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Spring Program in Neuroscience


FACULTY

MURPHY


DR. TIMOTHY H. MURPHY

Ph.D. 1989 (Johns Hopkins Univ.)
B.Sc. 1984 (Saint Mary's College Maryland)

  • Professor
  • Psychiatry
  • Cellular and Physiological Sciences
  • E-mail:
    Phone: (604) 822-0705

                  

    General areas of research:

    Structure-function of brain circuits in relation to stroke.  High resolution imaging of individual synapses and sensorimotor circuits in live mice to provide insight into mechanisms of initial stroke damage and stroke recovery.  We are currently focusing on understanding how sensory and motor circuits compensate after stroke.

    CNS synaptic plasticity/physiology:  in vivo imaging of synaptic interactions and sensorimotor processing, novel brain mapping procedures using optogenetics, and single synapse biophysical approaches.

    Imaging neurochemistry during stroke.  Monitoring of oxidative stress and ionic disturbances in vivo and testing of novel antioxidant treatments during stroke.


    New insights, strategies, and tools to study and promote recovery after stroke that were highlighted within the media and concerned our lab:

    Using light to probe and facilitate sensory and motor circuit recovery after stroke:  see Globe and Mail.

    Stroke damage occurs begins in less than 3 minutes: see Science Daily.

    Commentary on Ron Frostig's lab's work using sensory stimulation to restore blood flow: see Neurology Today.

    Globe and mail article about "connectome"  see Globe and Mail.


    New work: mapping canonical long-range structural and functional circuits in the mouse brain.

    Spontaneous cortical activity alternates between motifs defined by regional axonal projections.  Mohajerani MH, Chan AW, Mohsenvand M, Ledue J, Liu R, McVea DA, Boyd JD, Wang YT, Reimers M, Murphy THNat Neurosci. 2013 Aug 25. doi: 10.1038/nn.3499. [Epub ahead of print]  We define consensus pathways for activity flow across wide regions of mouse cortex using voltage sensitive dye imaging in mouse cortex. Patterns of activity flow strongly resembled connectivity maps for intracortical monsynaptic projections derived from assessment of the connectivity database made by the Allen Institute for Brain Science.  Activity sources and sinks can be observed in the video clip that shows the average response to C2 whisker stimulation below. If you are interested the first author PDF Dr. Majid Mohajerani now has a new lab at the Univ. of Lethbridge AB.

     

    Allen Institute Brain Exploer Visualization

    New tools for charting the mouse intracortical connectome from the Allen Institute for Brain Science.   Projection from barrel cortex to motor cortex is shown (see arrow).  Parietal association area makes strong midline projection (see arrow).  For functional connectivity strengths in and out of these areas see Lim et al. 2012 and Mohajerani et al. 2013.  We have used the raw data from the Allen Institute Conectivity Atlas to compare functional to structural connectivity.

     

    Three methods for functional mapping of mouse barrel cortex yield similar results (left whisker movement, middle spontaneous activity, right Channelrhodopsin-2 direct cortical stimulation). Functional connectivity maps were compared to patterns of axonal projections from the Allen Institute database (not shown).  A small island of labeling is present in motor cortex (inset).

     



    A new review article:

    Lim DH, Ledue J, Mohajerani MH, Vanni MP, Murphy TH.  (2013) Optogenetic approaches for functional mouse brain mapping. Front Neurosci. 7:54. doi: 10.3389/fnins.2013.00054. This paper describes recent approaches to map function within mouse brain in vivo with optogenetics and highlights mesoscale imaging that our lab has developed.

    optogenetic brain mapping from Front. Neurosci. 2013

    In vivo large-scale cortical mapping using channelrhodopsin-2 stimulation in transgenic mice reveals asymmetric and reciprocal relationships between cortical areas.  Diana H.Lim, Majid H.Mohajerani, Jeffrey LeDue, Jamie Boyd, Shangbin Chen and Timothy H.Murphy  Front. Neural Circuits 2012 6:11. doi: 10.3389/fncir.2012.00011 

     

    A new approach to assess connections between cortical areas that will be applied to study plasticity after stroke.  Note similarities between maps evoked by visual stimulation and channelrhodopsin stimulation of visual cortex in video clip.

         
     

     

     


    Loss of synaptic structure during stroke occurs despite deep hypothermia.

      New work: Moderate or deep local hypothermia does not prevent the onset of ischemia-induced dendritic damage. Tran S, Chen S, Liu RR, Xie Y, Murphy TH. 2012  J Cereb Blood Flow Metab.  [Epub] While post-ischemic hypothermia reduces neuronal injury following global ischemia, spared neurons may still show ultrastructural abnormalities in the days after the initial insult (Colbourne et al 1999). We have evaluated hypothermia’s effects on dendrite morphology in the immediate phase of ischemia and in early reperfusion using repeated 2-photon in vivo imaging. Ischemia-induced dendritic blebbing could not be prevented even with deep hypothermic treatment and may be an obligate effect of energy failure and impaired ionic homeostasis. These findings have implications for clinical practice since deep hypothermia is used in some cardiac surgery procedures.  JCBFM Tran et al. 2012



    Distinct cortical circuit mechanisms for complex forelimb movement and motor map topography. Harrison TC, Ayling OG, Murphy TH.    Neuron. 2012 Apr 26;74(2):397-409.  The motor cortex has long been known to play a central role in the generation of movement, but fundamental questions remain about the functional organization of its subregions and their neuronal circuits. Results from electrical brain stimulation have traditionally been interpreted with an emphasis on a cortical body map, but the utility of this principle has diminished with the discovery of multiple representations of the body that could overlap in cortical space. Despite the detailed knowledge gleaned from these efforts, our understanding of the macroscopic organization of motor cortex remains incomplete. Much of our understanding about the motor cortex comes from experiments in which stimulation or recording is performed at a few cortical points. Recently, we and others have developed a novel method for rapid automated multi-point motor mapping based on light activation of the recombinant ion channel Channelrhodopsin-2. We apply light-based motor mapping to investigate the functional subdivisions of the motor cortex and their dependence on intracortical synaptic activity.

    Upper image shows setup for movement measurement and mouse laser brain stimulation. Center left image shows cortical targets where Channelrhodopsin-2 stimulation was performed.  Center right image show examples of general movement direction bias over the cortical surface, abduction versus adduction areas indicated. Lower image shows an example of a complex movement evoked by prolonged stimulation.

    The ability to repeatedly map the motor cortex over timescales ranging from minutes to months has allowed us to appreciate the dynamic nature of movement representations and facilitated the comparison of motor maps generated before and after pharmacological perturbations of the intracortical circuitry. We have exploited the predominant expression of Channelrhodopsin-2 in layer 5 pyramidal neurons of Thy-1 transgenic mice to target this class of cortical output cells directly, exposing their contribution to motor cortex topography and identifying a functional subdivision of the mouse forelimb representation based on general movement direction. Prolonged trains of light or electrical stimulation revealed that activation of these subregions drives movements to distinct positions in space. To identify mechanisms that could account for the different movement types evoked by stimulation of these cortical subregions, we performed pharmacological manipulations of the intracortical circuitry and targeted anatomical tracing experiments. 

    Blocking excitatory cortical synaptic transmission did not abolish basic motor map topography (directional bias of movement), but the site-specific expression of complex movements was lost. Our data suggest that the topography of movement maps arises from their hard-wired segregated output projections, whereas complex movements evoked by prolonged stimulation require intracortical synaptic transmission.


    Targeted mini-strokes produce changes in interhemispheric sensory signal processing that are indicative of disinhibition within minutes.  Mohajerani MH, Aminoltejari K, Murphy TH. 

     


    Proc Natl Acad Sci U S A. 2011 May 31;108(22):E183-91. Epub 2011 May 16.  Pseudocolor images of voltage-sensitive dye signals in response to tactile stimulation of the left forepaw before (A) and after (B) targeted focal stroke within the right hemisphere (forelimb sensory cortical area). The stroke area is outlined by the white circle. (C and D) Cartoon illustrating the re-routing of sensory processing for the affected forelimb within the first hours of targeted ischemia. See also author summary in more simple terms.

     

    Stroke-induced changes in circuit use can  extend to both hemispheres within 1 hour, indicating that existing cortical circuits may be able to re-route sensory signals over long distances.  The brain routes sensory signals to both hemispheres.  Most processing is crossed or contralateral, but a minority of ipsilateral or un-crossed processing occurs.  After stroke the ipsilateral non-crossed signal from the stroke-affected limb is preserved despite loss of the response in the contralateral cortex (the contralateral  cortex is normally the source of the ipsilateral signal).  Our results suggest that stroke leads to a switch in the mechanism of ipsilateral cortical processing and a relative enhancement of a normally latent (completely non-crossed) ipsilateral signal from the left forepaw to the left cortex (see right video). Disinhibiton was dependent on the contralateral thalamus.  Other evidence for disinhibtion is from observations of enhanced responses to the non-stroke-affected paw (video below).  While we do not yet know the behavioral significance of re-routing sensory information to preserve the affected-paw ipsilateral response after stroke (or to enhance responses to the non-affected paw), these results do suggest the potential for rapid engagement of latent long-range cortical circuits. 
    Movie of the left "affected" forelimb response before/after stroke (preservation of ipsilateral response to stroke-affected paw).  Stroke center is indicated by a circle, bregma (skull landmark) by a dot.  The ipsilateral response (left side) is seen well before any residual response in the stroke affected hemisphere.  A new mechanism of ipsilateral signal routing was observed after stroke with an apparent enhancement of non-crossed input from the stroke-affected paw leading to a preserved ipsilateral cortex response.  In some cases the amplitude of this novel ipsilateral cortex response was even enhanced see ~100 ms after stimulation in video above.

     

     

    Movie of the "right" non-affected forelimb response before/after stroke on right side (see circle) (enhancement of contralateral response to non-stroke-affected right paw), both hemispheres are shown in voltage sensitive dye response movie. Signifcantly enhanced contralateral and ipsilateral responses were observed for the non-stroke affected paw.

     


    Li and Murphy 2008 J Neurosci.

    Stroke rapidly damages neuronal structural circuitry.  If the blood supply is promptly restored (reperfusion) the structure within the stroke penumbra (area near the border with partial flow) can recover. However, the stroke core is less likely to recover even with reperfusion. The lost function of the core region may be compensated for by related brain areas through a process termed "plasticity". Li and Murphy J. of Neurosci. 2008


    Murphy and Corbett, 2009Stroke-induced plasticity made simple (hopefully). Major points are made below, see review for the details.  FL and HL indicate forelimb and hindlimb somatosensory cortex respectively.  When stroke affects the forelimb (FL) cortex activity can re-map to related areas such as hindlimb (HL) cortex or motor cortex.  Murphy, T.H. and Corbett D.(2009) Plasticity during stroke recovery: from synapse to behaviour. Nat. Rev. Neurosc. 10:861-872.

    layer 5 neuron"Reader's Digest" condensed version of the review.

    1)  Reductions in blood flow to the brain of sufficient duration and extent lead to stroke, which results in damage to neuronal networks and impairment of sensation, movement, or cognition.  We find that apparent damage to networks can occur after only 2-3 min of ischemia where blood flow drops to less than 20% of basal values.

    2)  A time-limited window of neuroplasticity opens over weeks following stroke in the adult brain, during which partial behavioral recovery can occur, that can be further augmented by rehabilitative therapy.

    3)  Enhanced sensory and motor performance that occur after stroke is referred to as ‘recovery’, although re-emergent post-stroke behaviour is unlikely to be identical to the pre-stroke state, therefore a more accurate term is behavioural compensation provided by remaining and newly developed brain circuits that result in altered and/or new response strategies.

    4)  Plasticity in the adult brain after stroke is enabled by a surprising amount of diffuse and redundant synaptic connectivity within the CNS, and the ability of new structural and functional circuits to form through re-mapping between related cortical regions.

    5)  Many of the molecular mechanisms underlying stroke recovery are similar to those involved during development, a "critical period" of heightened neuroplasticity akin to that occurring during visual system development may exist after stroke.  For successful rehabilitation after stroke it is critical to align behavioral interventions with critical periods.

    6)  It is possible to conceptualize synaptic learning rules after stroke into two broad classes and temporal phases: occurring first are mechanisms that ensure that each neuron receives an adequate amount of synaptic input akin to homeostatic plasticity, occurring later are Hebbian mechanisms in which synaptic strength is redistributed to favor coincident activity and properly functioning circuits.  Although these are concepts and mechanisms that have been described in other systems whether they occur in the stroke affected brain is currently unclear.

    How can basic knowledge aid stroke victims?  It may be possible to develop drugs that stimulate neurotransmitter action or circuit sprouting. However, the most practical means (and immediately applicable) of facilitating stroke recovery may be innovative rehabilitation strategies or even brain stimulation protocols that promote the proper use of remaining circuits using insight gathered from basic research. It is even possible that the vehicle for accomplishing rehabilitation may be already in the grandkids bedroom in the form of movement-based gaming systems (Wii) that can be easily adapted to stroke recovery as recently done by some clinical institutions including the University of Toronto.  Other means of brain stimulation include transcranial magnetic stimulation (TMS) that is currently used on stroke patients that our colleagues at UBC use in research. One of our goals is to employ brain stimulation in animal models using light and channelrhodopsin-2 to establish the timing and other parameters that will be important for effective treatment of the patient with methods such as TMS.


    We use two-photon imaging to assess how individual neurons and their dendritic arbors are affected by stroke within the mouse brain, see animation below based on this data.

    Nov. 2009: Neurons with partially lesioned dendritic arbors survive within the peri-infarct zone and undergo growth within their remaining dendrites.  from Brown CE, Boyd JD, Murphy TH (2010) Longitudinal in vivo imaging reveals balanced and branch-specific remodeling of mature cortical pyramidal dendritic arbors after stroke. J Cereb Blood Flow Metab. (EPub)

    selective peri-infarct dendrite plasticity
    Selective re-modeling of peri-infarct dendrites observed by longitudinal 2-photon imaging in mice weeks after stroke. Left cartoon
    showing events that occur just after stroke induction and the weeks that following including dendritic blebbing, cell death, spine production, changes in vasculature (angiogenesis) and selective maintenance of dendrites that project away from the infarct and loss those in the most ischemic areas.  Above data from Brown et al. 2010, apical dendritic arbor of a single somatosensory neuron shown at 3 different timepoints.

    We determine using two-photon imaging how the mitochondrion (energy generating capacity of the cell) is affected during the first minutes after a stroke.

    Nov. 2009: Surprisingly, in vivo imaging of mitochondria depolarization suggests mediators of delayed cell death may be activated within 5 min of stroke onset.  from Liu R.R. and Murphy T.H. (2009) Reversible cyclosporine A sensitive mitochondrial depolarization occurs within minutes of stroke onset in mouse somatosensory cortex in vivo. A two-photon imaging study.  EPub, J. of Biol. Chem. 

    Cartoon stroke, structure recovers but cell death triggered  Liu_Murphy_JBC_mito_depolar_stroke

    Mitochondria (form wordpress)

    Left panel and above cartoon movies showing changes in dendrite structure and possible initiation of cell death mechanisms after stroke.  Although neurons can partially recover from structural damage when blood flow is restored (reperfusion), they may still be subject to delayed cell death via a process called apoptosis.  The mitochondria (left) normally supplies the cell with energy, but its depolarization through the mitochondrial permeability transition pore can trigger apoptosis and delayed cell death. Here we image mitochondria in living mice during stroke (above images) using a dye called Rh-123 and 2-photon microscopy. We show that mitochondrial depolarization that has hallmarks of this mechanism occurs within 5 min of stroke induction (brighter image on right). These results suggest that treating both loss of structure and initiation of cell death mechanisms are important for maintaining function. Fortunately for stroke victums not all neurons die from apoptosis and neurons with partially intact synaptic structure continue to function. Rehabilitation and synaptic plasticity ensure that remaining circuits make up for some of the losses due to stroke.

     

    Using regional imaging techniques and analysis of synaptic structure within living mice we show that new structural and functional cortical circuits form within functionally related cortical areas areas that are close to the stroke core.

    Feb. 2009: Stroke effects on brain circuit structure-function assessed with millisecond level brain functional imaging.  from Publication: In vivo voltage-sensitive dye imaging in adult mice reveals that somatosensory maps lost to stroke are replaced over weeks by new structural and functional circuits with prolonged modes of activation within both the peri-infarct zone and distant sites. Brown C.E., Aminoltejari K., Erb H., Winship I.R., and Murphy T.H., J. Neurosci. 2009 29:1719-1734.








    Cover of J.Neurosci. 29 (6); 11 Feb 2009.  New Structural and Functional Circuits After Recovery from Stroke Two-photon image of a GFP-labeled layer 5 dendrite superimposed onto a montage showing whole-brain cortical responses to forelimb stimulation after stroke.



    Regional imaging techniques show that existing redundant cortical circuits may begin to compensate for the effects of stroke even sooner than previously anticipated (within hours).

    July 2009: Imaging rapid redistribution of sensory-evoked depolarization through existing cortical pathways after targeted stroke in mice.  Results of Voltage Sesitive Dye (VSD) imaging revealed that patterns of sensory-evoked depolarization redistribute within hours after an ischemic stroke in the forelimb region of the somatosensory cortex in mice. Published in Proceedings of the National Academy of Sciences of the United States of America 106 (2009): 11759-11764 .
    see also:
    * Supporting Information (PDF) .

    Rapid sensory response redistributionIllustration of rapid sensory response redistribution mechanisms for somatosensory cortex function after stroke. Forelimb derived sensory signals are routed to the forelimb somatosensory cortex (FL), but also to a lesser extent to nearby cortical regions such as the sensory hindlimb (HL) representation. Within hours after stroke to the FL area, diffuse off-target FL derived signals that are present in HL cortex are spared from stroke damage and are well positioned to aid in re-mapping of circuits in the recovering animal.  

    We also study the basic properties of cortical circuits with the hope of applying this knowledge to understand how brain circuits recover after stroke.

    Mar. 2010: Fast imaging of bilateral spontaneous activity reveals local cortical circuit patterns.  Mirrored Bilateral Slow-Wave Cortical Activity within Local Circuits Revealed by Fast Bihemispheric Voltage-Sensitive Dye Imaging in Anesthetized and Awake Mice.  Mohajerani MH, McVea DA, Fingas M, Murphy TH.  J Neurosci. 2010 30(10):3745-51. 

    Regional patterns in spontaneous activity. Bilateral imaging of spontaneous fluctuations in cortical membrane potential measured with 20 milliseconds between images in wildtype (left columns) and acallosal (right) mice superimposed on traces of cortical EEG activity.  Wildtype mice show bilaterally synchronous activity while acallosal mice oscillate asynchronously. Sequences of spontaneous activity are shown in 7 image columns corresponding to 140 ms of data.  Clear patterns emerge in the WT mice that favor midline areas and may represent cortical default networks. Knowledge of these circuits may be important for understanding basic brain function and for recovery after disease such as stroke.

    wild type mouse            acallosal mouse

    Regional patterns in spontaneous activity


    Novel automated tools to study the motor system that will be applied to stroke.

    Feb. 2009: In vivo synaptic physiology and optogenetic brain mapping. 

    Publication: Ayling OG, Harrison TC, Boyd JD, Goroshkov A, Murphy TH. Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.  Nature Methods 6 (2009): 219-224.

    Laser activation of Channelrhodospin-2, fixed brain shown.   Maps of mouse forelimb motor cortex

    Optical stimulation of motor cortex fixed brain example shown, left.  Maps of mouse forelimb motor cortex derived from light-evoked muscle activity, middle raw EMG thumbnails, and right grayscale map of EMG amplitude.



    Stroke brain circuit structure-function research questions/plan:

    Close-up view of dendritic spine synapse vessel relationship in mouse brain in vivo.  On average synapses are 13 micrometers from a flowing capillary and are supplied by about 100 red blood cells per second. J Neurosci (2005) 25:5333-8.
    Image showing Texas-Red labeled vasculature and green dendrites taken from a green fluorescent protein transgenic mouse before induction of ischemia in vivo. 

    Our aim is to help stroke victims regain brain function through understanding of how synaptic networks are damaged and recover from interruptions in brain blood flow.  We anticipate that advances learned in our lab will translate into new treatments, treatment guidelines, and hope.

    There are two major goals for our research:

    1) Reveal the key chemical and electrical events that lead to early stroke-induced damage to synaptic networks in intact animals by employing high-resolution two-photon and other forms of in vivo imaging. The hope is that if we can better understand these processes we might be able to stop them.

    2) Understand the structural and functional basis of adaptive changes to brain circuitry that accompany stroke recovery. A key component of stroke recovery is the re-mapping of function from damaged brain areas to surviving areas. Although new areas of activation occur, how information flows in and out reorganized cortical networks on the millisecond timescale and by what circuitry is unclear. Our goal is to understand these changes with the aim of using this knowledge to promote the recovery of human stroke patients.


    Cerebral vessels labeled in vivo using FITC-dextran permits monitoring blood flow at the single capillary level using 2-photon microscopy.
    Sectioning through live brain with 2-photon microscopy, dendrites receiving end of synaptic inputs are labeled green (YFP line H transgenic) and blood vessels red (slice in a box view). J Neurosci (2005) 25:5333-5338.




    Tools for mapping brain circuit function.

    Intrinsic Map
    Intrinsic optical signal imaging/mapping provides a means of assessing cortical circuits involved in processing touch and permits linking microscopic structure of vessels and synapses to function PLoS Biol (2007) 5:e119.

     

    Brain mapping of forelimb somatosensory cortex using intrinsic signal imaging (IOS), darkened area indicates the response to contralateral forelimb stimulation.

    For free software and hardware description see: Harrison TC, Sigler A, and Murphy TH. (2009) Simple and cost-effective hardware and software for functional brain mapping using Intrinsic Optical Signal imaging as well as our software page. J Neurosci Methods 182:211-218.

    Tools for creating targeted ischemia (stroke).

      Rose Bengal induced photothrombosis

    Laser induced small artery clotting by photoactivation of Rose Bengal. This process occurs over 2 min PLoS Biol (2007) 5:e119. It permits targeted stroke in mouse somatosensory cortex. The diameter of the shown arteriole is ~40 µm. See also Sigler A, Goroshkov A, and Murphy TH (2008) Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope. J Neurosci Methods 170:35-44 .


    Stroke damage to synapses is apparent within minutes, but can be reversed with prompt reperfusion of blood.

    Rapid and reversible dendritic damage

    Rapid and reversible dendritic damage. Reversible damage to dendrites during ischemia and reperfusion (images shown are from layer I cortex) J Neurosci (2008) 28:1756-1772.


    Rapid and reversible dendritic damage

    Imaging waves of stroke damage. Changes in reflected light signal (ratio to preischemic condition, scaled between 95-105% of baseline reflectance) associated with ischemic depolarization over a 5 min period within a 3.1 mm wide brain window. The star on the upper right marks the time when ischemic depolarization was observed by monitoring the EEG . The time per frame is 1s and the movie starts 28s before the induction of ischemia as defined by EEG suppression. At the beginning of the movie the image is a uniform gray that corresponds to 100% of preischemic reflected light levels. Within 30s the surface vessels darken followed by the surrounding tissues indicating deoxygenation. ~170s after occlusion a wave of brightening (increased light scattering) begins to move across the cortex starting with the most anterior and lateral tissues.  This wave leads to damaged synaptic networks J Neurosci (2008) 28:1756-1772.



    Changes to the structure and function of somatosensory circuits support stroke recovery weeks after ischemia.


    Synapse elimination in vivo (time in hours).

    Synapse addition in vivo. J Neurosci (2007) 27:4101-4109.



    Changes to the function of single neurons that support re-mapping of sensory function weeks after ischemia.


    Layer 2 neurons that are normally responsive to only a single limb exhibit broader receptive fields and can respond to all 4 limbs as sensory responses are re-mapped. Winship IR and Murphy TH (2008) In vivo calcium imaging reveals functional rewiring of single somatosensory neurons after stroke.
    J. Neurosci. 28:6592-6606




    Circuit and cellular level re-mapping model after stroke based on un-masking of sub-threshold connections between related regions of cortex.
    Winship IR and Murphy TH (2009) Re-mapping the somatosensory cortex brain after stroke: insight from imaging the synapse to network.
    The Neuroscientist 15, 507-524.



    Detailed Research Plans and Background

    • Aim 1. Determine the relationship between local blood flow and reversible changes in synaptic structure during ischemia and reperfusion in vivo.
    • Stroke is the most common neurological disorder affecting our aged population currently. Stroke is defined as a sudden loss of blood flow to the brain making it a disorder of vascular plumbing. However, it is apparent that the stroke-plumbing problem evolves to affect the brain's electrical system through the ischemia-induced loss of synapses and circuits. It would be a great advantage to have approaches that can stop damage to neurons and brain synaptic networks from occurring during stroke. The first objective of this work is to uncover the key events that lead to stroke-induced damage of synaptic networks in intact animals by employing high-resolution two-photon and other forms of in vivo imaging. Originally developed by Winfred Denk and Watt Webb (1990), two-photon microscopy uses pulsed infrared light to excite fluorophores by the combined power of two long wavelength photons. One of the key advantages of this approach is that it allows one to image structures deep within thick biological specimens, whilst achieving micron level resolution. Combining this imaging technique with transgenic mice engineered to express fluorescent proteins within a subset of neurons (Feng et al., 2000) has enabled us and other investigators to track changes in neuronal structures in living animals over days, weeks and even months (Grutzendler et al., 2002; Trachtenberg et al., 2002). Here we describe recent results using this approach to examine the acute vulnerability of synaptic circuits to stroke, as well as the remarkable degree of plasticity (ability to change) in dendritic spines in regions adjacent to tissues lost to stroke damage (ie. the peri-infarct zone) (see Aim 2). Dendrites play a fundamental role in cell to cell communication in the brain, as they are the post-synaptic targets of most synapses in the brain. A large percentage of dendrites are decorated with tiny protrusions known as dendritic spines, which usually possess at least one excitatory synapse (Arellano et al., 2007).

      When dendrites become ischemic, they undergo a relatively stereotyped pattern of degeneration with the most salient morphological feature being the appearance of varicosities or beads along the shaft of the dendrite (Zhang et al. 2005; Hasbani et al., 2001; Park et al., 1996). We have recently shown the dendritic wiring of neurons can be degraded within 1-3 minutes following stroke. Surprisingly, the structure and function of the brain's synaptic wiring system was severely compromised only 1-3 minutes after stroke by an event termed ischemic depolarization. However, if blood flow was promptly restored, as can occur using stroke treatments with clot-busting drugs, the synapses bounced back from severe deformation (Murphy et al. 2008). These data suggest that ischemic depolarization is the major damaging event associated with stroke's effect on synapses, and suggest that strategies that control this process would be fruitful avenues for stroke prevention and treatment.

      A number of damaging events have already been identified in a variety of different systems that could underlie stroke damage and therapies for protecting brain circuitry and neurons are largely based on these studies. However, it is unclear what the exact timing and sequence of damaging events is after stroke? Currently we are producing an accurate timeline of potentially damaging events that occur in intact brain in response to stroke at the level of individual synapses and brain circuits.

      The two major classes of events that could damage brain neurons and synaptic circuitry are over-excitation of neurons (excitotoxicity), and a production of reactive oxygen metabolites produced by normal metabolism gone wrong (oxidative stress). In excitotoxicity messages which are normally involved in communication between neurons (and rely on the passage of ions such as calcium) are now increased in volume to the point where they damage neurons. In oxidative stress byproducts of normal cellular metabolism such as reactive oxygen species are now either over-produced or over-accumulate resulting in damage to brain proteins, membranes, and loss of cells. Although these two classes of mechanisms have been identified, if, when, and where they occur is not fully understood in intact animals and is the subject of our acute stroke imaging work.

      Our experiments will accurately describe the early events associated with stroke damage in an intact animal model. Although these studies themselves do not aim to treat stroke, we will identify potentially damaging events in intact animal models and will help to prioritize future treatment strategies and may be even suggest re-examining some current practices. By using a mouse stroke model we can also take advantage of mutant mice that can model aspects of human stroke and cardiovascular disease, allowing the experiments to be translated to those at risk for stroke through heritable disorders.

      Why is it important to describe the early events of stroke? We believe that an accurate description of the events that underlie stroke damage will be critical for stroke therapeutics. For example, if we find that the damaging effects of too much synaptic communication are largely over with within minutes following ischemia it may not be appropriate to design neuroprotective strategies that try to block these damaging events hours after ischemic onset. Furthermore, if we determine that reactive oxygen species only accumulate when blood flow is restored we may recommend that patients be treated with appropriate therapies only upon restoration of blood perfusion.

    • Aim 2. Determine structural and functional changes in brain circuits that underlie the plasticity associated with recovery of function over days to weeks after stroke.
      Our lab has taken the approach of using in vivo two photon microscopy to monitor real-time changes in the brain's fine synaptic wiring during the initial ischemic episode (within the first hours), and over the days to weeks during stroke recovery (Brown et al., 2007; Zhang et al., 2005; Zhang and Murphy, 2007). We have described using this approach a remarkable degree of plasticity in dendritic spines in regions adjacent to tissues lost to stroke damage that occurs weeks after stroke (Brown et al. 2007). We then relate these new imaging results to previous histological and functional studies to suggest that dendritic remodelling in peri-infarct regions plays an integral role in the process of recovery from stroke damage.

    • Surrounding the death and destruction of the ischemic core, is a region of hypo-perfused tissue known as the peri-infarct zone (Hossmann, 2006). Currently, there is some debate as to what exactly constitutes the "peri-infarct zone" or how large this area extends. For the sake of simplicity we will define it as the region of surviving tissue immediately surrounding the core of the infarct, which in a rodent could be less than a millimeter whereas in humans, this region may be several orders of magnitude larger (Carmichael, 2005). In the days to weeks following an ischemic insult, accumulating data suggest that the peri-infarct region is a hot spot for neuronal plasticity that subserves functional recovery from stroke (Witte, 1998). In particular, peri-infarct zones undergo significant changes in the expression of growth promoting and inhibitory factors that are essential for neuronal re-wiring, angiogenesis and neurogenesis (Carmichael, 2006). Furthermore, anatomical studies have shown that for several weeks after focal stroke, peri-infarct regions show increased synaptogenesis, are enriched with histochemical markers of axonal sprouting such as GAP-43, and receive new intracortical projections (Dancause et al., 2005; Ito et al., 2006; Stroemer et al., 1995).

      Recovery from brain injury such as stroke is dependent upon how well surviving brain tissues assume the responsibilities of those lost to injury. Given the topographic organization of the cerebral cortex, functionally homologous areas situated in close proximity to the site of injury (i.e. peri-infarct regions) are most likely to adopt new functional roles after stroke. Despite considerable progress in our understanding of cortical plasticity after stroke, many fundamental questions remain. For instance, we do not know the spatiotemporal dynamics with which sensory information flows in and out of re-organized cortical networks or the precise structural changes that underlie these changes. We are currently combining network-imaging approaches with 2-photon imaging to address these questions.

    Imaging miniature synaptic activity at single synapses using calcium imaging.
    Mackenzie et al.
    J. Neurosci (1999) 19:RC13.


    The video clip shows miniature synaptic calcium transients visualized with the fluorescent calcium probe (fluo-3) in a spiny cultured rat cortical neuron dendrite. The image shows activity over a 10 sec period.


    3D reconstructions of cortical synapses in culture. Shown below are a 3-D reconstruction of a rat cortical neuron spine from electron microscopic images. Analysis of spine function using calcium imaging suggests that larger more complex spines (having larger and multiple PSDs; indicated in dark color) have a greater quantal amplitude suggesting that structural changes to dendrites may be required for synaptic plasticity.

    3D reconstructions of cortical synapses 3D reconstructions of cortical synapses



    Our funding sources:

    Our funding sources: