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
Automated mouse brain imaging and brain stimulation:
we develop models of neurological and psychiatric
disease that employ internet enabled mouse homecages
that are used to manipulate and assess brain activty.
Connectivity mapping based on
resting-state activity in mice has revealed functional
motifs of correlated activity. However, the rules by
which motifs organize into larger functional modules
that lead to hemisphere wide spatial-temporal activity
sequences is not clear. We used in vivo
wide-field calcium imaging of the indicator GCaMP6 awake
mice to characterize the organization of spontaneous
cortical activity at different spatiotemporal scales.
Correlation analysis defines the presence of two to
three superclusters of activity that span traditionally
defined functional territories and were frequency
Event triggered mesoscale GCaMP6 imaging: how
wide-scale cortical spontaneous activity is linked to
the firing of single neurons (potentially anywhere in
brain). Xiao D, Vanni MP, Mitelut
CC, Chan AW, LeDue JM, Xie Y, Chen AC, Swindale NV,
Murphy TH Mapping cortical
mesoscopic networks of single spiking cortical or
eLife 2017 Feb 4;6. pii: e19976. doi:
RFID mouse tracking open source
Python software and Raspberry Pi interface:
Bolanos, F. and LeDue J., Murphy TH. (2017) Cost effective
raspberry pi-based radio frequency identification tagging of
mice suitable for automated in vivo imaging.
J. Neurosci. Meth.276:79-83
Automation of animal experimentation improves
consistency, reduces potential for error while
decreasing animal stress and increasing
Radio frequency identification (RFID)
tagging can identify individual mice in group
housing environments enabling animal-specific
tracking of physiological parameters.
The Python software can serve as the basis of
any behavioral or physiological task where users
will need to identify and track specific
get software here
Automated mouse homecage imaging:
Mouse home cage cortical mesoscopic
imaging supports 5 mice at a time and requires minimal
investigator intervention. Murphy
TH, Boyd JD, Bolaños F, Vanni MF, Silasi G, Haupt D, andLeDue JM “High-throughput automated home-cage
mesoscopic functional imaging of mouse cortex” (2016)
data automated collection: cages located remotely in animal facility, cortical response to brief light flashes.
Collaborative project (led by AM Craig UBC)
linking altered cortical dynamics to synaptic suppressor
Ammendrup-Johnsen I, Chan AW, Kishimoto Y, Murayama C,
Kurihara N, Tada A, Ge Y, Lu H, Yan R, LeDue JM,
Matsumoto H, Kiyonari H, Kirino Y, Matsuzaki F, Suzuki
T, Murphy TH,
Wang YT, Yamamoto T, Craig AM. (2016) Altered
Cortical Dynamics and Cognitive Function upon
Haploinsufficiency of the Autism-Linked Excitatory
Synaptic Suppressor MDGA2. Neuron.
2016 Sep 7;91(5):1052-68. doi:
Stroke is restricted
to the right side of the mouse brain (localized to the
forelimb area), but deficits (red) in connection strength at 7
days and gains (green) from 1 week to 8 weeks recovery are felt
throughout the network. see
Lim et al. 2014 J. of Neurosci. see below for
tutorial on making these connectivity diagrams. Want to make
your own connectivity diagram using Matlab? see
Lim et al. 2015 Neurophotonics
We developed a mouse model of small-vessel disease where
occlusions are produced through endovascular injection
of fluorescent microspheres that target ~12 μm diameter
penetrating arterioles and can be localized in
histology. Using Thy1-GFP transgenic mice, we visualized
the impact of microocclusions on neuronal structure.
Microocclusions in the hippocampus produce cell loss or
neuronal atrophy (~7% of lodged microspheres led to
microinfarcts), while axons within white matter tracts,
as well as the striatum and thalamus became blebbed or
Detailed protocol and
Cortical mapping article and interactive tool:
Vanni, M. and Murphy T.H. (2014) Mesoscale transcranial
spontaneous activity mapping in GCaMP3 transgenic mice reveals
extensive reciprocal connections between areas of somatomotor
J. of Neurosci.
34(48):15931-46 Imaging through intact bone using a
chronic window reveals functional symmetries between M1 and S1.
Maps are made using correlated sponataneous activity
with a partcular seed location. To assess your own
seeds of interest click here and a local
correlation viewer will open (be patient takes
30-60 sec) move the mouse over window to see
different local maps within the 9x9 mm bilateral window
(the viewer only works in Internet Explorer or
Chrome no Firebox). We thank Caroline
Rougier for assistance with HTML code. The interactive
maps represent both hemispheres with a field of view
similar to the images below on the right.
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
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.
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
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).
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.
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,
Murphy TH. 2012
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
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
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.
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.
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
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 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.
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.