Craig E. Brown

Sequential phases of cortical specification involve Neurogenin‐dependent and ‐independent pathways

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early‐born (lower‐layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later‐born (upper‐layer) cortical neurons are specified in an Ngn‐independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.

Extensive Turnover of Dendritic Spines and Vascular Remodeling in Cortical Tissues Recovering from Stroke

Recovery of function after stroke is thought to be dependent on the reorganization of adjacent, surviving areas of the brain. Macroscopic imaging studies (functional magnetic resonance imaging, optical imaging) have shown that peri-infarct regions adopt new functional roles to compensate for damage caused by stroke. To better understand the process by which these regions reorganize, we used in vivo two-photon imaging to examine changes in dendritic and vascular structure in cortical regions recovering from stroke. In adult control mice, dendritic arbors were relatively stable with very low levels of spine turnover (<0.5% turnover over 6 h). After stroke, however, the organization of dendritic arbors in peri-infarct cortex was fundamentally altered with both apical dendrites and blood vessels radiating in parallel from the lesion. On a finer scale, peri-infarct dendrites were exceptionally plastic, manifested by a dramatic increase in the rate of spine formation that was maximal at 1–2 weeks (5–8-fold increase), and still evident 6 weeks after stroke. These changes were selective given that turnover rates were not significantly altered in ipsilateral cortical regions more distant to the lesion (>1.5 mm). These data provide a structural framework for understanding functional and behavioral changes that accompany brain injury and suggest new targets that could be exploited by future therapies to rebuild and rewire neuronal circuits lost to stroke.

In Vivo Voltage-Sensitive Dye Imaging in Adult Mice Reveals That Somatosensory Maps Lost to Stroke Are Replaced over Weeks by New Structural and Functional Circuits with Prolonged Modes of Activation within Both the Peri-Infarct Zone and Distant Sites

After brain damage such as stroke, topographically organized sensory and motor cortical representations remap onto adjacent surviving tissues. It is conceivable that cortical remapping is accomplished by changes in the temporal precision of sensory processing and regional connectivity in the cortex. To understand how the adult cortex remaps and processes sensory signals during stroke recovery, we performed in vivo imaging of sensory-evoked changes in membrane potential, as well as multiphoton imaging of dendrite structure and tract tracing. In control mice, forelimb stimulation evoked a brief depolarization in forelimb cortex that quickly propagated to, and dissipated within, adjacent motor/hindlimb areas (<100 ms). One week after forelimb cortex stroke, the cortex was virtually unresponsive to tactile forelimb stimulation. After 8 weeks recovery, forelimb-evoked depolarizations reemerged with a characteristic pattern in which responses began within surviving portions of forelimb cortex (<20 ms after stimulation) and then spread horizontally into neighboring peri-infarct motor/hindlimb areas in which depolarization persisted 300–400% longer than controls. These uncharacteristically prolonged responses were not limited to the remapped peri-infarct zone and included distant posteromedial retrosplenial cortex, millimeters from the stroke. Structurally, the remapped peri-infarct area selectively exhibited high levels of dendritic spine turnover, shared more connections with retrosplenial cortex and striatum, and lost inputs from lateral somatosensory cortical regions. Our findings demonstrate that sensory remapping during stroke recovery is accompanied by the development of prolonged sensory responses and new structural circuits in both the peri-infarct zone as well as more distant sites.

Rapid Morphologic Plasticity of Peri-Infarct Dendritic Spines After Focal Ischemic Stroke

Background and Purpose— Focal stroke is associated with cell death, abnormal synaptic activity, and neurologic impairments. Given that many of these neuropathologic processes can be attributed to events that occur shortly after injury, it is necessary to understand how stroke affects the structure of neurons in surviving peri-infarct regions, particularly at the level of the dendritic spines, which transmit normal and potentially abnormal and injurious synaptic signaling. Recently, we described ischemia-induced changes in the structure of layer 1 dendritic tufts of transgenic mice expressing YFP in layer 5 cortical neurons. However, these in vivo imaging experiments could not address ischemia-related phenomena that occur in deeper cortical structures/layers, other cortical regions, or submicron changes in dendritic spine structure. Methods— Focal stroke was induced in the forelimb sensorimotor cortex by the photothrombotic method. Two, 6, and 24 hours after stroke, brains were processed for Golgi-Cox staining to permit a detailed analysis of primary apical dendritic spine structure from layer 2/3 and 5 cortical pyramidal neurons. Results— Photothrombotic stroke caused a rapid deterioration of neurons, as revealed by Golgi-Cox labeling, in the infarct core that could be readily distinguished from surviving peri-infarct regions. Analysis of >15 000 dendritic spines revealed that although many spines were lost in the peri-infarct cortex during the first 24 hours after stroke (≈38% lost), spines that remained were significantly longer (≈25% at 6 hours). Furthermore, these effects were found in both layer 2/3 and 5 neurons and were restricted primarily to peri-infarct regions (<200 &mgr;m from the infarct border). Conclusions— These rapid changes in dendritic spine number and length may reflect an early adaptive response of potentially vulnerable peri-infarct neurons coping with postischemic spreading depression-like depolarizations and the loss of presynaptic contacts.

Longitudinal in vivo imaging reveals balanced and branch-specific remodeling of mature cortical pyramidal dendritic arbors after stroke.

The manner in which fully mature peri-infarct cortical dendritic arbors remodel after stroke, and thus may possibly contribute to stroke-induced changes in cortical receptive fields, is unknown. In this study, we used longitudinal in vivo two-photon imaging to investigate the extent to which brain ischemia can trigger dendritic remodeling of pyramidal neurons in the adult mouse somatosensory cortex, and to determine the nature by which remodeling proceeds over time and space. Before the induction of stroke, dendritic arbors were relatively stable over several weeks. However, after stroke, apical dendritic arbor remodeling increased significantly (dendritic tip growth and retraction), particularly within the first 2 weeks after stroke. Despite a threefold increase in structural remodeling, the net length of arbors did not change significantly over time because dendrite extensions away from the stroke were balanced by the shortening of tips near the infarct. Therefore, fully mature cortical pyramidal neurons retain the capacity for extensive structural plasticity and remodel in a balanced and branch-specific manner.

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.

Distribution of zincergic neurons in the mouse forebrain.

Synaptically released zinc is thought to play an important role in neuronal signaling by modulating excitatory and inhibitory receptors and intracellular signaling proteins. Consequently, neurons that release zinc have been implicated in synaptic plasticity underlying learning and memory as well as neuropathological processes such as epilepsy, stroke, and Alzheimers disease. To characterize the distribution of these neurons, investigators have relied on a technique that involves the retrograde transport of zinc–selenium crystals from axonal boutons to the cell bodies of origin. However, one major problem with this method is that labeling of cell bodies is obscured by high levels of staining in synaptic boutons, particularly within forebrain structures where this staining is most intense. Here, we used a modification of the retrograde labeling method that eliminates terminal staining for zinc, thereby enabling a clear and comprehensive description of these neurons. Zincergic neurons were found in all cerebral cortical regions and were arranged in a distinct laminar pattern, restricted to layers 2/3, 5, and 6 with no labeling in layer 4. In the hippocampus, labeling was present in CA1, CA3, and the dentate gyrus but not in CA2. Labeled cell bodies were also observed in most amygdaloid nuclei, anterior olfactory nuclei, claustrum, tenia tecta, endopiriform region, lateral ventricle, lateral septum, zona incerta, superior colliculus, and periaqueductal gray. Moreover, retrograde labeling was also noted in the dorsomedial and lateral hypothalamus, regions that previously were thought to be devoid of neurons with a zincergic phenotype. Collectively these data show that zincergic neurons comprise a large population of neurons in the murine forebrain and will provide an anatomical framework for understanding the functional importance of these neurons in the mammalian brain. J. Comp. Neurol. 479:156–167, 2004.

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.

Livin' on the Edge: Imaging Dendritic Spine Turnover in the Peri-Infarct Zone during Ischemic Stroke and Recovery:

The spontaneous recovery of sensory, motor, and cognitive functions after stroke is thought to be mediated primarily through the reorganization and rewiring of surviving brain circuits. Given that dendritic spine turnover underlies rewiring during normal development and plasticity, this process is likely to play a key role in mediating functional changes that occur during and after stroke. Recently, a new approach has been taken using two-photon microscopy to monitor, in real time, the temporal and spatial progression of dendritic plasticity in the living animal, both while it is experiencing the initial ischemic episode as well as during long-term recovery from stroke damage. Here, we highlight recent evidence showing that stroke can trigger extensive changes in the relatively hardwired adult brain. For example, when dendrites are challenged by acute ischemia, they can disintegrate within minutes of ischemia and rapidly reassemble during reperfusion. Over longer time scales, dendrites in the surviving peri-infarct zone show heightened levels of spine turnover for many weeks after stroke, thereby raising the possibility that future stroke therapies may be able to facilitate or optimize dendritic rewiring to improve functional recovery. NEUROSCIENTIST 14(2):139—146, 2008. DOI: 10.1177/1073858407309854

Experience-dependent regulation of synaptic zinc is impaired in the cortex of aged mice

Zinc plays an important role in synaptic signaling in the mammalian cerebral cortex. Zinc is sequestered into presynaptic vesicles of subpopulations of glutamatergic neurons and is released by depolarization, in a calcium-dependent manner. As the majority of mechanisms that have been suggested to participate in experience-dependent alterations in synaptic strength in the cerebral cortex implicate signaling by glutamate, it stands to reason that zincergic signaling might also be crucial. Here we show that synaptic zinc is rapidly and dynamically modulated in relation to alterations in sensory input and that this response is highly age-dependent. Juvenile, adult, and aged mice were subjected to whisker removal and levels of staining for synaptic zinc in deprived and non-deprived cortical barrels were quantitatively assessed at post-deprivation times ranging from 3 h to 21 days. In the first 12 h, zinc levels increased slightly, but significantly, in all groups. At later time points, zinc levels increased robustly (23%) in the youngest group by 24 h and remained elevated through 7 days. By contrast, deprivation-induced changes in zinc staining in aged animals, achieved their maximal levels at 12 h (approximately 10%) and steadily declined thereafter. Adult animals revealed a biphasic, intermediate change with time. In all age groups, levels of zinc staining returned to baseline by 21 days after whisker plucking. However, only in juvenile and adult mice did we observe that the level of zinc staining in deprived barrel hollows, was correlated with the length of whiskers as they regrew. Our data suggest that alterations in the regulation of synaptic zinc may be involved with decrements of synaptic plasticity that accompany senescence.