Optogenetic rewiring of thalamocortical circuits to restore function in the stroke injured brain.

To regain sensorimotor functions after stroke, surviving neural circuits must reorganize and form new connections. Although the thalamus is critical for processing and relaying sensory information to the cortex, little is known about how stroke affects the structure and function of these connections, or whether a therapeutic approach targeting these circuits can improve recovery. Here we reveal with in vivo calcium imaging that stroke in somatosensory cortex dampens the excitability of surviving thalamocortical circuits. Given this deficit, we hypothesized that chronic transcranial window optogenetic stimulation of thalamocortical axons could facilitate recovery. Using two-photon imaging, we show that optogenetic stimulation promotes the formation of new and stable thalamocortical synaptic boutons, without impacting axon branch dynamics. Stimulation also enhances the recovery of somatosensory cortical circuit function and forepaw sensorimotor abilities. These results demonstrate that an optogenetic approach can rewire thalamocortical circuits and restore function in the damaged brain.

Experience with the "good" limb induces aberrant synaptic plasticity in the perilesion cortex after stroke.

Following unilateral stroke, the contralateral (paretic) body side is often severely impaired, and individuals naturally learn to rely more on the nonparetic body side, which involves learning new skills with it. Such compensatory hyper-reliance on the "good" body side, however, can limit functional improvements of the paretic side. In rats, motor skill training with the nonparetic forelimb (NPT) following a unilateral infarct lessens the efficacy of rehabilitative training, and reduces neuronal activation in perilesion motor cortex. However, the underlying mechanisms remain unclear. In the present study, we investigated how forelimb movement representations and synaptic restructuring in perilesion motor cortex respond to NPT and their relationship with behavioral outcomes. Forelimb representations were diminished as a result of NPT, as revealed with intracortical microstimulation mapping. Using transmission electron microscopy and stereological analyses, we found that densities of axodendritic synapses, especially axo-spinous synapses, as well as multiple synaptic boutons were increased in the perilesion cortex by NPT. The synaptic density was negatively correlated with the functional outcome of the paretic limb, as revealed in reaching performance. Furthermore, in animals with NPT, there was dissociation between astrocytic morphological features and axo-spinous synaptic density in perilesion motor cortex, compared with controls. These findings demonstrate that skill learning with the nonparetic limb following unilateral brain damage results in aberrant synaptogenesis, potentially of transcallosal projections, and this seems to hamper the functionality of the perilesion motor cortex and the paretic forelimb.

Delayed inhibition of VEGF signaling after stroke attenuates blood-brain barrier breakdown and improves functional recovery in a comorbidity-dependent manner.

Diabetes is a common comorbidity in stroke patients and a strong predictor of poor functional outcome. To provide a more mechanistic understanding of this clinically relevant problem, we focused on how diabetes affects blood-brain barrier (BBB) function after stroke. Because the BBB can be compromised for days after stroke and thus further exacerbate ischemic injury, manipulating its function presents a unique opportunity for enhancing stroke recovery long after the window for thrombolytics has passed. Using a mouse model of Type 1 diabetes, we discovered that ischemic stroke leads to an abnormal and persistent increase in vascular endothelial growth factor receptor 2 (VEGF-R2) expression in peri-infarct vascular networks. Correlating with this, BBB permeability was markedly increased in diabetic mice, which could not be prevented with insulin treatment after stroke. Imaging of capillary ultrastructure revealed that BBB permeability was associated with an increase in endothelial transcytosis rather than a loss of tight junctions. Pharmacological inhibition (initiated 2.5 d after stroke) or vascular-specific knockdown of VEGF-R2 after stroke attenuated BBB permeability, loss of synaptic structure in peri-infarct regions, and improved recovery of forepaw function. However, the beneficial effects of VEGF-R2 inhibition on stroke recovery were restricted to diabetic mice and appeared to worsen BBB permeability in nondiabetic mice. Collectively, these results suggest that aberrant VEGF signaling and BBB dysfunction after stroke plays a crucial role in limiting functional recovery in an experimental model of diabetes. Furthermore, our data highlight the need to develop more personalized stroke treatments for a heterogeneous clinical population.

Age-dependent reorganization of peri-infarct "premotor" cortex with task-specific rehabilitative training in mice.

BACKGROUND: The incidence of stroke in adulthood increases with advancing age, but there is little understanding of how poststroke treatment should be tailored by age. OBJECTIVE: The goal of this study was to determine if age and task specificity of rehabilitative training affect behavioral improvement and motor cortical organization after stroke. METHODS: Young and aged mice were trained to proficiency on the Pasta Matrix Reaching Task prior to lesion induction in primary motor cortex with endothelin-1. After a short recovery period, mice received 9 weeks of rehabilitative training on either the previously learned task (Pasta Matrix Reaching), a different reaching task (Tray Reaching), or no training. To determine the extent of relearning, mice were tested once weekly on the Pasta Matrix Reaching Task. Mice then underwent intracortical microstimulation mapping to resolve the remaining forelimb movement representations in perilesion motor cortex. RESULTS: Although aged mice had significantly larger lesions compared with young mice, Pasta Matrix Reaching served as effective rehabilitative training for both age-groups. Young animals also showed improvement after Tray Reaching. Behavioral improvement in young mice was associated with an expansion of the rostral forelimb area ("premotor" cortex), but we failed to see reorganization in the aged brain, despite similar behavioral improvements. CONCLUSIONS: Our results indicate that reorganization of motor cortex may be limited by either aging or greater tissue damage, but the capacity to improve motor function via task-specific rehabilitative training continues to be well maintained in aged animals.

Compensatory limb use and behavioral assessment of motor skill learning following sensorimotor cortex injury in a mouse model of ischemic stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Thinking outside the brain: structural plasticity in the spinal cord promotes recovery from cortical stroke.

Neuroanatomically connected regions distal to a cortical stroke can exhibit both degenerative and adaptive changes during recovery. As the locus for afferent somatosensory fibres and efferent motor fibres, the spinal cord is ideally situated to play a critical role in functional recovery. In contrast to the wealth of research into cortical plasticity after stroke, much less focus has previously been placed on the role of subcortical or spinal cord plasticity in recovery of function after cortical stroke. Little is known about the extent and spatiotemporal profile of spinal rewiring, its regulation by neurotrophins or inflammatory cytokines, or its potential as a therapeutic target to improve stroke recovery. This commentary examines the recent findings by Sist et al. (2014) that there is a distinct critical period of heightened structural plasticity, growth factor expression, and inflammatory cytokine production in the spinal cord. They suggest that neuroplasticity is highest during the first two weeks after stroke and tapers off dramatically by the fourth week. Spinal cord plasticity correlates with the severity of cortical injury and temporally matches periods of accelerated spontaneous recovery of skilled reaching function. The potential of treatments that extend or re-open this window of spinal cord plasticity, such as anti-Nogo-A antibodies or chondroitinase ABC, to dramatically improve recovery from cortical stroke in clinical populations is discussed.

Diabetes augments in vivo microvascular blood flow dynamics after stroke.

Stroke usually affects people with underlying medical conditions. In particular, diabetics are significantly more likely to have a stroke and the prognosis for recovery is poor. Because diabetes is associated with degenerative changes in the vasculature of many organs, we sought to determine how hyperglycemia affects blood flow dynamics after an ischemic stroke. Longitudinal in vivo two-photon imaging was used to track microvessels before and after photothrombotic stroke in a diabetic mouse model. Chronic hyperglycemia exacerbated acute (3-7 d) ischemia-induced increases in blood flow velocity, vessel lumen diameter, and red blood cell flux in peri-infarct regions. These changes in blood flow dynamics were most evident in superficial blood vessels within 500 µm from the infarct, rather than deeper or more distant cortical regions. Long-term imaging of diabetic mice not subjected to stroke indicated that these acute stroke-related changes in vascular function could not be attributed to complications from hyperglycemia alone. Treating diabetic mice with insulin immediately after stroke resulted in less severe alterations in blood flow within the first 7 d of recovery, but had more variable results at later time points. Analysis of microvessel branching patterns revealed that stroke led to a pruning of microvessels in peri-infarct cortex, with very few instances of sprouting. These results indicate that chronic hyperglycemia significantly affects the vascular response to ischemic stroke and that insulin only partially mitigates these changes. The combination of these acute and chronic alterations in blood flow dynamics could underlie diabetes-related deficits in cortical plasticity and stroke recovery.

Skill learning induced plasticity of motor cortical representations is time and age-dependent.

Movement representations in the motor cortex can reorganize to support motor skill learning during young adulthood. However, little is known about how motor representations change during aging or whether their change is influenced by continued practice of a skill after it is learned. We used intracortical microstimulation to characterize the organization of the forelimb motor cortex in young and aged C57/BL6 mice after short (2-4 weeks) or long (8 weeks) durations of training on a skilled reaching task or control procedures. In young mice, a short duration of reach training increased the area of proximal forelimb movement representations at the expense of distal representations. Following a longer training duration, ratios of proximal to distal movements returned to baseline, even with ongoing practice and skill maintenance. However, lingering changes were evident in thresholds for eliciting distal forelimb movements, which declined over the longer training period. In aged mice, movement representations and movement thresholds failed to change after either duration of training. Furthermore, there was an age-related loss of digit representations and performance decrements on other sensorimotor tests. Nevertheless, in quantitative measures of reaching success, aged mice learned and performed the skilled reaching task at least as well as younger mice. These results indicate that experience-driven topographical reorganization of motor cortex varies with age, as well as time, and is partially dissociable from behavioral performance. They also support an enduring capacity to learn new manual skills during aging, even as more youthful forms of cortical plasticity and sensorimotor function are lost.

Diabetes impairs cortical plasticity and functional recovery following ischemic stroke.

Diabetics are at greater risk of having a stroke and are less likely to recover from it. To understand this clinically relevant problem, we induced an ischemic stroke in the primary forelimb somatosensory (FLS1) cortex of diabetic mice and then examined sensory-evoked changes in cortical membrane potentials and behavioral recovery of forelimb sensory-motor function. Consistent with previous studies, focal stroke in non-diabetic mice was associated with acute deficits in forelimb sensorimotor function and a loss of forelimb evoked cortical depolarizations in peri-infarct cortex that gradually recovered over several weeks time. In addition, we discovered that damage to FLS1 cortex led to an enhancement of forelimb evoked depolarizations in secondary forelimb somatosensory (FLS2) cortex. Enhanced FLS2 cortical responses appeared to play a role in stroke recovery given that silencing this region was sufficient to reinstate forelimb impairments. By contrast, the functional reorganization of FLS1 and FLS2 cortex was largely absent in diabetic mice and could not be explained by more severe cortical infarctions. Diabetic mice also showed persistent behavioral deficits in sensorimotor function of the forepaw, which could not be rescued by chronic insulin therapy after stroke. Collectively these results indicate that diabetes has a profound effect on brain plasticity, especially when challenged, as is often the case, by an ischemic event. Further, our data suggest that secondary cortical regions play an important role in the restoration of sensorimotor function when primary cortical regions are damaged.

The organization of the forelimb representation of the C57BL/6 mouse motor cortex as defined by intracortical microstimulation and cytoarchitecture.

The organization of forelimb representation areas of the monkey, cat, and rat motor cortices has been studied in depth, but its characterization in the mouse lags far behind. We used intracortical microstimulation (ICMS) and cytoarchitectonics to characterize the general organization of the C57BL/6 mouse motor cortex, and the forelimb representation in more detail. We found that the forelimb region spans a large area of frontal cortex, bordered primarily by vibrissa, neck, shoulder, and hindlimb representations. It included a large caudal forelimb area, dominated by digit representation, and a small rostral forelimb area, containing elbow and wrist representations. When the entire motor cortex was mapped, the forelimb was found to be the largest movement representation, followed by head and hindlimb representations. The ICMS-defined motor cortex spanned cytoarchitecturally identified lateral agranular cortex (AGl) and also extended into medial agranular cortex. Forelimb and hindlimb representations extended into granular cortex in a region that also had cytoarchitectural characteristics of AGl, consistent with the primary motor-somatosensory overlap zone (OL) characterized in rats. Thus, the mouse motor cortex has homologies with the rat in having 2 forelimb representations and an OL but is distinct in the predominance of digit representations.

The vermicelli and capellini handling tests: simple quantitative measures of dexterous forepaw function in rats and mice.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. & Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

Sensorimotor behavioral effects of endothelin-1 induced small cortical infarcts in C57BL/6 mice.

Mouse models have not paralleled rat models of stroke in advances in sensitive, species appropriate measures of neurological and behavioral recovery. Most available tests of mouse sensorimotor function are adaptations of those originally developed in rats and may not be as sensitive in detecting behavioral deficits after small cortical lesions in mice. Our purpose was to test the use of a vasoconstricting peptide, endothelin-1 (ET-1), to produce focal infarcts of the mouse sensorimotor cortex and to establish a behavioral test battery sensitive to resulting sensorimotor deficits. Young adult (3-5-month-old) male C57BL/6 mice received intracortical infusions of ET-1 that produced unilateral lesions of the forelimb region of the sensorimotor cortex, intracortical infusions of sterile saline, or sham surgeries. Pre-operatively and at various time points over 3 weeks post-surgery, they were administered a test battery that included measures of sensorimotor asymmetry (Corner and Bilateral Tactile Stimulation Tests), coordinated forepaw use (Cylinder and Ladder Rung Tests), and dexterous forepaw function (Pasta Matrix Reaching Test). ET-1 infusions resulted in consistently placed, focal cortical infarcts and forelimb impairments as measured with the Ladder Rung, Bilateral Tactile Stimulation, and Pasta Matrix Reaching Tests. On the Bilateral Tactile Stimulation and Pasta Matrix Reaching Tests, impairments persisted throughout the time span of observation (26 days). These results support ET-1 as a viable option for creating small, reproducible lesions of anatomical subregions in the mouse neocortex that result in lasting functional impairments in the forelimb, as observed with sufficiently sensitive measures.