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Uncrossed cortical and thalamic circuits to mediate sensory processing weeks after stroke Aminoltejari, Khatereh 2011

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UNCROSSED CORTICAL AND THALAMIC CIRCUITS TO MEDIATE SENSORY PROCESSING WEEKS AFTER STROKE. by Khatereh Aminoltejari B.Sc., The University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2011  © Khatereh Aminoltejari, 2011  Abstract One of the enduring principles in neurobiology is that most sensory activity is processed in the hemisphere opposite (contralateral) to the sensation. After a relatively small stroke, function can re-map to related peri-infarct cortical areas. However, after a large stroke, areas with spared and related function may only be present within the “unaffected” hemisphere leading to ipsilateral (uncrossed) sensory processing. We investigated the mechanism of the ipsilateral cortical representation of tactile information before, and 1 and 8-10 weeks after stroke using voltage sensitive dye imaging of forelimb-evoked sensory responses in mice. In control animals before stroke, we observed bilateral cortical activation in response to unilateral stimulation. Ipsilateral forelimb responses before stroke were dependent on the contralateral cortex and presumably mediated through transcallosal fibers that homotopically connect the somatosensory cortices. Large strokes that affected both sensory and motor cortices led to a shift in processing of forelimb sensory stimuli to predominantly the unaffected hemisphere. Pharmacological silencing of the peri-infarct cortex or thalamus within the strokeaffected hemisphere failed to affect prominent non-crossed responses in animals 8-10 weeks after stroke, yet these treatments blocked ipsilateral responses in control animals. Ipsilateral forelimb responses after 8-10 weeks of recovery from a large stroke were attributed to non-crossed ascending circuits that were dependent on the ipsilateral thalamus and apparently substitute for damaged transcallosal cortico-cortical connections. These results indicate that ipsilateral sensory responses present in animals after 8-10 weeks of recovery from stroke  ii  are through a fundamentally different mechanism that involves circuits well upstream of the peri-infarct tissue with little dependence on the thalamus or cortex within the stroke-affected hemisphere.  iii  Preface This thesis presents work that is not previously published. Dr. Timothy Murphy supervised the project, provided financial support, and aided in writing the manuscript. Alexander Goroshkov provided assistance with optics and fabrication of the hardware. Dr. Jamie Boyd and Dr. Albrecht Sigler developed the software necessary to control the hardware. Mr. Pu Min Wang assisted with the surgeries. Dr. Mohajerani consulted with regards to figures and data analysis.  For this paper I conducted the majority of the experiments including intrinsic optical signal (IOS) imaging and voltage sensitive dye (VSD) imaging as well as electrophysiology and pharmacology experiments, analysed data and wrote the manuscript.  Ethical Approval was obtained from the University of British Columbia Animal Care Committee, the certificate number is A10-0140.  iv  Table of Contents Abstract ................................................................................................................ii Preface .................................................................................................................iv Table of Contents ................................................................................................v List of Figures ....................................................................................................vii Acknowledgements ..........................................................................................viii INTRODUCTION ...................................................................................................1 Neuroplasticity............................................................................................................ 1 Rodent models of focal ischemic stroke.................................................................. 2 Organization of the sensorimotor cortex ................................................................. 3 Cortical reorganization in response to ischemic changes to local blood flow .... 6 Stroke: size matters, and so does the phase of recovery ...................................... 8 Lateralization of sensory-evoked signal processing ............................................ 13 The hurdle: investigating subthreshold signals that contribute to sensory signal processing in a biologically relevant manner........................................................ 14 Rationale and hypothesis ........................................................................................ 16  METHODS ...........................................................................................................19 Animal model ............................................................................................................ 19 Intrinsic optical signal (IOS) Imaging ..................................................................... 19 Targeted photothrombotic stroke procedure ........................................................ 20 VSD imaging.............................................................................................................. 21 Data Analysis ............................................................................................................ 22 Cortical EEG recording ............................................................................................ 23 Local field potential recording ................................................................................ 23 Local pharmacology................................................................................................. 24 Histology ................................................................................................................... 24 Statistical analysis.................................................................................................... 25  RESULTS ............................................................................................................26 Bilateral processing of sensory information and interhemispheric reorganization after stroke ...................................................................................... 26 Unilateral pharmacological silencing of the cortex does not mimic the interhemispheric effect of stroke.................................................................................... 29 Bilateral sensory-evoked responses can exist in the absence of transcallosal connections............................................................................................................... 30  v  The effects of unilateral pharmacological inactivation of thalamic nuclei on sensory-evoked responses after stroke................................................................. 31  DISCUSSION ......................................................................................................33 Multiple circuits contribute to bilateral activation of the somatosensory cortices in response to unilateral stimulation ...................................................................... 34 What is the function of ipsilateral activation after a unilateral ischemic lesion?35 Mechanistic insight into ipsilateral responses in stroke-recovering animals .... 36 Future directions and conclusions ......................................................................... 38  FIGURES .............................................................................................................41 REFERENCES ....................................................................................................55  vi  List of Figures  Figure 1 Experimental timeline and schematics of sensory circuitry and targeted stroke location........................................ 41 Figure 2 Assessment of bilateral sensory processing within the contralateral (stroke-affected hemisphere) and ipsilateral Hemisphere using VSD ............................................................... 42 Figure 3 Quantification of the VSD response amplitude, integral, and latency to peak following 8-10 weeks of recovery from stroke ................................................................................... 44 Figure 4 Laterality of signal processing before and 8-10 weeks after stroke ................................................................................... 45 Figure 5 Unilateral cortical silencing with Lidocaine does not affect the ipsilateral response 8-10 weeks after a large stroke ............................................................................... 47 Figure 6 Bilateral somatosensory responses can exist in the absence of a corpus callosum ................................................... 49 Figure 7 LFP recording and pharmacological manipulation of thalamic nuclei reveals un-crossed pathways as the source of ipsilateral activation after large strokes ................... 50 Figure 8 Pharmacological manipulation of ipsilateral thalamic nuclei blocks ipsilateral activation after stroke ........................ 52 Figure 9 Model for cortical and subcortical changes in inhibitory and excitatory connections after stroke.................................... 53  vii  Acknowledgements Thank you Dr. Tim Murphy, my supervisor, who gave me the opportunity to learn a great deal. Thank you members of the Murphy lab. I am grateful for all the help and support. Dr. Swindale, Dr. McKeown and Dr. Boyd for all your guidance and support throughout the years. Last and certainly not least, thanks to my family for their support and love every day, especially my mom.  viii  INTRODUCTION When blood flow to a circumscribed region of the brain is interrupted, it leads to an incapacitating neurological condition called stroke. Stroke is the third leading cause of death in North America and the leading cause of adult disability (Carmichael, 2005; Murphy and Corbett, 2009). With a 75% survival rate, stroke disables more than it kills thus propelling the search for strategies for neuronal repair after stroke. A deeper understanding of structural and functional changes in the brain after stroke will lead to the development of targeted treatments for stroke as well as other traumatic brain injuries.  Neuroplasticity Oxygen and nutrient deprivation of neurons leads to massive structural and functional injury within seconds where for every minute delay in treating a stroke, the average patient loses 1.9 million brain cells, 13.8 billion synapses, and 12 km of axonal fibers (Saver, 2006) leading to behavioral and functional deficits in survivors. Despite the initial devastating loss, many patients undergo some spontaneous recovery and with a combination of therapies can further improve their behavioral and functional performance. This recovery is a result of altered synaptic activity in response to external stimuli or injury which is commonly referred to as plasticity (Nudo, 2003). The result of processes that bring about plasticity is to restore synaptic structural elements and function towards target levels, namely, the pre-stroke capacity. Plasticity throughout the central nervous system is the cornerstone of functional and behavioral learning as well as recovery.  1  Rodent models of focal ischemic stroke Experimental stroke models are well developed in rodents and have been valuable for the in vivo study of the effects of stroke both acutely and chronically and determining structural and functional changes in response to stroke in the brain. While the development of primate and higher mammal stroke models is important, they are not cost effective and cannot be conducted on the same scale as rodent model investigations. Therefore, rodent models provide an effective alternative and are the basis of the large body of stroke research data at present. Although many rodent focal models of stroke, such as the intraluminal suture model produce large infarcts that closely resemble the malignant and fatal human infarction than the average sized human stroke (Carmichael, 2005), there are a few techniques, including the photothrombosis model that more closely resemble human stroke in the rate of recovery and have provided extensive data on changes in neuronal physiology and neurotransmission in the peri-infarct and contralateral cortex. Stroke models such as proximal or distal middle cerebral artery (MCA) occlusions and MCA embolisms can produce effective infarcts resembling that of a human ischemic stroke but they tend to vary in size and be more challenging to reproduce. In the endothelin 1 (ET 1) vasoconstriction model that produces ischemia by constricting the blood vessels, the location of the stroke can be localized easily and lesion size can be adjusted using different volumes of ET 1. However, in all of the abovementioned models, the surgical procedure is invasive in which the skull has to be breached thus increasing the risk of physical damage to the cortex as well as the  2  possibility of post-operative infections. The photothrombosis model has been streamlined so that a photosensitive dye can be injected intraperitoneally in an animal that has had a section of its skull removed or thinned. With a green laser or an epifluorescent light source directed at the underlying blood vessels, microvascular occlusions are formed leading to ischemia in the cortex. Infarcts can then be produced in any area of the cortex without invasive surgery (Watson et al., 1985; Brown et al., 2009; Mohajerani et al., 2011b). Here we have utilized the photothrombotic model of stroke in order to create reproducible strokes, and have developed a method of also modifying the size of the ischemic lesion by varying laser beam and thinned skull dimensions, which allowed us to investigate the effects of stroke size on mechanisms of recovery. Previous studies have shown that the size of the stroke can greatly influence the extent and mechanism of recovery (Bayona et al., 2005; Carmichael, 2005). Volume of injury is often used to describe the extent of brain damage which assumes cortical equivalency and ignores the special importance of certain cortical circuits in generating behavior (Crafton et al., 2003). Another advantage of using the photothrombosis model is that these circuits can be exclusively targeted so as to identify the molecular, cellular, and structural changes that result after a localized ischemic assault in specific circuitry.  Organization of the sensorimotor cortex The complexity of neuronal organization in the cortex is a well-established fact. It has been shown that neurons that contribute to complex functions, such as a memory trace, are not localized in a single brain region but are distributed throughout  3  the cortex (Lashley, 1950). Therefore, when considering mechanisms and routes of rewiring after brain injury such as stroke, it is important to consider that despite its defined circuit structure, the brain functions as a spatially distributed computational machine that routes signals along multiple pathways, each capable of adapting to changes in transmission fidelity. This redundancy in neuronal processing along with diffuse connectivity throughout the cortex might facilitate recovery from stroke damage. The motor and sensory cortices are loosely organized by functional map of body parts that can be modified by experience (Nudo et al., 1996). In the somatosensory cortex, this phenomenon referred to as somatotopy denotes that specific motor cortex neurons are paired with muscles and in the sensory cortex maps reflect body part pairings to neurons in the cortex (Kleim et al., 1998; Nudo, 2006). Typically, these specific circuits are formed during the development of the central nervous system (CNS) and adjust, strengthen and form functional and structural relationships with other circuits via mechanisms of plasticity with training or environmental interfacing (Heddings et al., 2000). However, these circuits do no operate in isolation but also form specialized and redundant connections with near and far circuits throughout the cortex (Lashley, 1950). Redundancy in neuronal processing together with diffuse connectivity might facilitate recovery from stroke damage. Many of the mechanisms of recovery are in fact similar to those found in the intact brain (Kleim and Jones, 2008); therefore, a study of stroke recovery mechanisms must encompass both structural and functional changes in the strokeeffected area as well as circuits that have a functional and structural relationship with  4  those damaged by ischemia. In each hemisphere of the brain, the mouse forelimb (FL) and hindlimb (HL) somatosensory cortices are adjacent to their motor representations of the FL and HL (Murphy and Corbett, 2009) (Figure 1B). There is an approximately 50 percent overlap between the sensory and motor cortices in mice (Ayling et al., 2009), which correlates to diffuse connectivity between the two functionalities. Also, a seminal study by Matyas and colleagues (2010) demonstrated that the somatosensory cortex exerts control over motor movements. They found an additional and equally direct pathway for cortical motor control driven by the primary somatosensory cortex (Matyas et al., 2010). Further research is needed in primates and humans to show the existence of such pathways; however, in stroke research, the motor cortex is often highlighted and investigated because of the blatant deficits that injury to this circuitry induces. These recent findings reveal the need to reevaluate the functional organization of cortical maps, in particular in area of stroke recovery, which is why we have focused our investigation on the somatosensory cortex. Sensory pathways are typically contralateral, such that signals from the left limb go to the right cortex. Although the contralateral processing of sensory and motor signals is a dogmatic view, anatomical and physiological studies in various mammalian species, including humans show that a small degree of ipsilateral (uncrossed pathways) processing of sensory information may be apparent (Armand and Kuypers, 1980; Jones et al., 1989; Gonzalez et al., 2004) (Figure 1C). There are various studies ranging in scope from physiological to behavioral in support of the role of the ipsilateral hemisphere, the hemisphere on the same side of the body from  5  which sensation arises, in processing information (Price and Fowler, 1981; Sunderland et al., 1999). For example, it has been demonstrated that ischemic injury to one side of the somatosensory cortex causes deficits to sensorimotor function on both sides of the body (Jones et al., 1989; Sunderland et al., 1999). As such, the role of the ipsilateral hemisphere in sensory and motor processing after stroke must also be highlighted and investigated. The sensory and motor cortical representations are also interhemispherically linked to their homotopic counterparts in the opposite hemisphere via fibers that traverse the corpus callosum (Barbaresi et al., 1994; Manzoni, 1997; Fabri et al., 1999). The prevailing view is that in an intact brain, most ipsilateral activity is relayed from the contralateral hemisphere and is dependent on afferents that connect homotopic regions via the corpus callosum (Manzoni, 1997; Petersen, 2007; Petreanu et al., 2007) (Figure 1C). Therefore, functionally hierarchical upstream and downstream regions from the ischemic area are potential targets for plasticity resulting in functional and behavioral recovery. Clarifying the pathways involved in somatosensory processing and identifying functional and structural changes in this circuitry in response to ischemic damage could help provide more clear targets for therapeutic interventions, both pharmacological and rehabilitative.  Cortical reorganization in response to ischemic changes to local blood flow When a clot blocks the flow of blood into a region of the brain, it forms a stroke core, which has less then 20% of baseline blood flow and fails to regain its fine dendritic structure, even if there is reperfusion and is eventually the site of glial scar  6  tissue (Li and Murphy, 2008). The area directly surrounding the core is termed the penumbra or peri-infarct cortex and experiences reduced blood flow but can also experience a reversal of damage (Hossmann, 2006; Zhang and Murphy, 2007). This area is also defined as a perfusion-diffusion mismatch (perfusion lesion at least 20% larger than the lesion on diffusion-weighted imaging), which indicates ischemically threatened but viable tissue (Staroselskaya et al., 2001). The peri-infarct cortex is a hotbed of structural and functional remodeling after stroke in which the seeds of recovery can be sown. Cortical space is at a premium and there is continuous competition for cortical map territory. It has been shown in differentiation studies that when a single digit is removed in an adult animal, the cortical area previously dedicated that digit swiftly remaps to represent the intact digits represented in the neighboring areas (Merzenich et al., 1983; Jones, 2000). This evidence clearly demonstrates that functional remapping can occur even in an adult brain in response to a lack of input. After stroke, instead of an expansion of existing maps into the deafferented cortical territory, there is competition between recovering peri-infarct regions and adjacent healthy tissue to remap function of the lost cortical area onto surrounding regions as well as more distant areas directly and indirectly connected to the injured cortical area (Nudo et al., 1996; Metz et al., 2005). This data is largely derived from anterograde and retrograde tracing studies and visualization of local dendritic spine turnover within the peri-infarct cortex and the contralesional hemisphere (Wall, 1977; Dancause et al., 2005a; Takatsuru et al., 2009). In these studies, the tracers indicate connections to the peri-infarct zone from regions within the affected hemisphere as well as  7  projections from the peri-infarct zone to other sites (Carmichael and Chesselet, 2002; Brown et al., 2009). The question arises then as to which factors contribute to recruitment of local circuits, or more distal connections. Cortical remapping after stroke is dependent on both activity and competition both of which can have a profound influence on the extent of recovery from stroke and both of which are influenced by the size of the ischemic insult and the phase of recovery (Stroemer et al., 1995; Biernaskie and Corbett, 2001; Carmichael et al., 2001; Biernaskie et al., 2005).  Stroke: size matters, and so does the phase of recovery Recruiting peri-infarct tissue as apposed to more distal circuitry appears to be dependent on the size of the stroke. In animal studies where small stroke models that resemble the relative size of a survivable human stroke were utilized (those that affect 5-15% of the hemisphere) (Murphy and Corbett, 2009), it was primarily the surviving healthy tissue with similar function that was recruited (Cramer et al., 2006; Winship and Murphy, 2008b). Therefore, recovery after a small stroke is likely to involve peri-infarct tissue that has a similar function. After a large stroke however, tissue that has a similar function might be found only at more distant sites, such as the premotor cortex (for strokes that affect the primary motor cortex) (Frost et al., 2003) or regions in the contralateral hemisphere (Biernaskie et al., 2005), where structural remodeling can occur (Takatsuru et al., 2009). A recent in vivo investigation from our laboratory revealed the sequence and kinetics of the activation of peri-infarct cortical circuits after stroke in mice using voltage-sensitive dye imaging (Brown et al., 2009). Eight weeks after stroke in the forelimb sensory cortex, the surviving portion of  8  forelimb sensory cortex, which falls within the peri-infarct region actively relays enhanced sensory signals to the motor cortex as well as the surviving HL somatosensory representation. Notably, the remapped responses last significantly longer in animals that have recovered from stroke than in normal controls and show a greater degree of spreading from the motor cortex to distant cortical regions, including the retrosplenial cortex (Brown et al., 2009). These findings indicate that the recovery of sensorimotor functions after stroke and brain remapping involve changes in the temporal and spatial spread of sensory information processing across local and distant sites and that the size of the stroke influences the range of functional and structural changes throughout the brain. Mechanisms suggested for mediating cortical reorganization involve the formation of novel circuits, the unmasking of existing, but latent, synaptic connections, and modulation of synaptic efficacy in active connections (Sanes and Donoghue, 2000; Butefisch, 2004). While redistribution of circuit function is clearly the final result of the above processes, mechanism and route of sensory signal transduction may be different in the first hours to days after stroke versus weeks to months after which the system may reach a homeostatic balance. Moreover, the size of the ischemic damage may determine which circuits are recruited for immediate and chronic compensation. Immediately after an ischemic insult, the local environment in the cortex may be altered to induce changes in the expression of specific genes to permit residually active stroke-affected input to compete more efficiently for connections within the surrounding intact tissue (Stroemer et al., 1995; Liauw et al., 2008; Murphy and  9  Corbett, 2009). For example, Liauw et al. (2008) found that production of glial-derived synaptogenic thrombospondin 1 and 2 are induced in the peri-infarct region, which might give the stroke-affected tissue a competitive edge over healthy circuitry. In a way, the acute stroke recovery process can be viewed not as a response to damaging events such as apoptosis and necrosis but as a form of compensation (homeostatic plasticity) because of loss of function (Liepert et al., 2003; Murphy and Corbett, 2009). For example, previous studies report neuronal hyperexcitability after stroke through changes in excitation and inhibition balance (Luhmann et al., 1995; Centonze et al., 2001). Electrophysiological studies for acute slices showed that oxygen deprivation causes an immediate reduction of GABAergic synaptic transmission (Luhmann et al., 1995; Centonze et al., 2001). Concurrently, the expression of functional GABAA receptors is reduced (Hagemann et al., 1998; Qu et al., 1998), synaptic glutamate release becomes elevated (Benveniste et al., 1984; Wang, 2003), and NMDA receptor activity is enhanced (Hagemann et al., 1998; Kozlowski and Schallert, 1998) leading to the removal of surrounding inhibition and formation of new patterns of activation using existing disinhibited spared circuits. These altered electrophysiological properties could induce potentiation of excitatory inputs in the peri-infarct cortex within the first hour after stroke (Fujioka et al., 2004) and form an environment suitable for later rewiring during the chronic stages of recovery (Carmichael, 2003; Brown and Murphy, 2008; Winship and Murphy, 2008a) leading to functional and structural reorganization (Zepeda et al., 2004; Carmichael, 2006; Kolb et al., 2007). Altogether, the physiological and molecular changes within and around the  10  stroke-affected cortical region leads to a process termed “diaschisis” which is defined by reversible suppression of function in regions adjacent to and physiologically connected to the infarct (Garraghty et al., 1990; Maggiolini et al., 2008). A study by Sigler and colleagues (2009), showed that after a targeted stroke induced by unilateral blockage of an arteriole feeding into the forelimb somatosensory cortex that leads to loss of partial function within the forelimb somatosensory cortex, the focus of forelimb-specific somatosensory cortex activity could be rapidly redistributed to the neighboring hindlimb and motor cortex. Considering that structural mechanisms of early changes to circuits hours after stroke are not rapid, synaptogenesis is unlikely since the formation of new synaptic connections in the adult cortex would likely take longer (Zito and Svoboda, 2002). Therefore, existing but less active connections (Di Filippo et al., 2008) could mediate short-term reorganization of the somatosensory cortex after focal ischemia and prime the neighboring circuitry to mediate recovery of function lost due to stroke. In fact, this particular pattern sensory-evoked activation has been observed in mice recovering from stroke after 8 weeks (Brown et al., 2009). The question then remains as to which circuit is recruited in the acute stages of stroke recovery if the stroke is large enough to damage the entire somatosensory cortex. In a recent in vivo investigation in our lab, we were able to show that within minutes after a large stroke causing ischemic damage to all nearby tissue of similar function, the contralesional hemisphere continues to respond to ipsilateral stimulation (Mohajerani et al., 2011a). Using targeted photothrombotic stroke, the forelimb somatosensory cortical representation underwent irreversible ischemic damage and using voltage sensitive dye imaging, the contralesional hemisphere was shown to  11  remain responsive to ipsilateral stimulation for minutes to hours after stroke (Mohajerani et al., 2011b). As time points are too early for sprouting new connections, especially interhemispheric connections, processes such as disinhibition and diaschesis both upstream and downstream of the stroke-affected tissue can reconcile this evidence to the observed rearrangement in the patterns of sensory-evoked activity (Schiene et al., 1996; Redecker et al., 2002; Centonze et al., 2007). It is possible that, just as in the case of small, partial strokes in the somatosensory cortex, these processes prime the physiologically related tissue, in more distant areas to support recovery. Interestingly, it appears that the processes of hyperexcitability and increased GABA activity in the patterns that are observed are unique to stroke and do not occur in the same way if at all if the loss of activity is due to non-injurious events such as cooling, pharmacological or magnetic silencing of cortical tissue (Clarey et al., 1996; Blankenburg et al., 2008; Mohajerani et al., 2011b). Therefore, these processes and the resulting patterns of activity are unique to stroke injury. They facilitate sensoryevoked signal processing in the contralesional hemisphere without the use of corpus callosal fibers (Fifkova, 1966) by means of waves of depolarization that originate in areas adjacent to a stroke core and propagate into homotopic brain regions soon after injury onset (Mun-Bryce et al., 2006; Risher et al., 2010). The depolarizing waves reported traversing across the cerebral cortex after stroke may be a means of regulating subcortical, upstream control of signal transmission. Bures et al. (1963) reported increased firing of reticular neurons in the ipsilesional thalamus of rats that were subject to cortical damage. The thalamic  12  reticular nucleus, often referred to as the thalamic pacemaker, is a net-like structure comprised of inhibitory neurons that covers the thalamus and can gate thalamic relays via connections to the ventroposterior lateral (VPL) and ventroposterior medial (VPM) nuclei of thalamus which are sensory relay nuclei (Fuentealba and Steriade, 2005). Evidence from electrophysiological and neuroanatomical (Rinvik, 1984; Raos and Bentivoglio, 1993; Battaglia et al., 1994; Timofeev and Steriade, 1996) investigations in rodents, cats and primates reticular nuclei in the two thalami provide ample support for reticuloreticular connections between the two hemispheres; therefore, increased firing of ipsilesional, inhibitory reticular nucleus neurons could inhibit the contralesional reticular nucleus and thus lead to disinhibition of contralesional thalamic relay nuclei (VPL and VPM). We hypothesize that disinhibition of the ipsilateral sensory relay circuit could increase the responsiveness of the contralesional (ipsilateral) hemisphere to sensory input and with time and usedependent competition, allow remapping of contralateral sensory function onto ipsilateral cortical territory. This may be accomplished by subcortical reorganization to provide midline crossover points to re-establish sensory processing lost to ischemic damage.  Lateralization of sensory-evoked signal processing As mentioned previously, the anatomical organization of the sensory processes dictates that stimulus-evoked signals from one side of the body are processed in the cerebral cortex on the opposite side, with a small number of fibers that maintain ipsilateral connectivity (Armand and Kuypers, 1980; Gonzalez et al., 2004). A number of studies have presented evidence for bilateral processing of  13  sensory-evoked stimuli in the intact brain (Petersen et al., 2003; Cramer, 2008; Brown et al., 2009). In addition, data from human imaging studies suggests that after stroke, there’s a distinct shift from primarily contralateral to ipsilateral (contralesional) signal processing, resulting in a shift in the laterality of sensory processing (Ward et al., 2003a; Cramer, 2008). An emerging consensus from these studies is that patients with larger stroke exhibit more extensive and longer lasting ipsilateral activation and show less complete recovery, leading clinicians and scientists to correlate ipsilateral activation with a poorer prognosis. However, the debate over whether ipsilateral activation after stroke is a compensatory mechanism or a warning sign that recovery is out of reach is yet inconclusive.  The hurdle: investigating subthreshold signals that contribute to sensory signal processing in a biologically relevant manner Sensory perception and higher cortical functions such as cognition and memory emerge from intricate, dynamic interactions in very large cortical networks. To understand the functioning of a cortical area, therefore, necessitates the use of techniques that permit following the dynamics of neuronal population activity with high spatial and temporal resolution. In the spatial domain, techniques relying on the metabolic consequences of changes in neuronal activity have excellent spatial resolution, but they share a common limitation in the time domain. This group includes 2-deoxyglucose mapping (2-DG), positron emission tomography (PET) intrinsic optical signal (IOS) imaging (Haglund and Hochman, 2007), near infrared spectroscopy (NIRS), and functional magnetic resonance imaging (fMRI)(Shoham et al., 1999; van Meer et al., 2010). The  14  metabolic signals to which these techniques are sensitive are slow relative to the neuronal activity. Thus, none of the existing metabolism-based imaging techniques at present offers temporal resolution better than hundreds of milliseconds. On the other hand, conventional techniques that do provide temporal resolution in the millisecond range suffer from serious limitations in spatial resolution. Some are essentially electrophysiological recordings at particular points (intracellular recordings, extracellular single or multiunit recordings, or local field potentials) (Mittmann et al., 1998; Frost et al., 2000). For others, such as electroencephalography (EEG) (Iijima et al., 1992) and magnetoencephalography (MEG) (Bardouille and Ross, 2008), identifying the sources that give rise to the observed signals is fundamentally difficult due to signal spread and spatial limitations of recording sites. Another potentially fast imaging technique could be based on the fast light scattering intrinsic signal, in the millisecond time domain, that has been routinely observed in vitro (Hill and Keynes, 1949), but as this signal is much smaller than the intrinsic signal, it cannot be used for high resolution in vivo imaging. To identify the anatomical and functional factors that govern sensory stimulus representation in the somatosensory cortex before and after stroke a technique that can overcome both the temporal and spatial limitations at once is necessary. Petersen et al. (2003) showed that the subthreshold synaptic input to L2/3 on deflection of a single whisker rapidly changes in the millisecond time scale across the cortical surface. One shortcoming that most of these techniques share is their inability to measure subthreshold activity that is an important component of the ipsilateral response (Petersen et al., 2003; Berger et al., 2007). Thus, to better study ipsilateral  15  processing, we have used voltage-sensitive dye (VSD) imaging which provided a sensitive means of assaying both sub-threshold and supra-threshold activity with a biologically relevant temporal resolution (ms timescale) (Petersen et al., 2003; Brown et al., 2009; Ghosh et al., 2010; Mohajerani et al., 2010). VSDs insert into the plasma membrane and change their fluorescence intensity dependent on the potential across the lipid bilayer (Shoham et al., 1999; Grinvald and Hildesheim, 2004) and reveals both subthreshold and suprathreshold activity (Shoham et al., 1999). Using VSD imaging, we are able to resolve the “where” and “when” components of neuronal sensory processing, when the imaging technique was combined with breakthrough surgical techniques that exposed nearly the entire dorsal surface of the mouse brain. This is particularly useful in the investigation of the changes that a unilateral stroke induces on the contralesional side.  Rationale and hypothesis The field of stroke research has extensively investigated the changes that occur in the peri-lesional cortex in response to stroke at acute and chronic time points (Brown et al., 2007; Winship et al., 2007; Li and Murphy, 2008). However, even though changes in the contralesional hemisphere in response to stroke have been reported (Shanina et al., 2006; Schaechter et al., 2008) the mechanism and circuitry of these adaptive alterations have not been investigated. Understanding the role of the contralesional (ipsilateral to affected limb) hemisphere in functional and behavioral recovery after stroke is important for targeting therapeutic interventions (Jones et al., 2009). However, here we do not attempt to understand the functional role of ipsilateral processing, since from a behavioral aspect this is a complex area  16  where increased ipsilateral activity has been attributed to both successful, as well as poor recovery (Cramer et al., 1997; Dijkhuizen et al., 2001; Schaechter et al., 2008). In contrast, our goal was to understand the mechanism by which ipsilateral signals are relayed to the cortex within animals that are undergoing re-mapping of forelimb sensory area function up to 8-10 weeks after stroke. In animal models and humans, shifts in the laterality of sensory processing are apparent, with increased ipsilateral processing of sensory signals occurring following stroke (Nelles et al., 1999; Dijkhuizen et al., 2001; Feydy et al., 2002; Schaechter and Perdue, 2008). There is evidence for interhemispheric transcallosal pathways (Vanderwolf et al., 1987; Manzoni et al., 1989; Petit et al., 1990; Quigley et al., 2003; Li and Ebner, 2006; van Meer et al., 2010), as well as subcortical (Iwayama et al., 1988), and uncrossed pathways (Armand and Kuypers, 1980) that contribute to ipsilateral activation. The question remains, however, about how much each pathway contributes to ipsilateral activation, and if the contribution is weighted differently before and after weeks of recovery from stroke. Therefore, this study tests the following hypotheses. Firstly, we predict that small, focal strokes that partially damage the FL somatosensory cortex and leave the surrounding tissue intact should allow remapping to occur primarily within the ipsilesional cortex. Secondly, we propose that large strokes that damage most of the FL and HL, and part of the motor cortical representations unilaterally, will not allow remapping to occur as extensively within the peri-lesional cortex and therefore, lead to a switch in laterality of sensory-evoked signal processing. Thirdly, we propose that after a large unilateral stroke, there is cortical and subcortical rewiring that leads to  17  ipsilateral processing of sensory-evoked stimuli. We predict that the ipsilateral signal will not originate from activation of the contralateral hemisphere, nor through signal conduction via the corpus callosum, but will undergo subcortical rewiring such that the latent, uncrossed ipsilateral fibers will directly cause ipsilateral processing of sensory-evoked stimuli.  18  METHODS Animal model Adult male C57BL6J mice (> 2 months of age) were used, for most experiments, while (n=6) I/LnJ acallosal mice (the Jackson Laboratory) were used for a subset of these experiments. The University of British Columbia’s Animal Care Committee approved all animal protocols. Anesthesia was induced with urethane (0.12 % w/w) and body temperature was maintained at 37±0.5 C with the use of a feedback controlled heating pad. A large (7×8 mm; bregma 2.5 to −4.5mm, lateral -4 to 4 mm) bilateral cranial window was surgically created and the dura matter was carefully removed to expose a large region of cortex as described previously (Mohajerani et al., 2010).  Intrinsic optical signal (IOS) Imaging All animals belonging to sham control or stroke groups underwent IOS imaging to identify the forelimb and hindlimb somatosensory representations in the cortex. Mice were lightly anesthetized with isoflurane gas (1.5% for induction, 1% for maintenance; mixed in air) while maintaining body temperature at 37°C. For prestroke or sham surgery, intrinsic optical signal imaging of FL and HL representations, a 3x3 mm region of skull was carefully thinned to ~50% of original thickness using a high-speed dental drill and a surgical scalpel (Surgistar #6400). After applying 1.3% low-melt agarose [at 37–38°C; Type 3-A Sigma; A9793, dissolved in a HEPESbuffered artificial CSF (ACSF)] and a coverslip over the skull, the cortical surface was illuminated by red and green sets of light-emitting diodes (LEDs) mounted around the microscope objective driven by a regulated direct current power supply (Circuit Test 19  Electronics). The green LED light source was used for visualizing the surface of the cortex and pattern of vessels. The red LED light source (center at 635 nm) was used to detect sensory-evoked changes in light reflectance in cortical regions in which levels of deoxyhemoglobin and neuronal activity is increased (Frostig et al., 1990). Before functional imaging, the plane of focus was set to 300µm below the cortical surface to blur the contribution of large surface vessels. Image acquisition was performed using XCAP standard version 2.2 imaging software (EPIX) with a Dalsa M60 Pantera 12-bit camera mounted on a video macroscope setup that used a short focus front-to front dual-video lens system (3.8x3.8mmfield, 7.5 µm per pixel). Each data collection session consisted of 20–40 trials, each taken 20 s apart. During each trial, 15 image frames were collected over 1.5 s before and after contralateral limb stimulation using a piezoelectric device (100 Hz for 1 s).  Targeted photothrombotic stroke procedure Unilateral ischemic stroke was induced in the forelimb sensorimotor cortex (outlined with a water insoluble black marker) using the photothrombotic method (Watson et al., 1985). This model was selected because it is minimally invasive and produces highly localized and reproducible lesions (Maxwell and Dyck, 2005). Briefly, the skull over the forelimb area was illuminated with a collimated beam of green laser light (532 nm, 17 mW; ~1.5 mm diameter) for 15 min after an injection of 1% Rose Bengal solution (100 mg/kg, i.p., in PBS). Age-matched sham surgery controls were treated in an identical manner with the omission of either laser illumination or Rose Bengal injection. In either case, sham surgery did not produce any signs of damage (i.e., scarring) or structural alterations to neuronal dendrites (Brown et al., 2007). After  20  surgery, mice were returned to their home cages in which they were housed either singly or in pairs and were allowed to recover. The stroke size (small or large) was altered by prolonging laser exposure time to 20 min in case of large strokes as compared to 10 min for small lesions. The size of the beam was also increased from 1.5 mm to 2.5 mm for small to large strokes respectively.  VSD imaging For in vivo voltage sensitive dye (VSD) imaging, the dye RH1692 (Optical Imaging, New York, NY) (Shoham et al., 1999), was dissolved in the HEPES-buffered saline with optical density of 6-8 (measured at 550 nm) and applied to the exposed cortex for 90 min. The dye was then thoroughly washed off and the surface of the brain was covered with 1.3% low-melt agarose dissolved in a HEPES-buffered ACSF and then sealed with a custom-cut glass coverslip to minimize movement artifacts due to respiration. Two different VSD setups were used for data acquisition and their results were pooled. 18 mice were imaged with one setup where 12-bit images were captured every 5 ms using a Dalsa 1M-60 Pantera 12-bit camera controlled by XCAP version 2.2 imaging software (EPIX). Voltage-sensitive dyes were excited with red light (Luxeon K2 LED, 627 nm) focused 300 µm below the surface through either an Olympus XLfluor 4X (NA 0.28) or 2X (NA 0.14) objective. Other animals were imaged where 12-bit images were captured with 6.67 ms resolution with a CCD camera (1M60 Pantera, Dalsa, Waterloo, ON) and EPIX E1DB frame grabber with XCAP 3.1 imaging software (EPIX, Inc., Buffalo Grove IL). In this second system images were taken through a macroscope composed of front-to-front video lenses (8.6 x 8.6 mm field of view, 67 µm per pixel). For both imaging setups, to measure cortical activity  21  evoked by sensory stimulation, we stimulated the hind- and forelimbs with a brief movement using a piezoelectric device (Q220-AY-203YB, Piezo Systems) with single 5 ms taps. In some cases as indicated for thalamic recordings electrical stimulation (1ms, 2.5mA) was given to the paw instead of a tap.  Data analysis IOS imaging. 20-40 trials were collected during each imaging session taken 20 s apart. Each trial consisted of 15 image frames taken over 1.5 s before and after the contralateral limb stimulation by a piezo-electric device (100 Hz for 1 s). Maps of the forelimb and hindlimb cortical representations were generated by summing and filtering trials for each limb and mean filtering (radius=3) using NIH Image J software. Dividing all image frames taken 1.5 s after stimulation by those taken before then identified responsive areas. Recent work from our laboratory using in vivo single-cell calcium imaging (Winship and Murphy, 2008) has shown that the functional border between forelimb and hindlimb responsive neurons is very sharp. Therefore, we chose to threshold maps at two-thirds of maximal response amplitude that most routinely delineated forelimb and hindlimb responsive areas with minimal overlap. Response maps were then assigned a particular color and merged onto an image of the surface vasculature to create color-coded response maps. VSD responses to sensory-evoked stimulation in forelimb, hindlimb and motor cortex in both hemispheres represent the average of 10–20 stimulation trials. For each trial, images were collected for 205 ms before and 500 ms after a single 5 ms mechanical deflection (300 µm amplitude) of the contralateral forelimb or hindlimb delivered through a piezoelectric device or electrical stimulation. The images collected during  22  stimulation trials were divided by null stimulation trials to correct for dye bleaching. The amplitudes of VSD responses were expressed as the percentage change in VSD signal (ΔF/Fo) by dividing image frames taken after stimulation by the average of those taken before (~100 ms baseline). Evoked cortical responses were quantified by placing a square region of interest (600 x 600 µm) over the forelimb, hindlimb, and motor cortex, using NIH ImageJ software. Motor cortical areas were identified based on stereotaxic coordinates (Franklin and Paxinos, 1997) and their stereotyped position relative to the functionally defined hindlimb area.  Cortical EEG recording A Teflon-coated silver wire (0.125 mm) with a bare chloride-coated tip was placed on the cortical surface. A reference electrode was placed on the nasal bone. The cortical signal was amplified and filtered (0–1000 Hz) using a DC amplifier.  Local field potential recording The local field potential (LFP) was recorded by inserting a glass pipette (4-7 MΩ) within one of three thalamic nuclei (the ventral posterolateral (VPL), the ventral posteromedial (VPM) and the posterior medial nucleus (PoM)). The signal was amplified by an Axopatch 200A (Axon Instruments) and filtered from DC to 1000 Hz. The forelimb sensory cortex was identified based on the VSD fluorescence signal. The thalamic nuclei were localized based on stereotaxic coordinates (Paxinos and Franklin, 2004), and their stereotyped position relative to the Bregma (for PoM 1.055 mm posterior, 1.5 mm lateral, depth=3.5mm; for VPL 1.655 mm posterior, 1.5mm lateral, depth=3mm; for VPM, 1.655 mm posterior, 2.0 mm lateral, depth=3.75 mm).  23  The recording positions were confirmed by recording the extracellular synaptic potentials evoked by electrically stimulating the contralateral forelimb (1 ms pulse, 2.5 mA). Furthermore, in a subset of animals we injected biocytin into thalamic regions from which we recorded and confirmed the locations of the injections into the specific nuclei using histology in a subset of animals.  Local pharmacology Local silencing was performed by pressure injection (30–60 s, 20–40 psi; Picospritzer II; General Valve Corporation, Fairfield, NJ) of 30 µM tetrodotoxin dissolved in a ACSF solution or 2% lidocaine.  Histology Glass pipettes used for recording from thalamic nuclei were filled with biocytin (5%) (Sigma) dissolved in 0.05M Tris, pH 7.6 along with 30 µM TTX in 140 mM NaCl. Using the pressure ejection method, described above for pharmacological silencing of the thalamic nuclei, we injected biocytin. Following survival period of 4 h mice were transcardially perfused with 0.1 M phosphate buffer (PB, pH 7.4) followed by 4% paraformaldehyde. After perfusion, the brain was removed and stored in 4% paraformaldehyde for one day. Coronal sections (50 µm) were cut using a vibratome. For HRP reaction we used diaminobenzidine (DAB) according to the procedure of King et al. (King et al., 1989). Sections of the injection sites were washed twice in two changes of 0.1 M PB containing 0.3% Triton X-100 over 10 min. The washed sections were incubated for 3 h in avidin D-231 HRP (Vector) dilution (1:500 in 0.1 M PB, containing 0.3% Triton X-100). Then, the sections were washed in three changes of  24  0.1 M PB for 3 x 10 min and pre-incubated for 10 min in 0.05% DAB in 0.1 M PB with DAB. The pre-incubated sections were transferred into the incubation medium (150ml 1% H20 2 to 50 ml DAB solution) and reacted until reaction products were evident when monitored while viewing through a stereomicroscope (approximately 5-20 min). After washing in three changes of 0.1 M PB (3 X10 min) the reacted sections were mounted on gelatin-coated slides.  Statistical analysis One-way ANOVA analysis, adjusted by Bonferroni corrections were used to compare the amplitude, time to peak, and the integral of the VSD evoked response. All p values 0.05 were considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001). All data are expressed as mean ± SE.  25  RESULTS To assess the long-term effect of stroke of varying size on cortical processing we compared the effects of small (1.1±0.03 mm2; n=7) or large (2.4±0.02 mm2; n=14) photothrombotic strokes targeted to the center of the forelimb somatosensory representation of one hemisphere (Figure 1). Adult male C57BL/6 wild-type mice were allowed to recover from stroke for 1 or 8-10 weeks (Figure 1A). Small strokes were targeted to the right forelimb somatosensory cortex, and large strokes encompassed the right forelimb and hindlimb somatosensory cortex as well as part of the primary motor cortex (M1) (Figure 1B). Photothrombosis effectively produces lesions that extend through all cortical layers without damaging the underlying white matter (Watson et al., 1985; Witte et al., 2000).  Bilateral processing of sensory information and interhemispheric reorganization after stroke Sensory-evoked VSD signals were imaged bilaterally in animals without stroke and in animals with small and large strokes respectively. In naïve and sham-operated animals, a single 5 ms mechanical stimulus delivered to the forepaw or hindlimb caused a prominent depolarizing signal within 13±3 ms (n=7) in the contralateral somatosensory cortex (Figure 2A.I.). Signal onset was obtained from interpolation of the plotted data and reflected the time to reach 50% of peak amplitude. This signal was then followed (with a latency of 18±5 ms (n=7) from the initial stimulus) by a depolarization in the homotopic region of the hemisphere that was ipsilateral to stimulation (Figure 2A.I.).  26  Consistent with previous data (Brown et al., 2009), one week after the induction of a small stroke, little or no forelimb stimulated VSD response could be detected within peri-infarct areas (p<0.013; n=7 mice; Fig 2A.II.). Further, the ipsilateral cortical response, which normally followed the activation of the contralateral cortex, was also undetectable (Fig 2A.II.). However, 8-10 weeks of recovery from a relatively smaller stroke, as described before (Brown et al., 2009), was associated with the re-emergence of sensory evoked depolarizations in the posterior and medial aspects of the peri-infarct cortex (Fig 2A.III.). The peak amplitude of the cortical response within the peri-infarct region (0.08±0.01 ΔF/Fο, p <0.0069; n=7) was ~30% of the amplitude observed in control animals (0.25%±0.01 ΔF/Fο, p < 0.0089; n=7) (Fig 2B.II, 3D). Along with the re-emergence of the contralateral peri-infarct signal, the ipsilateral cortical response in small and large stroke-affected animals returned to a similar peak amplitude as observed within the non-stroke control group (control ipsilateral amplitude=0.083±0.012, small stroke= 0.087±0.014, large stroke=0.091±0.016 ΔF/Fο; each group n=7; Figure 2B.III., 3E). After 8 weeks of recovery from a large stroke that unilaterally targeted the forelimb and the hindlimb somatosensory cortex, more widespread sensory activation was observed, with signals attributed to forepaw stimulation mainly present in the ipsilateral hemisphere (same side as the stimulated paw) instead of the contralateral hemisphere (Figure 2A.IV.). The amplitude of ipsilateral activation was similar in control animals versus 8-week recovery for both small and large stroke cases (n=7; Figure 2B.III, 3E), yet it was the contralateral (stroke-affected) hemisphere where the response amplitude was significantly decreased after a large stroke (p < 0.009)  27  (Figure 2B.II, 3D). In animals with large strokes we observed that ipsilateral responses were slowed when compared to the ipsilateral signal in control animals. The latency to peak of the ipsilateral response was significantly increased from 25±2 ms in control animals to 133±45 ms in the case of large strokes (Figure 2A.I, IV, 2B.III). The activation period in stroke animals was prolonged to 300±12 ms post stimulation (Figure 2B.III), whereas in control animals, the signal terminated within the first 120±16 ms post stimulation (Figure 2B.II) (n=7). Termination was defined as the point at which the signal reached 25% of peak amplitude. In analyzing VSD responses, we also measured both the peak VSD signal as well as the response integral over 500 ms. The response integral agreed well with the measurements of peak amplitude where the area under the ipsilateral hemisphere response curve was significantly smaller in control animals as compared to animals that received large or small strokes (p< 0.034; n=7 in all cases). This is consistent with previous findings for small strokes (Brown et al., 2009) (Figure 3F). To quantitatively compare the relative activity of the ipsilateral FL area versus contralateral and peri-infarct FL before and after stroke, a laterality index (LI) measure was used (Wilke and Lidzba, 2007). The LI is a measure of the sensory processing preference between hemispheres and was defined as in Figure 3. The possible range for the LI is from +1.0 (all activity in the contralateral hemisphere) to 1.0 (all activity in the ipsilateral hemisphere) (Cramer, 2004; Wilke and Lidzba, 2007; Seghier, 2008). The neo-forelimb (nFL) refers to a new area of the cortex that, after stroke, activates in response to forepaw stimulation apparently in place of the damaged forelimb cortex. In unlesioned animals, the laterality index, as measured by  28  the value of the peak amplitude within each hemisphere, was +0.51±0.07 indicating a modest, but detectable level of ipsilateral activation (Figure 3). After a small stroke the LI score was significantly changed to +0.05±0.02 (p<0.0081) indicating similar amplitude of the sensory signal in both hemispheres (Figure 3). After a large stroke, the LI index underwent a significant shift to -0.43±0.08 (p<0.0092; as compared to controls) reflecting mainly ipsilateral signal processing. Given that large strokes cause a greater change in inter-hemispheric sensory processing all subsequent mechanistic experiments were done on animals with large strokes.  Unilateral pharmacological silencing of the cortex does not mimic the inter-hemispheric effect of stroke Studies in a number of different mammals have established the existence of functional interconnections between related sensory and motor areas within the two hemispheres of the brain via transcallosal fibers (Ezrokhin and Grechushnikova, 1979; Jones et al., 1979; Smits et al., 1991) resulting in bilateral activation of the somatosensory cortices by sensation (Manzoni, 1997; Fabri et al., 1999). We attempted to mimic the interhemispheric effects of stroke acutely (within 2 h) by pharmacologically silencing the granular layer of the forelimb somatosensory cortex in unlesioned (control) animals using lidocaine (n=7). In unlesioned animals, blocking the contralateral forelimb somatosensory representation led to silencing of the ipsilateral VSD signal as well (p< 0.0083; n=7) (Figure 5C.II, D.I). For controls, we injected a group of mice with vehicle (n=6) and observed no significant change in the amplitude of the forelimb-evoked response in either hemisphere as compared to noninjected controls (Figure 5C.I-II). In contrast, after 8 weeks of recovery from stroke,  29  silencing the peri-infarct cortex had only minimal effects on the ipsilateral cortical response (Figure 5C.II, D.II;). Together, these results suggest that ipsilateral responses that re-emerge after stroke are not driven by transcallosal connections that homotopically connect the two hemispheres. Rather, the re-emergence of ipsilateral sensory responses after stroke may originate from changes in subcortical and/or uncrossed ipsilateral connections.  Bilateral sensory-evoked responses can exist in the absence of transcallosal connections We next investigated the possible source of signals arriving at the cortex ipsilateral to the stimulated limb by using congenitally acallosal mice (I/LnJ) (Figure 6A,B). Interestingly, acallosal animals showed the same pattern of bilateral activation as wild-type controls, with similar latencies of interhemispheric activation and ipsilateral response peak amplitude (Figure 6A,D). The laterality index calculated for acallosal animals indicates that the balance of interhemispheric response processing was similar to wild type animals (LI values +0.42±0.04, 0.36±0.04 for acallosal and control animals respectively; acallosal n=6, control n=6; Figure 6C). This observation suggested that significant ipsilateral non-crossing fibers could exist in cases where transcallosal communication is disrupted either by underdevelopment of the callosum (I/LnJ mouse) or after a relatively large stroke. These results in acallosal animals establish a precedent for ipsilateral responses without prominent transcallosal connections.  30  The effects of unilateral pharmacological inactivation of thalamic nuclei on sensory-evoked responses after stroke To address the source of the ipsilateral response of the affected forepaw after stroke, we manipulated thalamic activity while performing electrophysiological characterization and VSD imaging (Figure 7A). Studies have shown that plasticity after stroke not only occurs near the infarct region, but also at more distant sites (Dancause et al., 2005b; Lu Li, 2006; Brown et al., 2009). Recordings were made from the VPL, VPM, and PoM of the thalamus while concurrent EEG recordings were made at the cortical surface during delivery of sensory stimulation to the forepaw. The location of the injections was confirmed using biocytin delivered alongside TTX in the LFP recording pipette (Figure 7B). Left forelimb stimulation in controls led to a robust LFP at all three nuclei as well as a strong cortical EEG response (Figure 7D.III-IV, 7F). In stroke animals, we observed a significantly diminished cortical EEG response and interestingly a significantly diminished sensory-evoked field potential within the aforementioned thalamic nuclei (p<0.0073) as compared to controls (Figure 7C.III-IV, 7F). The amplitude of the local field potential within the thalamic nuclei of control animals was decreased significantly after the TTX injection (Figure 7DIII-IV, 7F). TTX treatment did not further attenuate the diminished thalamic response in strokeaffected animals. When VPL, VPM and PoM thalamic nuclei were pharmacologically inhibited using 30 µM tetrodotoxin (TTX) in control mice, the VSD response was blocked in both the contralateral and ipsilateral hemisphere (ipsilateral ΔF/Fο before injection 0.225±0.067 versus after injection, 0.023±0.003, p < 0.0078 n=6 mice) (Figure 7C-D.I-II). This provided further evidence that before stroke, the ipsilateral signal was the result of transmission through trans-callosal fibers that homotopically 31  connect the two somatosensory cortices. When the same thalamic nuclei were injected with TTX in animals 8-10 weeks after a large stroke, the ipsilateral signal remained at the same amplitude as before the injection (Figure 7D.II), suggesting that ipsilateral activation after stroke is a result of subcortical or ipsilateral changes that unmask an ipsilateral pathway (ipsilateral ΔF/Fο before injection 0.16±0.041 vs. after injection, 0.15±0.026 p=0.8; n=6) (Figure 7E). To confirm that the ipsilateral activation of the contralesional hemisphere 8-10 weeks after stroke was attributed to uncrossed fibers we injected the ipsilateral (to stimulation) thalamic nuclei VLP, VPM, and PoM, of stroke recovering animals with TTX (n=4; Figure 8A). Following the injection we found that the ipsilateral VSD response present in the unaffected hemisphere was significantly decreased (peak amplitude (ΔF/Fo) 0.16±0.021, response integral ((ΔF/Fo).ms 10.4±1.3 before TTX injection; Peak amplitude 0.03±0.01 (p<0.00082), response integral 2.4±0.4 (p<0.0079) after injection) substantiating the role of uncrossed ascending fibers (Figure 8B). These results implicate the ipsilateral thalamus in relaying information to the ipsilateral cortex after 8-10 weeks of recovery from stroke.  32  DISCUSSION We have used in vivo VSD imaging and pharmacological manipulations of cortical and subcortical activity to determine the mechanism of ipsilateral activation of sensory cortex after stroke. Before stroke, transcallosal connections are the primary means by which ipsilateral responses are generated. In contrast, 8-10 weeks after a large cortical stroke, an uncrossed thalamocortical sensory pathway mediates ipsilateral activation. We have previously shown that 8 weeks following recovery from a focal forelimb somatosensory stroke, new sensory maps are formed within the peri-infarct motor and hindlimb somatosensory representations (Brown et al., 2009). In a recent study, we also showed that there is also evidence of rapid, within hours, interhemispheric redistribution of somatosensory cortex activity after ischemic damage that is distinct from what is observed here because it relies on different circuits dependent of the contralateral thalamus switching signal lateralization (Mohajerani et al., 2011a). This rapid redistribution may involve surviving redundant pathways within peri-infarct regions, or re-distributed activity at sub-cortical levels within the affected hemisphere (Murphy and Corbett, 2009; Sigler et al., 2009). However, previous findings also support the notion that lesion-induced plasticity can take place over extended periods and involve distant brain areas as addressed here (Carmichael and Chesselet, 2002; Lee et al., 2004; Biernaskie et al., 2005; Dancause et al., 2005a; Dancause, 2006; Li et al., 2010). Data has emerged in support of bihemispheric processing of sensory information in response to unilateral stimulation (Salmelin et al., 1995; Hansson and Brismar, 1999). Given that somatosensory  33  cortices in both hemispheres are homologous in function and homotopically connected (Ferezou et al., 2007; Devor et al., 2008; Marcano-Reik and Blumberg, 2008), the ipsilateral hemisphere could shoulder the computational burden of the contralateral lesioned hemisphere after a large stroke. Behavioral studies in rodents and primates suggest that even after a large stroke skilled motor behavior can improve over weeks (Stroemer et al., 1995; Frost et al., 2003). It is, therefore, imperative to understand the mechanism underlying ipsilateral activation to better evaluate its potential compensatory role following stroke.  Multiple circuits contribute to bilateral activation of the somatosensory cortices in response to unilateral stimulation Bilateral activation of the somatosensory cortex has been extensively investigated in humans and other mammals using techniques such as functional magnetic resonance imaging (fMRI) (Disbrow et al., 1998), positron emission tomography (PET) (Burton et al., 1993) and VSD imaging (Ferezou et al., 2007; Brown et al., 2009). A prevalent view is that most ipsilateral activity is relayed from the contralateral hemisphere and is dependent on afferents that connect homotopic regions via the corpus callosum (Manzoni, 1997; Petersen, 2007; Petreanu et al., 2007). Callosotomy studies in humans have demonstrated that ipsilateral activation of the somatosensory cortex to stimulation of the ipsilateral hand depends on the transcallosal crossing of sensory response from the contralateral cortex to the homotopic ipsilateral cortex (Fabri et al., 1999) consistent with our lidocaine injection results.  34  In addition to the corpus callosum, anatomical and physiological studies in mammals suggest that the ipsilateral hemisphere receives approximately 10% uncrossed afferent input from ipsilateral limbs (Armand and Kuypers, 1980). Behavioral studies have also shown that unilateral lesion to the somatosensory cortex can affect sensory function in the ipsilateral limb (Price and Fowler, 1981; Baskett et al., 1996; Sunderland et al., 1999). This evidence, together with laterality of sensory processing, shown in this study and clinical reports (Rossini et al., 2003) imply that each hemisphere exerts a degree of bilateral control over its specialized functions. Therefore, damage to one hemisphere may produce bilateral deficits in performance of lateralized functions (Robinson et al., 1990; Biernaskie et al., 2005), and conversely ipsilateral processing could in part compensate for contralateral deficits caused by ischemic lesions.  What is the function of ipsilateral activation after a unilateral ischemic lesion? Is the return of an ipsilateral response after a large stroke a measure of recovery, or the result of irrevocable injury? Some studies suggest that patients that retain relatively normal lateralization of sensory processing after stroke show better recovery. In contrast, patients exhibiting bilateral cortical activation, typically after a stroke that affects relatively larger cortical and subcortical regions show less recovery (Ward et al., 2003b; Cramer, 2008). These studies presume that the ipsilateral activation is a result of the failure of compensatory mechanisms to restore lateralized sensory activity. However, it must be noted that in case of a large stroke, as reported in these studies, the ischemic lesion also destroys most of the tissue within the  35  affected hemisphere that has similar function (Figure 9A.II). As a result, sensory function maybe unable to remap within the affected hemisphere and new patterns of activation observed may be the result of inter-hemispheric compensation (Schiene et al., 1996; Redecker et al., 2002; Murphy and Corbett, 2009). Our results demonstrate that wide-scale bi-hemispheric circuit changes occur after stroke in response to the loss of contralateral sensory processing. Although it is not possible to firmly conclude that ipsilateral activation after stroke signals behavioral recovery, we can extrapolate from the data that the ipsilateral signal must be important as large-scale circuits undergo changes to restore it. Other spinal cord injury (Ghosh et al., 2010) and stroke research provide evidence for large-scale changes in circuitry following injury (Strittmatter et al., 1992; Carmichael et al., 2001; Lee et al., 2004; Clarkson et al., 2010). In support of our findings, behavioral studies in rats have shown that the undamaged (ipsilateral) circuitry can contribute to recovery of the stroke-affected forelimb (Biernaskie et al., 2005).  Mechanistic insight into ipsilateral responses in stroke-recovering animals While the appearance of a maintained ipsilateral signal independent of the contralateral hemisphere weeks after a large stroke was evident, the underlying mechanisms are not entirely clear. After stroke, changes in GABAergic inhibition at cortical and subcortical sites are a well-documented phenomenon (Schiene et al., 1996; Redecker et al., 2002; Clarkson et al., 2010). Therefore, a contralateral hemisphere-independent ipsilateral signal may reflect a combination of acute and chronic processes such as a loss of inhibition within cortical or thalamic circuits  36  leading to facilitation of existing ipsilateral fibers over time (Figure 9B,C). It must be noted that the absolute amplitude of the ipsilateral signal did not change after stroke (Figure 2B.III). This may be due to the fact that the percentage of fibers contributing to the ipsilateral pathway are far less than those which traverse the corpus callosum; therefore, disinhibition or increased excitation of subcortical and cortical circuits only brings the resulting signal to a level comparable with the pre-stroke ipsilateral VSD response amplitude. Alternatively, the composition of excitatory receptors could change after stroke in the peri-infarct zone, as well as distant sites leading to relatively smaller ipsilateral responses (Nudo et al., 2001; Liu et al., 2007). This change may be reflected in the spatial and temporal profile of the VSD response and the resulting differences in the ipsilateral response integral after stroke. In control animals, sensory responses were more localized to the center of the forelimb or hindlimb area and decayed rapidly. In contrast, sensory responses in stroke recovering animals were prolonged and spread over wider cortical areas (Figure 2B.III). Importantly, the recovered ipsilateral forelimb sensory response was not altered within off-targets areas such as the barrel field/trunk sensory region after stroke suggesting that stroke recovery does not reflect general disinhibition (Figure 2B.V). If ipsilateral activation after stroke is through a non-crossed pathway we may expect it to have shorter onset latency given it does not need to traverse the corpus callosum. However, we did not detect a shorter onset latency possibly due to the changes in receptor expression or inhibition discussed above, or due to a lack of methodological sensitivity.  37  It is also conceivable that disinhibition (Redecker et al., 2002; Reinecke et al., 2003) can affect interhemispheric activity. The effects of a loss of feed-forward inhibition are best studied in paradigms where activity is transiently blocked either by pharmacological means (Vanderwolf et al., 1987; Lavallee et al., 2005), or recently in our work with a targeted photothrombotic stroke. We showed that <1 h after stroke the inter-hemispheric balance of activity can change through a different mechanism than reported here after 8-10 weeks (Sigler et al., 2009; Mohajerani et al., 2011a). Within an hour after stroke we demonstrated that bilateral rearrangement of activity involved novel intra- and inter-thalamic crossing points, and not the dedicated noncrossed ipsilateral circuits as is observed here up to 10 weeks after stroke. Presumably, interthalamic re-routing of signals observed 1 h after stroke is a temporary way of relaying information to the non-stroke affected hemisphere. Over 810 weeks of recovery, this pathway changes so that a completely non-crossed pathway is used with little involvement of the thalamus or cortex with the strokeeffected hemisphere. Interestingly, our work suggests that the responsiveness of the contralateral thalamus may be diminished 8-10 weeks after stroke further supporting an entirely uncrossed mechanism of ipsilateral cortical activation (Figure 7F;8 ;9).  Future directions and conclusions In conclusion, we have used a number of experimental approaches to show bilateral changes in the sensory cortex after stroke. Our data indicates that a targeted small stroke in the somatonsensory cortex leads to an initial loss of function followed by remapping of function onto viable, peri-infarct tissue with similar function, such as the HL somatosensory cortex and the motor representation of FL and HL. Secondly,  38  we were able to show that the spatiotemporal dynamics of sensory-evoked responses become prolonged and more spread out over the cortex after stroke and this phenomenon is observed bilaterally. Thirdly, we demonstrated that after a large stroke, there’s extensive rewiring, consisting of unmasking of latent pathways which lead to ipsilateral somatosensory processing via uncrossed sensory fibers as compared to transcallosal fibers from the contralateral hemisphere, in an intact brain. These findings offer new insight into widespread changes in functional circuitry after stroke and provide a new comprehensive circuit model for understanding how the adult brain adapts to damages induced by stroke. Although functional consequences of the un-crossed ipsilateral sensory processing pathway we describe are unknown, this work provides important clues into the mechanism by which remapping of the ipsilateral signal occurs weeks after stroke. A monumental challenge in this field, linking functional and structural changes in circuitry to behavioral and functional recovery remains unsolved. New genetically targeted tools, such as optogenetic excitation and inhibition of neuronal function (Arenkiel et al., 2007; Airan et al., 2009; Gradinaru et al., 2009) in combination with promoters that are activated during stroke recovery (Li et al., 2010) will need to be used to unambiguously link specific changes in circuitry to improvements in behavior. Evidence from spinal cord injury research has shown that after a brief period of network priming using light-activated channelrhodopsin-2, rhythmic activity associated with breathing can be restored (Alilain et al., 2008). There’s also clinical evidence for behavioral improvement in stroke patients after the use of less welltargeted stimulation techniques such as transcranial magnetic stimulation (TMS)  39  (Plow et al., 2009). Therefore, identifying circuitry that undergoes changes after stroke can help provide a viable target for interventions such as TMS and in a not-sodistant future, optogenetic stimulation.  40  FIGURES  Figure 1. Experimental timeline and schematics of sensory circuitry and targeted stroke location. (A) Timeline of experimental procedures. Mice are imaged either before stroke, at 1 week after or 8-10 weeks after stroke. (B) The forelimb has afferent fibers that travel to the contralateral hemisphere as well as a small percentage of ipsilateral fibers that travel to the hemisphere on the same side of the body. A small stroke affects only the forelimb (FL) somatosensory cortex whereas a large stroke targets the forelimb, hindlimb and part of the primary motor cortex. (C) Each hemisphere receives input from the contralateral limb as well as ipsilateral fibers from the limb on the same side. The two hemispheres are also connected homo-topically via the corpus callosum. The thalamic sensory nuclei, including VPL/VPM and PoM are also connected via intra-thalamic fibers. Together, these serve as potential pathways for plasticity.  41  42  Figure 2. Assessment of bilateral sensory processing within the contralateral (stroke-affected hemisphere) and ipsilateral hemispheres using VSD. (A) I. In control animals, there is bilateral activation in response to a single, unilateral tap. II. 1 week after stroke, the signal has disappeared bilaterally. III. 8 wks after a small stroke, the peri-infarct tissue responds to stimulation. The ipsilateral signal also returns. iv. 8wks following a large stroke, there is very small peri-lesional activation, but the ipsilateral signal is present and diffuse throughout the hemisphere. The LI measure for the samples from each group is calculated based on the peak amplitude measure of the regions of interest in each hemisphere. (B) I. The stimulation of the left (affected) forelimb by a single tap to the paw. II. Graph showing contralateral forelimb area response before and after small and large stroke. The responses within the affected forelimb cortex is significantly diminished (p < 0.001) after large and small stroke. III. The responses within the contralesional hemisphere in response to ipsilateral stimulation is not changed in amplitude compared to controls. The spatio-temporal dynamics of the ipsilateral response change after stroke. iv. The green square indicates the region of interest posterior to the forelimb representation encompassing the primary sensory truck and barrel field cortices. v. Ipsilateral truck and barrel field primary sensory response to ipsilateral stimulation. The responses are similar in amplitude before and after stroke indicating a lack of general dis-inhibition.  43  Figure 3. Quantification of the VSD response amplitude, integral, and latency to peak following 8-10 weeks of recovery from stroke. (A) A single, unilateral tap was delivered to the forepaw of the mouse. (B) The data was collected and statistics calculated based on the response curve. (C-E) After both small and large strokes, the time to peak latency is significantly increased and the peak amplitude is significantly decreased in the contralateral (affected hemisphere) (p <0.001). The peak amplitude of the ipsilateral forelimb area response remains unaffected after stroke. (F) Response integral was calculated for both the contralateral and the ipsilateral responses in control and stroke animals. The contralateral response integral was significantly larger in control animals as compared to small or large stroke animals (p<0.001). In the ipsilateral hemisphere, the response integral is significantly larger in small and large stroke animals as compared to control animals (p<0.05).  44  Figure 4. Laterality of signal processing before and 8-10 weeks after stroke. (A) Laterality calculations based on the peak amplitude, this laterality index indicates that in control animals, the signal is mainly processed in the hemisphere contralateral to the stimulation. After a small stroke, the amplitude of the sensory signal was nearly equal in both hemispheres. After a large stroke, the signal amplitude in the ipsilateral hemisphere is nearly equivalent, while contralateral signal decreases in amplitude, shifting the symmetry of signal processing towards the ipsilateral hemisphere. Positive values are indicative of a contralateral processing and negative values designate ipsilateral processing. (B) Laterality calculations based on the response integral, this laterality index indicates that in control animals, the signal is more robust in amplitude showing a 45  larger area under the response curve. After a small stroke, the response integral is nearly equal in both hemispheres. After a large stroke, the response integral in the ipsilateral hemisphere indicates a longer lasting response while the contralateral signal is almost completely diminished, shifting the symmetry of the laterality index towards the ipsilateral hemisphere. Positive values are indicative of a contralateral processing and negative values designate ipsilateral processing. (C) VSD responses in control, small stroke and large stroke animals at 2 time points. This data is a representation of the data from which the laterality indices were calculated.  46  Figure 5. Unilateral cortical silencing with lidocaine does not affect the ipsilateral response 8-10 weeks after a large stroke. (A) Left forelimb is stimulated using a piezo-electric device. (B) Lidocaine is applied locally to the cortex (200-300 µm below the surface) to transiently silence activity in response to left FL stimulation in the right FL somatosensory cortex. (C) i&ii. In control animals, silencing the contralateral hemisphere also silences the ipsilateral response. In stroke animals, lidocaine silences the remaining contralateral 47  response, but the ipsilateral response remains unaffected (p < 0.01). (D) VSD responses to stimulation before and after lidocaine injection. The white circles indicate location of lidocaine injection. I. In control animals, the ipsilateral response is silenced after the local application of lidocaine. II. 8 weeks after a large stroke, silencing any remnant peri-infarct activity using lidocaine does not affect the ipsilateral response and the amplitude of the signal does not change when compared to ipsilateral signal amplitude in controls.  48  Figure 6. Bilateral somatosensory responses can exist in the absence of a corpus callosum. (A) VSD response to left forelimb stimulation reveals bilateral somatosensory forelimb cortex activation. (B) Coronal section of the brain of an acallosal mouse (Nissl stained) reveals the absence of a corpus callosum. (C-D) In comparison to the forelimb-stimulated sensory responses in controls, the acallosal animals show the same level of laterality of evoked responses. In addition, the peak amplitude of the ipsilateral forelimb responses is comparable between the control animals and the acallosal mice. Altogether, this suggests that other pathways aside from the corpus callosum, perhaps ipsilateral or subcortical, provide bilateral circuitry for cortical sensory processing.  49  Figure 7. LFP recording and pharmacological manipulation of thalamic nuclei reveal un-crossed pathways as the source of ipsilateral activation after large strokes. VSD responses in control and stroke animals before and after silencing thalamic nuclei VPL, VPM and PoM with TTX (30 µM). (A-B) The location of the thalamic injections. The injection site was visualized using biocytin staining. The coordinates were obtained using the work of Franklin and Paxinos (2004). (C) In control animals, silencing of the contralateral thalamic nuclei silences both the contralateral and ipsilateral signal. In stroke animals, TTX injection into the  50  aforementioned subcortical structures does not silence the ipsilateral (to stimulated paw) hemisphere signal. (D) I. In controls, VSD response within the ipsilateral hemisphere is silenced after TTX injection (p < 0.001). II. In stroke animals, the ipsilateral response is not changed in amplitude or spatiotemporal dynamics after the TTX injection. III-IV. EEG and LFP recordings in control and stroke animals reflect the results found by the VSD signal. (E) Peak amplitude of the ipsilateral response before and after TTX injection into thalamic nuclei in control and stroke animals. (F) The amplitude of the LFP recordings from thalamic nuclei VPL, VPM and PoM were significantly larger in control animals as compared to stroke animals, before or after inactivation with TTX.  51  Figure 8. Pharmacological manipulation of ipsilateral thalamic nuclei blocks ipsilateral activation after stroke. (A) Schematic of TTX injection into the ipsilateral VPL, VPM and PoM thalamic nuclei to find the source of the ipsilateral signal. (B) VSD response before and after injection of TTX into the ipsilateral thalamus. The ipsilateral signal is significantly dampened which suggest that the signal travels through ipsilateral fibers that remain uncrossed after the thalamus (p < 0.01).  52  53  Figure 9. Model for cortical and subcortical changes in inhibitory and excitatory connections after stroke. (A) I. Before stroke, the signal is routed contralaterally and transcallosal fibers mediate bilateral somatosensory activation. The predominant route of signal transduction is shown in bold lines. II. After stroke, the ipsilateral sensory signal is mediated through, uncrossed (ipsilateral) fibers, whereas before stroke, transcallosal fibers mediated bilateral somatosensory activation (as shown by bold lines mapping the route of sensory processing). This diagram is representative of the route of sensory transmission before and after stroke. (B) The sensory pathways before and after stroke. Excitatory connections travel contralaterally (1a) and ipsilaterally (1b) from the limb to the thalamus. VPL, VPM and PoM nuclei of the thalamus make excitatory projections to the somatosensory cortex (2) and from the cortex, excitatory connections travel via the corpus callosum to the homotopic contralateral cortex (3a). There is also excitatory feedback from the cortex to the reticular nucleus of the thalamus in the same hemisphere (3b) (Golshani et al., 2001) along with cortical feedback to the VPL and VPM nuclei (Iizuka et al., 1990). RT inhibits the VPL and VPM nuclei of the thalamus (Minderhoud, 1971) (4). VPL nuclei are connected via commissural fibers and exert excitatory influences on to each other (Landry et al., 1984) (5). After stroke the excitatory efferents from the cortex to the RT of thalamus are lost which translates to disinhibition of the VPL and VPM nuclei within the same hemisphere. This disinhibition may lead to potentiation of the unaffected thalamic nuclei which are transhemispherically connected (Landry et al., 1984). Lack of inhibition from the contralateral RT nucleus after stroke may be the underlying cause of the increased excitation of the ipsilateral VPL nucleus (6). During recovery from stroke, the influences of the VPL/M and RT nuclei of the thalamus within the affected hemisphere is reduced (Iizuka et al., 1990). As the cortico-cortical connection via the corpus callosum is also lost (8), the ipsilateral circuit remains the only source of input onto the ipsilateral cortex. 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