Visualization of mouse barrel cortex using ex-vivo track density imaging

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo and T2-weighted imaging and gradient echo Ti/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3 mm)3 block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system. (C) 2013 Elsevier Inc. All rights reserved

Reward circuitry is perturbed in the absence of the serotonin transporter

The serotonin transporter (SERT) modulates the entire serotonergic system in the brain and influences both the dopaminergic and norepinephrinergic systems. These three systems are intimately involved in normal physiological functioning of the brain and implicated in numerous pathological conditions. Here we use high-resolution magnetic resonance imaging (MRI) and spectroscopy to elucidate the effects of disruption of the serotonin transporter in an animal model system: the SERT knock-out mouse. Employing manganese-enhanced MRI, we injected Mn^(2+) into the prefrontal cortex and obtained 3D MR images at specific time points in cohorts of SERT and normal mice. Statistical analysis of co-registered datasets demonstrated that active circuitry originating in the prefrontal cortex in the SERT knock-out is dramatically altered, with a bias towards more posterior areas (substantia nigra, ventral tegmental area, and Raphé nuclei) directly involved in the reward circuit. Injection site and tracing were confirmed with traditional track tracers by optical microscopy. In contrast, metabolite levels were essentially normal in the SERT knock-out by in vivo magnetic resonance spectroscopy and little or no anatomical differences between SERT knock-out and normal mice were detected by MRI. These findings point to modulation of the limbic cortical–ventral striatopallidal by disruption of SERT function. Thus, molecular disruptions of SERT that produce behavioral changes also alter the functional anatomy of the reward circuitry in which all the monoamine systems are involved

Lamina-specific AMPA receptor dynamics following visual deprivation in vivo.

Regulation of AMPA receptor (AMPAR) expression is central to synaptic plasticity and brain function, but how these changes occur in vivo remains elusive. Here, we developed a method to longitudinally monitor the expression of synaptic AMPARs across multiple cortical layers in awake mice using two-photon imaging. We observed that baseline AMPAR expression in individual spines is highly dynamic with more dynamics in primary visual cortex (V1) layer 2/3 (L2/3) neurons than V1 L5 neurons. Visual deprivation through binocular enucleation induces a synapse-specific and depth-dependent change of synaptic AMPARs in V1 L2/3 neurons, wherein deep synapses are potentiated more than superficial synapses. The increase is specific to L2/3 neurons and absent on apical dendrites of L5 neurons, and is dependent on expression of the AMPAR-binding protein GRIP1. Our study demonstrates that specific neuronal connections, across cortical layers and even within individual neurons, respond uniquely to changes in sensory experience

Novel Endoscope for Surgical Guidance and Functional Brain Imaging

Optical imaging methods have become very powerful tools in biomedical research since they can achieve both high spatial and temporal resolutions. However, due to the effects of light scattering, the penetration depth of optical imaging methods is usually limited. Gradient refractive index (GRIN) lens that are 350–2,000 μm in diameter and provide micron-scale resolution have been shown to enable minimally invasive in vivo imaging of deep tissues. Based on GRIN lens, we developed a small hand-held optical coherence tomography (OCT) forward-imaging needle device for real-time epidural anesthesia surgery guidance and demonstrated its feasibility through ex vivo and in vivo animal experiments. Besides, to access subcortical brain structures, we combined voltage-sensitive dye imaging (VSDi) with GRIN lens to image neural activities evoked in thalamic barreloids by deflection of whiskers in vivo in the whisker system of rodents

Experience-dependent structural plasticity at pre- and postsynaptic sites of layer 2/3 cells in developing visual cortex

The developing brain can respond quickly to altered sensory experience by circuit reorganization. During a critical period in early life, neurons in the primary visual cortex rapidly lose responsiveness to an occluded eye and come to respond better to the open eye. While physiological and some of the molecular mechanisms of this process have been characterized, its structural basis, except for the well-known changes in the thalamocortical projection, remains obscure. To elucidate the relationship between synaptic remodeling and functional changes during this experience-dependent process, we used 2-photon microscopy to image synaptic structures of sparsely labeled layer 2/3 neurons in the binocular zone of mouse primary visual cortex. Anatomical changes at presynaptic and postsynaptic sites in mice undergoing monocular visual deprivation (MD) were compared to those in control mice with normal visual experience. We found that postsynaptic spines remodeled quickly in response to MD, with neurons more strongly dominated by the deprived eye losing more spines. These postsynaptic changes parallel changes in visual responses during MD and their recovery after restoration of binocular vision. In control animals with normal visual experience, the formation of presynaptic boutons increased during the critical period and then declined. MD affected bouton formation, but with a delay, blocking it after 3 d. These findings reveal intracortical anatomical changes in cellular layers of the cortex that can account for rapid activity-dependent plasticity

The Role Of The Nmda Receptor In Shaping Cortical Activity During Development

Currently, it is estimated that neuropsychiatric disorders will affect 20-25% of humans in their lifetime. These disorders are a major cause of mortality, suffering, and economic cost to society. Within this broad class, neurodevelopmental disorders (NDDs), including intellectual disability, autism spectrum disorder, and schizophrenia, are estimated to affect 2-5% percent of the world population. Devastatingly, we lack fundamental treatments for NDDs, which have proved some of the most imposing disorders to understand scientifically. The challenge is twofold: first, NDDs affect the most complex aspects of human cognition; second, pathogenesis begins early in neural circuit development, but we lack predictive biomarkers before overt behavioral deficits are apparent. Although we have identified many genes associated with these disorders, how underlying genetic disruptions lead to pathological neural network development and function remains unclear. The overarching framework of this dissertation is that all NPDs are disorders of distributed neural networks, and pathophysiology must be understood at this level to effectively intervene clinically. The cerebral cortex is necessary for complex human capacities, and cortical dysfunction is hypothesized to be central to the pathophysiology of NDDs. NMDA glutamate receptors (NMDARs) are important for the development of local circuit features in the cortex, for normal neurocognitive function, and are strongly implicated in NDDs. However, the role of NMDARs in the development of the large-scale cortical network dynamics that underly higher cognition has not been well examined. Understanding the role of NMDARs at this network level is critical because large-scale “functional connectivity” patterns are thought to be hallmarks of normal cortical function, are hypothesized to be disrupted in NDDs, and may be detectable in humans using non-invasive neuroimaging or electrophysiology. In the studies presented in this dissertation, I (in collaboration and with the support of my colleagues) tested the role of the NMDAR in shaping large-scale cortical network organization using in vivo widefield imaging of whole cortex spontaneous activity in developing mice. I found that NMDAR function in the lineage that includes cortical excitatory neurons and glia, specifically, was critical for the elaboration of normal cortical activity patterns and dynamic network organization. In the first set of experiments, NMDARs were deleted in glutamatergic excitatory neurons (Emx1-cre+/WT/Grin1f/f ; referred to as EX-NMDAR KO mice) or GABAergic inhibitory neurons (Nkx2.1+/WT/Grin1f/f; referred to as IN-NMDAR KO mice). The developing cortex normally exhibits a diverse range of spatio-temporal patterns, reflecting the emergence of functionally associated sub-networks. In EX-NMDAR KO mice, normal patterns of spontaneous activity were severely disrupted and reduced to a nearly one-dimensional dynamic space dominated by large, cortex-wide events. Interestingly, in IN-NMDAR KO mice, the structure and complexity of spontaneous activity was largely normal. In the next set of experiments, I tested the role of extrinsic thalamic neurotransmission on cortical activity during development. Deleting the vesicular glutamate transporter from thalamic neurons while leaving cortical NMDARs intact (Sert-Cre+/−,vglut1−/−,vglut2fl/fl; referred to as TH-VG KO mice) led to a shift in cortical activity patterns towards large domains of activity, reminiscent of patterns observed in EX-NMDAR KO mice. This manipulation also reduced the dimensionality of cortical activity, though not as severally as in EX-NMDAR KO mice. In a final set of experiments, I tested cortical activity in three established mouse models of mono-genetic causes of NDDs in humans: the FMR1-KO mouse based on Fragile X Syndrome, the CNTNAP2-KO mouse, and the TS2-neo mouse based on Timothy Syndrome. In all three of these mouse models, I found that large-scale cortical activity patterns were largely normal, but there was a statistically significant shift towards reduced cortex-wide synchrony and increased dimensionality of spontaneous activity, which may be consistent with the disconnectivity hypothesis of autism. In a final set of experiments, we tested our hypothesis, based on past literature and our results in EX-NMDAR KO and TH-VG KO mice, that the disruptions in cortical activity was predominantly due to the developmental loss of activity-dependent wiring of circuits. To test the developmental versus acute role of NMDAR function in shaping cortical activity, I blocked NMDAR pharmacologically in wild-type mice. I found that acute NMDAR blockade shifted cortical activity to a restricted dynamic space similar to that observed in EX-NMDAR KO mice and more extreme than that observed in TH-VG KO mice. These results strongly reinforce the critical role of NMDAR in shaping cortical activity during development, and suggest that a substantial component of that may be through NMDAR’s role in synaptic transmission and moment to moment cortex-wide circuit function. Overall, these results provide critical insight into the role of NMDARs and the glutamatergic system in cortical network functional organization during development. Specifically, they highlight the essential role of NMDARs in excitatory neurons on the functional connectivity and dynamic repertoire of the cortical network during development. These results make novel contribution to our understanding of how NMDARs may contribute to the pathophysiology of NDDs. Specifically, they contribute powerful new insight into to a critical mechanistic question about the cell-specific role of NMDARs in the pathophysiology of schizophrenia and the mechanisms of NMDAR antagonists, which have transformed psychiatry recently due to their rapid-acting anti-depressant and anti-suicidal properties. Furthermore, they identify a patterns of large-scale network dysfunction that might be detectable in humans using noninvasive functional imaging or electrophysiology

NOVEL TECHNOLOGIES AND APPLICATIONS FOR FLUORESCENT LAMINAR OPTICAL TOMOGRAPHY

Laminar optical tomography (LOT) is a mesoscopic three-dimensional (3D) optical imaging technique that can achieve both a resolution of 100-200 µm and a penetration depth of 2-3 mm based either on absorption or fluorescence contrast. Fluorescence laminar optical tomography (FLOT) can also provide large field-of-view (FOV) and high acquisition speed. All of these advantages make FLOT suitable for 3D depth-resolved imaging in tissue engineering, neuroscience, and oncology. In this study, by incorporating the high-dynamic-range (HDR) method widely used in digital cameras, we presented the HDR-FLOT. HDR-FLOT can moderate the limited dynamic range of the charge-coupled device-based system in FLOT and thus increase penetration depth and improve the ability to image fluorescent samples with a large concentration difference. For functional mapping of brain activities, we applied FLOT to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. We utilized FLOT to investigate the cell viability, migration, and bone mineralization within bone tissue engineering scaffolds in situ, which allows depth-resolved molecular characterization of engineered tissues in 3D. Moreover, we investigated the feasibility of the multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity FLOT for structural and molecular imaging of colon tumors, which has demonstrated more accurate diagnosis with 88.23% (82.35%) for sensitivity (specificity) compared to either modality alone. We further applied the multi-modal imaging system to monitor the drug distribution and therapeutic effects during and after Photo-immunotherapy (PIT) in situ and in vivo, which is a novel low-side-effect targeted cancer therapy. A minimally-invasive two-channel fluorescence fiber bundle imaging system and a two-photon microscopy system combined with a micro-prism were also developed to verify the results

A platform for brain-wide imaging and reconstruction of individual neurons

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.Howard Hughes Medical InstitutePublished versio