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 protective mechanisms of
hypothermia and 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.
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
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,
J Cereb Blood Flow Metab.
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
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
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
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
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.
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
Stroke-induced plasticity made simple (hopefully).
"Reader's Digest" condensed version of the review.
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.
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
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 protocolsthat 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
that can be easily adapted to stroke recovery as recently done by some
clinical institutions including the University
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.
We determine using two-photon imaging how the
mitochondrion (energy generating capacity of the cell) is
affected during the first minutes after a stroke.
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.
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.
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.
Stroke effects on brain circuit structure-function assessed
with millisecond level brain functional imaging.
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
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).
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
We also study the basic properties of
cortical circuits with the hope of applying this knowledge
to understand how brain circuits recover after stroke.
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
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
Novel automated tools to study the motor
system that will be applied to stroke.
Feb. 2009: In vivo synaptic physiology and optogenetic brain mapping.
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.
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)
Brain mapping of forelimb somatosensory cortex using intrinsic signal imaging
(IOS), darkened area indicates the response to contralateral
For free software and hardware description see:
Harrison TC, Sigler A, and Murphy TH. (2009) Simple and cost-effective hardware and software
for functional brain mapping using Intrinsic Optical Signal imaging
as well as our software page.
J Neurosci Methods 182:211-218.
Tools for creating targeted ischemia (stroke).
Laser induced small artery clotting
by photoactivation of Rose Bengal.
This process occurs over 2 min
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
Rapid and reversible dendritic damage.
Reversible damage to dendrites during ischemia and reperfusion (images shown are from layer I cortex)
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
the structure and function of somatosensory circuits support
stroke recovery weeks after ischemia.
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.
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.