.,	  &	  Diemer,	  N.	  H.	  (1984).	  Selective	  dendrite	  damage	  in	  hippocampal	  CA1	  stratum	  radiatum	  with	  unchanged	  axon	  ultrastructure	  and	  glutamate	  uptake	  after	  transient	  cerebral	  ischaemia	  in	  the	  rat.	  Brain	  Research,	  291(2),	  373–377.	  doi:10.1016/0006-­‐8993(84)91272-­‐1	  Kalaska,	  J.	  F.,	  Cohen,	  D.	  A.,	  Hyde,	  M.	  L.,	  &	  Prud’homme,	  M.	  (1989).	  A	  comparison	  of	  movement	  direction-­‐related	  versus	  load	  direction-­‐related	  activity	  in	  primate	  motor	  cortex,	  using	  a	  two-­‐dimensional	  reaching	  task.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  9(6),	  2080–2102.	  54	  Kerr,	  A.	  L.,	  Cheng,	  S.-­‐Y.,	  &	  Jones,	  T.	  A.	  (2011).	  Experience-­‐dependent	  neural	  plasticity	  in	  the	  adult	  damaged	  brain.	  Journal	  of	  Communication	  Disorders,	  44(5),	  538–548.	  doi:10.1016/j.jcomdis.2011.04.011	  Khazipov,	  R.,	  Congar,	  P.,	  &	  Ben-­‐Ari,	  Y.	  (1995).	  Hippocampal	  CA1	  lacunosum-­‐moleculare	  interneurons:	  comparison	  of	  effects	  of	  anoxia	  on	  excitatory	  and	  inhibitory	  postsynaptic	  currents.	  Journal	  of	  Neurophysiology,	  74(5),	  2138–2149.	  Kiritani,	  T.,	  Wickersham,	  I.	  R.,	  Seung,	  H.	  S.,	  &	  Shepherd,	  G.	  M.	  G.	  (2012).	  Hierarchical	  connectivity	  and	  connection-­‐specific	  dynamics	  in	  the	  corticospinal-­‐corticostriatal	  microcircuit	  in	  mouse	  motor	  cortex.	  The	  Journal	  of	  Neuroscience,	  32(14),	  4992–5001.	  doi:10.1523/JNEUROSCI.4759-­‐11.2012	  Kleim,	  J.	  A.,	  Barbay,	  S.,	  Cooper,	  N.	  R.,	  Hogg,	  T.	  M.,	  Reidel,	  C.	  N.,	  Remple,	  M.	  S.,	  &	  Nudo,	  R.	  J.	  (2002).	  Motor	  learning-­‐dependent	  synaptogenesis	  is	  localized	  to	  functionally	  reorganized	  motor	  cortex.	  Neurobiology	  of	  Learning	  and	  Memory,	  77(1),	  63–77.	  doi:10.1006/nlme.2000.4004	  Kleim,	  J.	  A.,	  Barbay,	  S.,	  &	  Nudo,	  R.	  J.	  (1998).	  Functional	  Reorganization	  of	  the	  Rat	  Motor	  Cortex	  Following	  Motor	  Skill	  Learning.	  Journal	  of	  Neurophysiology,	  80(6),	  3321–3325.	  Lashley,	  K.	  S.	  (1931).	  MASS	  ACTION	  IN	  CEREBRAL	  FUNCTION.	  Science	  (New	  York,	  N.Y.),	  73(1888),	  245–254.	  doi:10.1126/science.73.1888.245	  Lay,	  C.	  C.,	  Davis,	  M.	  F.,	  Chen-­‐Bee,	  C.	  H.,	  &	  Frostig,	  R.	  D.	  (2010).	  Mild	  sensory	  stimulation	  completely	  protects	  the	  adult	  rodent	  cortex	  from	  ischemic	  stroke.	  PloS	  One,	  5(6),	  e11270.	  doi:10.1371/journal.pone.0011270	  	   	   55	  Lay,	  C.	  C.,	  Davis,	  M.	  F.,	  Chen-­‐Bee,	  C.	  H.,	  &	  Frostig,	  R.	  D.	  (2011).	  Mild	  sensory	  stimulation	  reestablishes	  cortical	  function	  during	  the	  acute	  phase	  of	  ischemia.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  31(32),	  11495–11504.	  doi:10.1523/JNEUROSCI.1741-­‐11.2011	  Lemon,	  R.	  N.	  (2008).	  Descending	  pathways	  in	  motor	  control.	  Annual	  Review	  of	  Neuroscience,	  31,	  195–218.	  doi:10.1146/annurev.neuro.31.060407.125547	  Lemon,	  R.	  N.,	  &	  Griffiths,	  J.	  (2005).	  Comparing	  the	  function	  of	  the	  corticospinal	  system	  in	  different	  species:	  organizational	  differences	  for	  motor	  specialization?	  Muscle	  &	  Nerve,	  32(3),	  261–279.	  doi:10.1002/mus.20333	  Levin,	  M.	  F.,	  Kleim,	  J.	  A.,	  &	  Wolf,	  S.	  L.	  (2009).	  What	  Do	  Motor	  “Recovery”	  and	  “Compensation”	  Mean	  in	  Patients	  Following	  Stroke?	  Neurorehabilitation	  and	  Neural	  Repair,	  23(4),	  313–319.	  doi:10.1177/1545968308328727	  Lim,	  D.	  H.,	  Mohajerani,	  M.	  H.,	  LeDue,	  J.,	  Boyd,	  J.,	  Chen,	  S.,	  &	  Murphy,	  T.	  H.	  (2012).	  In	  vivo	  large-­‐scale	  cortical	  mapping	  using	  channelrhodopsin-­‐2	  stimulation	  in	  transgenic	  mice	  reveals	  asymmetric	  and	  reciprocal	  relationships	  between	  cortical	  areas.	  Frontiers	  in	  Neural	  Circuits,	  6,	  11.	  doi:10.3389/fncir.2012.00011	  Lin,	  J.	  Y.,	  Knutsen,	  P.	  M.,	  Muller,	  A.,	  Kleinfeld,	  D.,	  &	  Tsien,	  R.	  Y.	  (2013).	  ReaChR:	  A	  red-­‐shifted	  variant	  of	  channelrhodopsin	  enables	  deep	  transcranial	  optogenetic	  excitation.	  Nature	  Neuroscience,	  16(10),	  1499–1508.	  doi:10.1038/nn.3502	  Lipton,	  S.	  A.,	  &	  Nicotera,	  P.	  (February).	  Calcium,	  free	  radicals	  and	  excitotoxins	  in	  neuronal	  apoptosis.	  Cell	  Calcium,	  23(2-­‐3),	  165–171.	  doi:10.1016/S0143-­‐4160(98)90115-­‐4	  56	  Li,	  Y.,	  Lei,	  Z.,	  &	  Xu,	  Z.	  C.	  (2009).	  Enhancement	  of	  inhibitory	  synaptic	  transmission	  in	  large	  aspiny	  neurons	  after	  transient	  cerebral	  ischemia.	  Neuroscience,	  159(2),	  670–681.	  doi:10.1016/j.neuroscience.2008.12.046	  Lo,	  E.	  H.	  (2008).	  A	  new	  penumbra:	  transitioning	  from	  injury	  into	  repair	  after	  stroke.	  Nature	  Medicine,	  14(5),	  497–500.	  doi:10.1038/nm1735	  Madisen,	  L.,	  Mao,	  T.,	  Koch,	  H.,	  Zhuo,	  J.,	  Berenyi,	  A.,	  Fujisawa,	  S.,	  …	  Zeng,	  H.	  (2012).	  A	  toolbox	  of	  Cre-­‐dependent	  optogenetic	  transgenic	  mice	  for	  light-­‐induced	  activation	  and	  silencing.	  Nature	  Neuroscience,	  15(5),	  793–802.	  doi:10.1038/nn.3078	  Mergenthaler,	  P.,	  Dirnagl,	  U.,	  &	  Meisel,	  A.	  (2004).	  Pathophysiology	  of	  stroke:	  lessons	  from	  animal	  models.	  Metabolic	  Brain	  Disease,	  19(3-­‐4),	  151–167.	  Mohajerani,	  M.	  H.,	  Aminoltejari,	  K.,	  &	  Murphy,	  T.	  H.	  (2011).	  Targeted	  mini-­‐strokes	  produce	  changes	  in	  interhemispheric	  sensory	  signal	  processing	  that	  are	  indicative	  of	  disinhibition	  within	  minutes.	  Proceedings	  of	  the	  National	  Academy	  of	  Sciences	  of	  the	  United	  States	  of	  America,	  108(22),	  E183–191.	  doi:10.1073/pnas.1101914108	  Mohajerani,	  M.	  H.,	  Chan,	  A.	  W.,	  Mohsenvand,	  M.,	  LeDue,	  J.,	  Liu,	  R.,	  McVea,	  D.	  A.,	  …	  Murphy,	  T.	  H.	  (2013).	  Spontaneous	  cortical	  activity	  alternates	  between	  motifs	  defined	  by	  regional	  axonal	  projections.	  Nature	  Neuroscience,	  16(10),	  1426–1435.	  doi:10.1038/nn.3499	  Moon,	  S.-­‐K.,	  Alaverdashvili,	  M.,	  Cross,	  A.	  R.,	  &	  Whishaw,	  I.	  Q.	  (n.d.).	  Both	  compensation	  and	  recovery	  of	  skilled	  reaching	  following	  small	  	   	   57	  photothrombotic	  stroke	  to	  motor	  cortex	  in	  the	  rat.	  Experimental	  Neurology,	  218(1),	  145–153.	  Murphy,	  T.	  H.,	  &	  Corbett,	  D.	  (2009).	  Plasticity	  during	  stroke	  recovery:	  from	  synapse	  to	  behaviour.	  Nature	  Reviews.	  Neuroscience,	  10(12),	  861–872.	  doi:10.1038/nrn2735	  Nagel,	  G.,	  Szellas,	  T.,	  Huhn,	  W.,	  Kateriya,	  S.,	  Adeishvili,	  N.,	  Berthold,	  P.,	  …	  Bamberg,	  E.	  (2003).	  Channelrhodopsin-­‐2,	  a	  directly	  light-­‐gated	  cation-­‐selective	  membrane	  channel.	  Proceedings	  of	  the	  National	  Academy	  of	  Sciences	  of	  the	  United	  States	  of	  America,	  100(24),	  13940–13945.	  doi:10.1073/pnas.1936192100	  Neumann-­‐Haefelin,	  T.,	  Wittsack,	  H.	  J.,	  Wenserski,	  F.,	  Siebler,	  M.,	  Seitz,	  R.	  J.,	  Mödder,	  U.,	  &	  Freund,	  H.	  J.	  (1999).	  Diffusion-­‐	  and	  perfusion-­‐weighted	  MRI.	  The	  DWI/PWI	  mismatch	  region	  in	  acute	  stroke.	  Stroke;	  a	  Journal	  of	  Cerebral	  Circulation,	  30(8),	  1591–1597.	  Nudo,	  R.	  J.	  (2006).	  Mechanisms	  for	  recovery	  of	  motor	  function	  following	  cortical	  damage.	  Current	  Opinion	  in	  Neurobiology,	  16(6),	  638–644.	  doi:10.1016/j.conb.2006.10.004	  Nudo,	  R.	  J.,	  Milliken,	  G.	  W.,	  Jenkins,	  W.	  M.,	  &	  Merzenich,	  M.	  M.	  (1996).	  Use-­‐dependent	  alterations	  of	  movement	  representations	  in	  primary	  motor	  cortex	  of	  adult	  squirrel	  monkeys.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  16(2),	  785–807.	  Nudo,	  R.	  J.,	  Plautz,	  E.	  J.,	  &	  Frost,	  S.	  B.	  (2001).	  Role	  of	  adaptive	  plasticity	  in	  recovery	  of	  function	  after	  damage	  to	  motor	  cortex.	  Muscle	  &	  Nerve,	  24(8),	  1000–1019.	  58	  Nudo,	  R.	  J.,	  Wise,	  B.	  M.,	  SiFuentes,	  F.,	  &	  Milliken,	  G.	  W.	  (1996).	  Neural	  substrates	  for	  the	  effects	  of	  rehabilitative	  training	  on	  motor	  recovery	  after	  ischemic	  infarct.	  Science	  (New	  York,	  N.Y.),	  272(5269),	  1791–1794.	  Nukada,	  H.,	  &	  Dyck,	  P.	  J.	  (1987).	  Acute	  ischemia	  causes	  axonal	  stasis,	  swelling,	  attenuation,	  and	  secondary	  demyelination.	  Annals	  of	  Neurology,	  22(3),	  311–318.	  doi:10.1002/ana.410220306	  Ohtsubo,	  J.,	  &	  Asakura,	  T.	  (1976).	  Velocity	  measurement	  of	  a	  diffuse	  object	  by	  using	  time-­‐varying	  speckles.	  Optical	  and	  Quantum	  Electronics,	  8(6),	  523–529.	  doi:10.1007/BF00620143	  Okamoto,	  Y.,	  Ihara,	  M.,	  Fujita,	  Y.,	  Ito,	  H.,	  Takahashi,	  R.,	  &	  Tomimoto,	  H.	  (2009).	  Cortical	  microinfarcts	  in	  Alzheimerʼs	  disease	  and	  subcortical	  vascular	  dementia:	  NeuroReport,	  20(11),	  990–996.	  doi:10.1097/WNR.0b013e32832d2e6a	  Orrenius,	  S.,	  Zhivotovsky,	  B.,	  &	  Nicotera,	  P.	  (2003).	  Regulation	  of	  cell	  death:	  the	  calcium-­‐apoptosis	  link.	  Nature	  Reviews.	  Molecular	  Cell	  Biology,	  4(7),	  552–565.	  doi:10.1038/nrm1150	  Pang,	  Z.-­‐P.,	  Deng,	  P.,	  Ruan,	  Y.-­‐W.,	  &	  Xu,	  Z.	  C.	  (2002).	  Depression	  of	  fast	  excitatory	  synaptic	  transmission	  in	  large	  aspiny	  neurons	  of	  the	  neostriatum	  after	  transient	  forebrain	  ischemia.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  22(24),	  10948–10957.	  Paninski,	  L.,	  Fellows,	  M.	  R.,	  Hatsopoulos,	  N.	  G.,	  &	  Donoghue,	  J.	  P.	  (2004).	  Spatiotemporal	  Tuning	  of	  Motor	  Cortical	  Neurons	  for	  Hand	  Position	  and	  	   	   59	  Velocity.	  Journal	  of	  Neurophysiology,	  91(1),	  515–532.	  doi:10.1152/jn.00587.2002	  Penfield,	  W.,	  &	  Boldrey,	  E.	  (1937).	  Somatic	  Motor	  and	  Sensory	  Representation	  in	  the	  Cerebral	  Cortex	  of	  Man	  as	  Studied	  by	  Electrical	  Stimulation.	  Brain,	  60(4),	  389–443.	  doi:10.1093/brain/60.4.389	  Richard	  Green,	  A.,	  Odergren,	  T.,	  &	  Ashwood,	  T.	  (2003).	  Animal	  models	  of	  stroke:	  do	  they	  have	  value	  for	  discovering	  neuroprotective	  agents?	  Trends	  in	  Pharmacological	  Sciences,	  24(8),	  402–408.	  doi:10.1016/S0165-­‐6147(03)00192-­‐5	  Risher,	  W.	  C.,	  Ard,	  D.,	  Yuan,	  J.,	  &	  Kirov,	  S.	  A.	  (2010).	  Recurrent	  spontaneous	  spreading	  depolarizations	  facilitate	  acute	  dendritic	  injury	  in	  the	  ischemic	  penumbra.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  30(29),	  9859–9868.	  doi:10.1523/JNEUROSCI.1917-­‐10.2010	  Rosen,	  A.	  S.,	  &	  Morris,	  M.	  E.	  (1993).	  Anoxic	  depression	  of	  excitatory	  and	  inhibitory	  postsynaptic	  potentials	  in	  rat	  neocortical	  slices.	  Journal	  of	  Neurophysiology,	  69(1),	  109–117.	  Schafer,	  D.	  P.,	  Jha,	  S.,	  Liu,	  F.,	  Akella,	  T.,	  McCullough,	  L.	  D.,	  &	  Rasband,	  M.	  N.	  (2009).	  Disruption	  of	  the	  axon	  initial	  segment	  cytoskeleton	  is	  a	  new	  mechanism	  for	  neuronal	  injury.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  29(42),	  13242–13254.	  doi:10.1523/JNEUROSCI.3376-­‐09.2009	  60	  Schmidt,	  S.,	  Bruehl,	  C.,	  Frahm,	  C.,	  Redecker,	  C.,	  &	  Witte,	  O.	  W.	  (2012).	  Age	  dependence	  of	  excitatory-­‐inhibitory	  balance	  following	  stroke.	  Neurobiology	  of	  Aging,	  33(7),	  1356–1363.	  doi:10.1016/j.neurobiolaging.2010.11.019	  Schneider,	  C.,	  Devanne,	  H.,	  Lavoie,	  B.	  A.,	  &	  Capaday,	  C.	  (2002).	  Neural	  mechanisms	  involved	  in	  the	  functional	  linking	  of	  motor	  cortical	  points.	  Experimental	  Brain	  Research.	  Experimentelle	  Hirnforschung.	  Expérimentation	  Cérébrale,	  146(1),	  86–94.	  doi:10.1007/s00221-­‐002-­‐1137-­‐2	  Scott,	  N.	  A.,	  &	  Murphy,	  T.	  H.	  (2012).	  Hemodynamic	  responses	  evoked	  by	  neuronal	  stimulation	  via	  channelrhodopsin-­‐2	  can	  be	  independent	  of	  intracortical	  glutamatergic	  synaptic	  transmission.	  PloS	  One,	  7(1),	  e29859.	  doi:10.1371/journal.pone.0029859	  Shih,	  A.	  Y.,	  Blinder,	  P.,	  Tsai,	  P.	  S.,	  Friedman,	  B.,	  Stanley,	  G.,	  Lyden,	  P.	  D.,	  &	  Kleinfeld,	  D.	  (2013).	  The	  smallest	  stroke:	  occlusion	  of	  one	  penetrating	  vessel	  leads	  to	  infarction	  and	  a	  cognitive	  deficit.	  Nature	  Neuroscience,	  16(1),	  55–63.	  doi:10.1038/nn.3278	  Sigler,	  A.,	  Goroshkov,	  A.,	  &	  Murphy,	  T.	  H.	  (2008).	  Hardware	  and	  methodology	  for	  targeting	  single	  brain	  arterioles	  for	  photothrombotic	  stroke	  on	  an	  upright	  microscope.	  Journal	  of	  Neuroscience	  Methods,	  170(1),	  35–44.	  doi:10.1016/j.jneumeth.2007.12.015	  Sigler,	  A.,	  Mohajerani,	  M.	  H.,	  &	  Murphy,	  T.	  H.	  (2009).	  Imaging	  rapid	  redistribution	  of	  sensory-­‐evoked	  depolarization	  through	  existing	  cortical	  pathways	  after	  targeted	  stroke	  in	  mice.	  Proceedings	  of	  the	  National	  Academy	  of	  Sciences	  of	  	   	   61	  the	  United	  States	  of	  America,	  106(28),	  11759–11764.	  doi:10.1073/pnas.0812695106	  Silasi,	  G.,	  Boyd,	  J.	  D.,	  LeDue,	  J.,	  &	  Murphy,	  T.	  H.	  (2013).	  Improved	  methods	  for	  chronic	  light-­‐based	  motor	  mapping	  in	  mice:	  automated	  movement	  tracking	  with	  accelerometers,	  and	  chronic	  EEG	  recording	  in	  a	  bilateral	  thin-­‐skull	  preparation.	  Frontiers	  in	  Neural	  Circuits,	  7,	  123.	  doi:10.3389/fncir.2013.00123	  Symon,	  L.,	  Branston,	  N.	  M.,	  Strong,	  A.	  J.,	  &	  Hope,	  T.	  D.	  (1977).	  The	  concepts	  of	  thresholds	  of	  ischaemia	  in	  relation	  to	  brain	  structure	  and	  function.	  Journal	  of	  Clinical	  Pathology.	  Supplement	  (Royal	  College	  of	  Pathologists),	  11,	  149–154.	  Tennant,	  K.	  A.,	  Adkins,	  D.	  L.,	  Donlan,	  N.	  A.,	  Asay,	  A.	  L.,	  Thomas,	  N.,	  Kleim,	  J.	  A.,	  &	  Jones,	  T.	  A.	  (2011).	  The	  organization	  of	  the	  forelimb	  representation	  of	  the	  C57BL/6	  mouse	  motor	  cortex	  as	  defined	  by	  intracortical	  microstimulation	  and	  cytoarchitecture.	  Cerebral	  Cortex	  (New	  York,	  N.Y.:	  1991),	  21(4),	  865–876.	  doi:10.1093/cercor/bhq159	  Tennant,	  K.	  A.,	  Adkins,	  D.	  L.,	  Scalco,	  M.	  D.,	  Donlan,	  N.	  A.,	  Asay,	  A.	  L.,	  Thomas,	  N.,	  …	  Jones,	  T.	  A.	  (2012).	  Skill	  learning	  induced	  plasticity	  of	  motor	  cortical	  representations	  is	  time	  and	  age-­‐dependent.	  Neurobiology	  of	  Learning	  and	  Memory,	  98(3),	  291–302.	  doi:10.1016/j.nlm.2012.09.004	  Teskey,	  G.	  C.,	  Flynn,	  C.,	  Goertzen,	  C.	  D.,	  Monfils,	  M.	  H.,	  &	  Young,	  N.	  A.	  (2003).	  Cortical	  stimulation	  improves	  skilled	  forelimb	  use	  following	  a	  focal	  ischemic	  infarct	  in	  the	  rat.	  Neurological	  Research,	  25(8),	  794–800.	  62	  Timothy	  H.	  Murphy.	  (2011).	  Two-­‐Photon	  Imaging	  of	  Neuronal	  Structual	  Plasticity	  in	  Mice	  during	  and	  after	  Ischemia.	  In	  Imaging	  in	  Neuroscience:	  A	  laboratory	  Manual	  (pp.	  949–960).	  New	  York:	  Cold	  Spring	  Harbor.	  Tudela,	  R.,	  Soria,	  G.,	  Pérez-­‐De-­‐Puig,	  I.,	  Ros,	  D.,	  Pavía,	  J.,	  &	  Planas,	  A.	  M.	  (2014).	  Infarct	  volume	  prediction	  using	  apparent	  diffusion	  coefficient	  maps	  during	  middle	  cerebral	  artery	  occlusion	  and	  soon	  after	  reperfusion	  in	  the	  rat.	  Brain	  Research,	  1583,	  169–178.	  doi:10.1016/j.brainres.2014.08.008	  Van	  Meer,	  M.	  P.	  A.,	  Otte,	  W.	  M.,	  van	  der	  Marel,	  K.,	  Nijboer,	  C.	  H.,	  Kavelaars,	  A.,	  van	  der	  Sprenkel,	  J.	  W.	  B.,	  …	  Dijkhuizen,	  R.	  M.	  (2012).	  Extent	  of	  bilateral	  neuronal	  network	  reorganization	  and	  functional	  recovery	  in	  relation	  to	  stroke	  severity.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  32(13),	  4495–4507.	  doi:10.1523/JNEUROSCI.3662-­‐11.2012	  Waagepetersen,	  H.	  S.,	  Sonnewald,	  U.,	  Larsson,	  O.	  M.,	  &	  Schousboe,	  A.	  (1999).	  Synthesis	  of	  vesicular	  GABA	  from	  glutamine	  involves	  TCA	  cycle	  metabolism	  in	  neocortical	  neurons.	  Journal	  of	  Neuroscience	  Research,	  57(3),	  342–349.	  Wang,	  J.	  H.	  (2003).	  Short-­‐term	  cerebral	  ischemia	  causes	  the	  dysfunction	  of	  interneurons	  and	  more	  excitation	  of	  pyramidal	  neurons	  in	  rats.	  Brain	  Research	  Bulletin,	  60(1-­‐2),	  53–58.	  Wang,	  M.,	  Iliff,	  J.	  J.,	  Liao,	  Y.,	  Chen,	  M.	  J.,	  Shinseki,	  M.	  S.,	  Venkataraman,	  A.,	  …	  Nedergaard,	  M.	  (2012).	  Cognitive	  deficits	  and	  delayed	  neuronal	  loss	  in	  a	  mouse	  model	  of	  multiple	  microinfarcts.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  32(50),	  17948–17960.	  doi:10.1523/JNEUROSCI.1860-­‐12.2012	  	   	   63	  Watson,	  B.	  D.,	  Dietrich,	  W.	  D.,	  Busto,	  R.,	  Wachtel,	  M.	  S.,	  &	  Ginsberg,	  M.	  D.	  (1985).	  Induction	  of	  reproducible	  brain	  infarction	  by	  photochemically	  initiated	  thrombosis.	  Annals	  of	  Neurology,	  17(5),	  497–504.	  doi:10.1002/ana.410170513	  Xie,	  Y.,	  Chen,	  S.,	  Anenberg,	  E.,	  &	  Murphy,	  T.	  H.	  (2013).	  Resistance	  of	  optogenetically	  evoked	  motor	  function	  to	  global	  ischemia	  and	  reperfusion	  in	  mouse	  in	  vivo.	  Journal	  of	  Cerebral	  Blood	  Flow	  &	  Metabolism.	  doi:10.1038/jcbfm.2013.89	  Xiong,	  W.,	  &	  Jin,	  X.	  (2012).	  Optogenetic	  field	  potential	  recording	  in	  cortical	  slices.	  Journal	  of	  Neuroscience	  Methods,	  210(2),	  119–124.	  doi:10.1016/j.jneumeth.2012.07.019	  Xu,	  Z.	  C.,	  &	  Pulsinelli,	  W.	  A.	  (1994).	  Responses	  of	  CA1	  pyramidal	  neurons	  in	  rat	  hippocampus	  to	  transient	  forebrain	  ischemia:	  an	  in	  vivo	  intracellular	  recording	  study.	  Neuroscience	  Letters,	  171(1-­‐2),	  187–191.	  Zhang,	  S.,	  Boyd,	  J.,	  Delaney,	  K.,	  &	  Murphy,	  T.	  H.	  (2005).	  Rapid	  reversible	  changes	  in	  dendritic	  spine	  structure	  in	  vivo	  gated	  by	  the	  degree	  of	  ischemia.	  The	  Journal	  of	  Neuroscience:	  The	  Official	  Journal	  of	  the	  Society	  for	  Neuroscience,	  25(22),	  5333–5338.	  doi:10.1523/JNEUROSCI.1085-­‐05.2005	  Zhang,	  S.,	  &	  Murphy,	  T.	  H.	  (2007).	  Imaging	  the	  impact	  of	  cortical	  microcirculation	  on	  synaptic	  structure	  and	  sensory-­‐evoked	  hemodynamic	  responses	  in	  vivo.	  PLoS	  Biology,	  5(5),	  e119.	  doi:10.1371/journal.pbio.0050119	  Zhao,	  G.,	  &	  Flavin,	  M.	  P.	  (2000).	  Differential	  sensitivity	  of	  rat	  hippocampal	  and	  cortical	  astrocytes	  to	  oxygen-­‐glucose	  deprivation	  injury.	  Neuroscience	  Letters,	  285(3),	  177–180.  


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