Sunil P. Gandhi, Ph.D. - US grants

Introduction: The ability to localize and differentiate between hypoxic and well-oxygenated tumors in vivo may aid in the staging of treatment and affect the choice of therapeutic agent. This potentially can be achieved through the use of magnetic resonance spectroscopy (MRS) to measure the relative concentrations of two molecules 1)lactate - a marker for non-oxidative glycolysis, and 2) choline - when enhanced, a marker for active tumor. To assess metastatic neck nodes resulting from primary occurances of squamous cell carcinoma, we have implemented a new J-difference editing scheme using band selective inversion with gradient dephasing (BASING) which was developed for water suppression and lactate rephasing. BASING applied to lactate editing is unique in that the four benefits are provided: 1) singlet suppression with resilience to B1-scaling errors, 2) easy incorporation into localization sequences such as point resolved spectroscopy (PRESS) 3) arbitrary echo times for TE >= 1/J, and 4) RF pulses with steep transition bands that are required to maintain visibility of the choline resonance. Methods: Data were collected from 8 patients and volunteers with the following scan parameters: TR = 2s, TE = 144 ms, number of acquisitions = 256. A 4-element head, neck and cervical phased-array coil (MRI Devices Corp., Waukesha, WI.) operating in receive-only mode was used to maximize coverage and the SNR. To achieve robust separation of lactate from the singlets, a novel phase regularization algorithm, which compensated for phase and frequency variations between successive excitations, was developed. Results: Data from the neck muscle of a normal volunteer showed that a lipid suppression factor of over 1000 was achievable with use of the phase regularization algorithm. The spectra from the neck tumor nodes demonstrated variable amounts of choline and lactate detected. However, the two spectra with the most lactate and choline had median pO2 values of approximately 0.7 mm Hg which is highly suggestive of hypoxia Conclusions: We have demonstrated the use of a new MRS technique capable of detecting both choline and lactate resonances in vivo in head and neck tumors. This has not previously been accomplished and may offer a noninvasive means of differentiating between necrosis, hypoxic and well-oxygenated tumors.

[unreadable] DESCRIPTION (provided by applicant): This proposal aims to investigate the relationship between the anatomical and functional plasticity of mammalian brain using ocular dominance plasticity in mouse visual cortex as a model system. Occluding an eye during early postnatal development leads to a rapid loss of vision through the deprived eye. The location and means of synaptic change in ocular dominance plasticity is a major outstanding problem in systems neuroscience. Recent evidence suggests that the earliest changes occur in the superficial layers (layer ll/lll) of primary visual cortex where some connections appear to rewire on a rapid timescale (< 2 days). By using new imaging techniques that facilitate the chronic imaging of single axons in the living brain, I aim to investigate further the hypothesis that visual deprivation stimulates a rapid rearrangement of synaptic connections in layer ll/lll. At the same time, I will use intrinsic signal imaging to keep track of the ocular dominance of the cortical territory containing the axons of study. I plan to apply these techniques to study the role of inhibition in opening the critical period for ocular dominance plasticity. Altogether, it is likely that this work will help elucidate the anatomical underpinnings of cortical plasticity. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Rewiring cortex using inhibitory neuron transplantation Childhood experiences shape our perceptions and behaviors. The same experiences as adults have little effect. Underlying the perceptual and behavioral malleability of youth is the pronounced capacity of the young brain to be rewired. One class of neurons, inhibitory interneurons, has been shown to play an active role in promoting the plasticity of the juvenile visual cortex. We recently discovered that transplantation of inhibitory interneurons reactivates plasticity in the visual cortex. In this proposal, we will use inhibitory neuron transplantation to gain insight into the mechanisms of cortical plasticity. We found previously that transplanted inhibitory neurons made direct synaptic connections with the host brain. We propose using in vivo calcium imaging of visual responses and in vivo optogenetic stimulation to study the input and output connections made by transplanted inhibitory neurons with cells in the host visual cortex. Our hypothesis is that transplanted cells add a pattern of variable inhibition o the host brain that makes existing connections in the visual cortex quicker to adapt to changes in sensory experience. We also propose to investigate whether the transplantation of inhibitory neurons reactivates plasticity when implanted into the fully adult brain. Therapeutic rewiring of adult cortex using transplantation would represent an entirely new avenue for reversing brain damage. Eventually, this research program may identify novel molecular interventions that promote brain plasticity by reactivating dormant developmental programs in endogenous inhibitory neurons.

The physiological aspects of experience-dependent critical period plasticity has been extensively studied starting with the pioneering studies of Hubel and Wiesel in the 1960s. However the molecular mechanisms that translate sensory deprivation into functional changes in circuit connections remain poorly understood. Neuregulin-1 (NRG1) signaling through its tyrosine kinase receptor ErbB4 is essential for the normal development of the nervous system, and has been linked to neuropsychiatric disorders such as schizophrenia. NRG1 is widely expressed in excitatory neurons, inhibitory interneurons and glial cells in the visual cortex, while ErbB4 expression is largely restricted to parvalbumin-expressing (PV) neurons. We discovered recently that NRG1/ErbB4 signaling in PV neurons is critical for the initiation of critical period visual cortical plasticity by controlling excitatory synaptic inputs onto PV neurons and thus PV-cell mediated cortical inhibition that occurs following visual deprivation. Building on the strong premise from the literature, this discovery and our data showing that NRG1 effects depend on specific neuronal types and are modulated further by deprivation duration, we propose to provide a detailed analysis of NRG1 signaling actions implicated in visual cortical plasticity at the cellular and circuit levels. We hypothesize that NRG1 signaling critically regulates functional circuit connections of PV inhibitory interneurons during short and prolonged visual deprivation that underlies the initiation and establishment of visual critical period cortical plasticity. We also hypothesize that manipulation of ErbB4 signaling in PV neurons is sufficient to extend the ocular dominance plasticity after the closure of the critical period. To test our hypotheses, in Aim1, we will use our established cell-type specific mRNA expression analysis and neurochemical immunostaining to map cellular NRG1 expression in normal and deprived cortex, and determine whether non-PV cell types contribute to the source of PV neuron NRG1. In Aim 2, we will combine ex vivo functional circuit mapping and in vivo 2-photon calcium imaging to test whether NRG1/ErbB4 signaling is required for maintenance of PV neuron excitatory inputs in normal cortex and for restoration of their excitatory inputs in deprived cortex. In Aim 3, we will use pharmacological and genetic approaches to manipulate ErbB4 signaling in PV neurons to extend and attempt to re-open the critical period window of cortical plasticity. Together the proposed research will advance our understanding of molecular mechanisms underlying visual cortical plasticity, and help to develop new therapeutic approaches to treat amblyopia and other neurodevelopmental disorders.

Project Summary To accomplish their diverse maintenance and protective roles, microglia must be extremely plastic, dynamically sensing and responding to specific local challenges throughout the brain. Recent results have called into question whether state-of-the-art models of microglial biology accurately capture the rich interactive environment encountered by human microglia in the living brain. To understand microglial biology, we need new robust and experimentally feasible technologies to systematically reveal the dynamic nature of these cells. Herein we aim to validate a new chimeric platform for exploring IPSC-derived microglial cells that we call the XMG platform. Initial transcriptional and histochemical analyses strongly support the notion that our that XMG cells have highly similar genetic profiles and activity to endogenous human microglia. Specific Aim 1 focuses on comparing the transcriptional landscape of our XMG system to endogenous microglia cells from mouse and human patients. Specific Aim 2 will rely upon cutting-edge in vivo multiphoton imaging experiments to establish the kinetic calcium activity and anatomical remodeling of XMGs in the context an acute, local laser-induced insult. Specific Aim 3 will create a novel XMG reporter system to image and isolate activated microglia for single-cell transcriptomic analyses. Additional efforts will focus on employing this system in the context of ß-amyloid plaque pathology (a hallmark of Alzheimer?s disease) and seek to identify any sex differences of such responses. Successful completion of these aims will result in multiple technological deliverables, such as novel microglial model systems, extensive genetic analysis of our XMG system in response to brain insults, publicly available transcriptomic web portals for easy public access, and novel human iPSC lines for easy dissemination to the field. Lastly, developing a robust and easily-manipulated technological platform to study human microglial cells in vivo will represent a quantum leap for research on human microglial biology.

The transplantation of embryonic inhibitory neurons has recently emerged as a promising avenue for cell-based brain repair. Previously, we and others have shown that transplanted inhibitory neurons restore juvenile plasticity to the circuits of adult visual cortex. In this study, we set out to elucidate the mechanisms for transplant-induced cortical plasticity. We hypothesize that transplanted inhibitory neurons reactivate plasticity by creating a new, disinhibitory microcircuit in recipient adult visual cortex. First, we will map out the effects of transplantation on the inter- and intralaminar balance of excitation and inhibition. Next, we will compare the cell-type specific effects of brief monocular deprivation on visual activity in host cortex. Lastly, we will test whether transplanted neurons make long-range connections with appropriate circuits. To evaluate these hypotheses, this proposal will take advantage of recent advances in optogenetic dissection of inhibitory circuits in brain slice, multiphoton imaging of defined cortical cell types using genetically encoded calcium indicators, chemical-genetic tools for testing the mechanisms of transplant- induced plasticity and viral tracing and whole brain clearing to identify the brain-wide connections onto transplanted cells. If successful, the proposed studies shed light on the mechanisms of transplant-induced cortical reorganization. These studies are also likely to give insight into the normal developmental regulation of cortical plasticity by inhibitory cells.