Carlos Portera-Cailliau - US grants

We want to investigate the mechanisms responsible for dendritic spine abnormalities in Fragile X syndrome (FXS). FXS is the most common inherited cause of autism and mental retardation. A clear link between the functional and structural (increased density and length of spines) abnormalities in FXS has not been established. A very similar defect in spines has been found in a knockout mouse model of FXS. Spines in FXS resemble dendritic filopodia, which are spine precursors. We show that in developing mouse neocortical neurons, filopodia are replaced by spines in the second postnatal week. Interestingly, the greatest differences in dendritic protrusions between wild type and fragile X mice occur at 1 week of age, and diminish thereafter. It is conceivable that anomaliesof filopodia in the first postnatal days are even more striking in the knockout mice, but this has not been explored. Our preliminary data also reveal that dendritic protrusions are longer and more densely packed when neuronal activity is blocked, so it is possible that spontaneous activity is reduced in FXS. Fragile X mice exhibit excessive group I metabotropic glutamate receptor (mGluR)-mediated long-term depression. But a direct link between abnormal mGluR signaling and spine dysgenesis has not yet been discovered. Here, we show that filopodia elongate in response to glutamate and note that others have shown that spines elongate with stimulation of group I mGluRs. We want to test the general hypothesis that a defect in filopodia, linked to abnormal group I mGluR signaling and/or to decreased neuronal activity occurs in FXS, and might impair their ability to mature into spines. Innovative and cutting-edge microscopy techniques will be used. First, we will look for abnormalities of filopodia in pyramidal neurons of fragile X mice with in vivo two-photon imaging in the first postnatal days. Next, we will examine whether spontaneous neuronal activity is reduced in neonatal fragile X mice, using two-photon calcium imaging of hundreds of neurons simultaneously. Finally, we will use two-photon glutamate uncaging to study whether glutamate-mediated elongation of filopodia is disrupted in FXS and whether mGluRs participate in this phenomenon. The experiments in this proposal are designed to identify novel molecular targets for therapeutics in FXS. Because spine abnormalities are common to several other types of mental retardation and autism disorders, these studies are of broad clinical significance.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) "Enabling Technologies" and specific challenge topic 06-AG-101* Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. It also applies to specific challenge topics 06-NS-101 (Developing minimally invasive measures of neural activity) and 06-NS-103 (Breakthrough technologies for neuroscience). One of the greatest challenges for neuroscience in the 21st century is to understand how the billions of neurons that form the brain communicate with one another to produce complex behaviors. The ultimate benefit from this type of research will come from deciphering how dysfunctional patterns of activity amongst neurons lead to devastating symptoms in a variety of neuropsychiatric disorders. Unfortunately, little is known regarding how neural computations in the brain interpret sensory inputs or generate behaviorally relevant responses. This is due in part to the current lack of tools to interrogate the activity of large numbers of neurons in the intact brain. Through an interdisciplinary research collaboration between physicists and neuroscientists, we have developed a high-speed 2-photon microscope for calcium imaging that combines fast resonant scanning mirrors and multi-beam imaging to achieve image acquisition rates more than 2 orders of magnitude faster than conventional 2-photon microscopes. To avoid the fundamental limitation of scattering ambiguity with multiple beams in deep-tissue 2-photon microscopy, we propose an innovative approach to detect and resolve scattered fluorescence emission from separate beams at different times. Specifically, we split the laser beam into four beam lets and then delay each beam optically from the others by 3 ns. We call this method Spatio- Temporal Excitation-emission Multiplexing (STEM). The signals from all four beams are detected by a state- of-the-art GHz bandwidth photodetector. Our microscope therefore preserves the unique advantages of 2- photon microscopy, including its ability to excite fluorophores deeper in the tissue, its reduced photo damage and its exquisite spatial resolution. We now propose to systematically optimize our STEM microscope in order to achieve fluorescence lifetime imaging (FLIM) capability and 4-color imaging. The ultimate goal is to achieve unprecedented 6-D (x, y, z, t, [unreadable], ") bio-imaging at the single cell level. In addition, we propose a series of in vivo calcium experiments to systematically dissect the micro-scale connectivity of neocortical circuits. First, we will calibrate our STEM system to demonstrate its superior action potential detection compared to conventional 2-photon calcium imaging. Next, we will examine the spatiotemporal dynamics of large ensembles of layer 2 and layer 3 neurons in barrel cortex in response to whisker deflections, by recording from hundreds of these neurons simultaneously in 2-D and 3-D at unprecedented speeds. Within 2 years, the instrument will be optimized and we will be able to characterize, for the first time, the functional wiring diagram of entire complement of neurons within a volume of neocortex. PUBLIC HEALTH RELEVANCE: We have recently developed a high-speed microscope to record the activity of neurons in the intact brain non-invasively. The goal of the proposed challenge grant is to optimize this instrument and then use it to investigate how brain circuits are assembled during development in areas important for emotion, cognition and creativity, as well as for learning and memory. This innovative tool will allow neuroscientists to design experiments that can generate new ideas regarding how subtle alterations in brain wiring could result in devastating neuropsychiatric disorders such as schizophrenia, autism, mental retardation or bipolar disorder.

DESCRIPTION (provided by applicant): To better understand the mechanisms of intellectual impairment and emotional disturbances in individuals with schizophrenia, bipolar disorder, mental retardation or autism, it is essential to first study how cortical circuits are assembled during normal brain development. Cajal-Retzius (CR) neurons have long been implicated in cortical development and are thought to play a role in several of these disorders. The best documented function of CR neurons is in orchestrating neuronal migration and cortical lamination. This is achieved through their secretion of reelin, though other reelin-producing cells in the cortex could also play a role. Interestingly, CR neurons show spontaneous correlated activity in the postnatal neocortex and are known to make both chemical and electrical synapses with apical dendritic spines of developing pyramidal neurons. Therefore, it is conceivable that CR neurons could play a role in synaptogenesis by pyramidal neuron dendrites and in the emergence of spontaneous oscillations in cortical neurons. We want to test this hypothesis using in vivo 2-photon imaging of the structure and function of immature cortical circuits. Specifically, we will image CR and pyramidal neurons expressing the green fluorescent protein (GFP) in different lines of transgenic mice, as well as cortical neurons loaded with fluorescent calcium indicators to detect neuronal firing. We have identified a line of transgenic mice (Ebf2-GFP) in which GFP is expressed in virtually all CR neurons. In addition, we obtained a line of mice (Ebf2:GFPiCre) that expresses the Cre recombinase in the same cells. Using state-of- the-art 2-photon microscopy, we intend to selectively kill CR neurons, or genetically silence their activity using optogenetics and Cre-Lox technologies, and then examine the repercussions of such targeted perturbations of CR neurons on neocortical development. We will investigate whether CR ablation/silencing affects the maturation of apical dendrites and spines of pyramidal neurons and/or the spontaneous activity of networks of pyramidal neurons. These studies address fundamental questions about cortical development. The experiments are designed to fill knowledge gaps about CR neurons and identify novel roles for these cells in cortical circuit assembly. These data should resolve longstanding controversies about their function that arose from methodological shortcomings of prior studies. Our work will also generate new ideas about how subtle defects in cortical circuits might contribute to several neuropsychiatric disorders and thereby help to find improved treatments for these devastating diseases. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate how brain circuits are assembled during development in areas important for emotion, cognition and creativity, as well as for learning and memory. The experiments are designed to generate new ideas about how subtle alterations in brain wiring could result in devastating neuropsychiatric disorders such as schizophrenia, autism, mental retardation or bipolar disorder.

DESCRIPTION (provided by applicant): Some of the sensory, motor and cognitive impairments caused by stroke eventually improve, suggesting that the brain has the ability to repair itself and restore lost functionalities. But large knowledge gaps still exist regarding the mechanisms of structural rewiring and functional remapping after stroke. Much of this brain plasticity takes place in the tissue surrounding the core of the ischemic lesion, known as the peri-infarct cortex, but when exactly these changes occur and which cells participate is not clear. In addition, the extent to which circuit remodeling correlates with functional improvement is not known. Recent in vivo imaging developments could help overcome previous limitations in experimental techniques used to record changes in neuronal structure and functional remapping. In particular, research on stroke plasticity and its role in functional recovery would benefit from the use of longitudinal in vivo imaging approaches that allow the investigator to track the dynamics of neuronal structure and function with exquisite spatial and temporal resolution, in the same neurons or circuits before and after stroke. We propose to use an innovative approach and cutting edge imaging techniques, including chronic in vivo two-photon microscopy, to monitor axonal/dendritic structure and record the remapping of lost functionalities, as well as optogenetics and pharmacological manipulations to perturb such remapping. We want to test the hypothesis that synaptic remodeling in pyramidal cell axons or GABAergic interneurons, also plays a role in brain repair. We want to test four hypotheses: 1) that pyramidal cell axons and dendrites of GABAergic interneurons in peri-infarct cortex also play a role in neural repair after stroke; 2) that the degree of structural plasticity correlates wth functional recovery; 3) that lost functionalities are consistently remapped according to pre-established circuits after stroke; and 4) that blocking tonic inhibition or using constraint therap improve recovery by enhancing plasticity. Our studies will focus on the clinically relevant middle cerebral artery occlusion model of stroke in adult mice and will directly examine the related issues of hemodynamics, collateral blood flow, and circuit plasticity. Our proposed work is intended to generate new knowledge about cortical circuit plasticity after stroke and other types of brain injury, with the hope that this will lead to better strategies for rehabilitation that enhnce functional recovery.

DESCRIPTION (provided by applicant): The mission of the UCLA-Caltech MSTP is to promote the education of outstanding physician-scientists. To fulfill this mission, our current goals are to 1) recruit exceptionally bright and accomplished students who exhibit an unusual degree of passion for scientific knowledge and a life-long commitment to research and leadership, 2) help guide admitted students toward outstanding training environments that encourage individual thinking and provide students with the tools needed to develop into accomplished physician-scientists, 3) provide a comprehensive support system to meet the trainees' needs and 4) play an increasingly prominent role in guiding the career development of undergraduate students from under-represented ethnic groups and disadvantaged backgrounds. To accomplish these goals as effectively as possible, the UCLA-Caltech MSTP is run by two equal Co-Directors, three Associate Directors, and a strong administrative team, all of whom are deeply committed to the Program. The Program is structured for an average of eight years of study. An integrated, problem-based medical school curriculum is particularly well suited for MSTP students, due to increased time for independent exploration and increased emphasis on research advances that contributed to current knowledge of disease etiology, diagnosis, and treatment. For their Ph.D. research, students choose mentors from a wide array of science and engineering Ph.D. Programs. The MSTP's commitment to excellence was perhaps most apparent when UCLA and Caltech entered into an affiliation agreement fifteen years ago. This affiliation, which provides an opportunity for two students per year to perform their thesis research at Caltech, not only has increased the number of outstanding mentors available to students, but also appears to have increased the Program's visibility and recruitment success. Substantial institutional support from the David Geffen School of Medicine at UCLA and from Caltech has permitted an increase in the size of the MSTP, with 97 students currently enrolled in the program. The MSTP derives great benefit from recent dramatic improvements in physical facilities at both UCLA and Caltech, from the financial health of the universities, and from the recruitment of a large number of outstanding new faculty members to UCLA and Caltech.

DESCRIPTION (provided by applicant): Neuropsychiatric disorders like schizophrenia, autism and bipolar disorder may be caused by changes in the maturation of connections between brain cells of the cerebral cortex during development. In mouse neocortex, we and others have shown that the 2nd postnatal week is a time when dendritic spines become stabilized, the density of synapses increases dramatically, and spontaneous activity becomes abruptly desynchronized. This is also a period when most Cajal-Retzius (CR) cells undergo cell death, although we recently showed that a small subset survives into adulthood. Although CR cells are well-known for their critical role in cortical lamination much less is known about their function as neurons. Indeed, considering that CR neurons are spontaneously active, fire synchronously, and make synapses with apical dendrites of pyramidal neurons, we envision that they might function as pacemakers of cortical network activity. Specifically, we propose to test the hypothesis that postnatal CR neurons can trigger synchronous activity in neocortex and that those that survive into adulthood continue to influence pyramidal neuron firing. Previous work in brain slices has been unable to demonstrate that CR neurons and pyramidal neurons are functionally connected probably because the integrity of axons and dendrites was disrupted. We intend to overcome this shortcoming by using in vivo two-photon Ca2+ imaging and electrophysiology to record from these cell types in the intact brain. We have identified a specific promoter for CR neurons that will allow us not only to specifically visualize these cells in Layer 1 but also to conditionaly express channel-rhodopsin using viral vectors and a Cre-Lox approach. In the first aim, we will examine morphological and electrophysiological properties of CR neurons at early postnatal vs. adult stages to determine whether surviving CR neurons in mature animals are distinct from those that are destined to die during early development. In the second aim, we intend to modulate the firing of cohorts of CR neurons with optogenetics while recording from their synaptic partners in deeper cortical layers. The goal is to test whether CR neurons can influence the firing of pyramidal neurons and contribute to the emergence of synchronous network activity in the developing neocortex. These experiments will lay the foundation for future studies exploring the mechanisms by which dysfunction of CR neurons could cause neuropsychiatric diseases.

DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) is the most common inherited form of intellectual impairment and the most common single gene cause of autism. Research in Fmr1 knockout (KO) mice, an animal model of FXS, has identified two major defects in the brain. The first is a structural abnormality in dendritic spines, the major recipiens of excitatory synapses in the cortex, and the second is a functional abnormality in synaptic and experience- dependent plasticity. Using in vivo two-photon microscopy, we and others have identified a developmental delay in the stabilization and maturation of dendritic spines of cortica pyramidal neurons in Fmr1 KO mice, which may be one of the earliest synaptic defects in FXS. Now, we will test the hypothesis that circuit remodeling triggered by sensory experience is intimately tied to the spine dynamics and size, thereby reconciling the structural and functional phenotypes of Fmr1 KO mice. We will also investigate synapse integrity at the ultrastructural level with electron microscopy, as well as the dynamics of axons and their boutons during cortical development, in order to ascertain whether they are also altered in mutant mice. In addition, using in vivo two-photon calcium imaging and electrophysiology to record neuronal activity in intact circuits, we have shown that pyramidal neurons in Fmr1 KO mice show abnormally high firing rates and synchrony, which could explain the deficits in learning and low seizure threshold in these mice. Here, we will test the hypothesis that this network hyperexcitability translates into problems with sensory-evoked activity and we will investigate whether these circuit-level problems in KO mice can be rescued with drugs that affect brainstem neuromodulation and inhibitory pathways. The experimental design employs cutting edge in vivo imaging techniques and seeks to address important knowledge gaps and controversial issues in FXS. Because dendritic spine abnormalities and many of the signaling pathways regulated by the fragile X mental retardation protein are also implicated in other neurodevelopmental disorders, we believe that our unique synapse-to- circuit approach has a very high significance and is likely to be of broad importance to many types of autism and mental impairment.

SUMMARY / ABSTRACT We plan to investigate circuit defects underlying sensory hypersensitivity in Fragile X syndrome (FXS), the most common inherited form of intellectual impairment and the most common single gene cause of autism. In hyperarousal to sensory stimuli, affected individuals are deeply troubled by sounds, smells, sights, or touches that seem normal to others. This leads to maladaptive behaviors, including avoidance responses, such as tactile defensiveness. Virtually all individuals with FXS suffer from tactile defensiveness and Fmr1 knockout (Fmr1-/-) mice, an animal model of FXS, exhibit clear signs of sensory hyperarousal. Elucidating the types of circuit dysfunction that cause fragile X mice to interpret certain stimuli as aversive/threatening, and how this eventually leads to an avoidance response, represents a major knowledge gap in FXS research. To address this, we propose a novel symptom-to-circuit-to-neuron approach in the Fmr1-/- mouse model of FXS in order to investigate disruptions at the circuit and single neuron levels that result in altered sensory processing. In a recent study (He et al., J Neurosci, 2017), we demonstrated how, in response to repetitive tactile stimulation of whiskers, Fmr1-/- mice display a sensory avoidance behavior analogous to tactile defensiveness in humans. Using in vivo calcium imaging in somatosensory (S1) barrel cortex, we then showed that repetitive whisker stimulation results in a gradual reduction in neuronal firing in 2-week-old and in adult wild-type (WT) mice, but not in Fmr1-/- mice. Thus, one of the circuit defects that could explain tactile defensiveness in FXS is a loss of neuronal adaptation in cortical neurons (simply put, neurons in S1 cortex of fragile X mice are not be able to tune out persistent tactile stimuli). We now propose to test whether this loss of neuronal adaptation results from a dysfunction in parvalbumin (PV) or somatostatin (SST) GABAergic interneurons in S1 cortex, and then to delineate circuit alterations in brain regions that are both upstream or downstream from S1 cortex. These studies will allow us to generate a more detailed wiring diagram of sensory hyperarousal in FXS, by examining three stages of sensory processing: thalamus (input), cortex (integration), and amygdala (output). Throughout, we will investigate whether manipulating neuronal activity at each of these stages of sensory processing might ameliorate maladaptive behaviors associated with sensory hyperarousal in Fmr1-/- mice. Our experimental design employs cutting edge techniques, including in vivo two-photon calcium imaging, silicon microprobes, DREADDs, and Cre-Lox genetics, and seeks to address important knowledge gaps in FXS. Because many of the signaling pathways that are dysregulated in FXS are also implicated in other neurodevelopmental disorders, we believe that our unique symptomàcircuit approach has a very high significance and is likely to be of broad importance to many types of autism and mental impairment.

ABSTRACT The mission of the UCLA-Caltech MSTP is to educate and train outstanding physician-scientists. To fulfill this mission, our current goals are to 1) recruit exceptionally bright and accomplished students who exhibit unusual passion for scientific knowledge and a life-long commitment to research, medicine, service, and leadership; 2) help guide admitted students toward outstanding training environments that encourage individual thinking and provide tools for development into accomplished physician-scientists; 3) provide a comprehensive support system to meet trainees' needs; and 4) enhance physician-scientist diversity by playing an increasingly prominent role in guiding the career development of current and future students from under-represented ethnic groups and disadvantaged backgrounds through mentoring and outreach. To accomplish these major goals as effectively as possible, the UCLA-Caltech MSTP is led by two equal Co-Directors, four Associate Directors, and a strong administrative team, all of whom are deeply committed to the Program. The Program is structured for an average of eight years of combined research and clinical training. Our integrated problem-based medical school curriculum emphasizes flexibility and is particularly well suited for MSTP students because it provides increased time for independent scientific exploration and encourages trainees to pursue research projects that will advance current knowledge of disease etiology, diagnosis, and treatment. For Ph.D. training, students choose mentors affiliated with a broad array of UCLA and Caltech graduate programs, most frequently in genetics & genomics, immunology, cell & developmental biology, neuroscience, bioinformatics, bioengineering, or our newly instituted social sciences track. A recent milestone achievement of our Program was the recent 20th anniversary of the affiliation between UCLA and Caltech into a joint MSTP (1997) for the mutual benefit of these preeminent campuses and our trainees. A second remarkable celebratory achievement will be the upcoming centennial of UCLA becoming a UC campus (1919) and its amazing rise to a world-ranked institution in research, education, and service in under 100 years of existence. A prominent goal of UCLA?s Centennial Campaign is to raise $4 billion, one-third of which is earmarked for research. Despite a competitive funding climate, the UCLA- Caltech MSTP is in a period of substantial growth because of generous institutional support from the David Geffen School of Medicine at UCLA and from Caltech. This has permitted an increase in the size of the Program, with 107 current students. The MSTP benefits tremendously from substantial improvements and expansion in physical facilities and research capabilities at UCLA and Caltech, from the strong financial health of both universities, and from the recruitment of many outstanding new faculty members to our campuses.

SUMMARY The underlying brain defects in Fragile X Syndrome (FXS) are not well understood. We have been investigating the specific circuit alterations that lead to a variety of symptoms in FXS, including attention deficit, anxiety, hyperarousal, sensory hypersensitivity and delayed learning. We focus on FXS, the most common inherited cause of autism and intellectual disability, because most investigators use the same Fmr1 knockout mouse model to investigate it, and because it lacks neuropathological features that often confound investigations in other neurodevelopmental disorders (e.g., severe epilepsy, neuronal migration defects, etc.). We strive to overcome limitations of previous studies by comparing the performance of humans and mice with FXS on analogous behavioral tasks. Our goal is to identify shared deficits in sensory processing and learning across both species that will hopefully improve outcomes of future clinical trials in FXS. Here, we will determine the impact of sensory distractors on behavioral performance in both humans and mice using a visual discrimination task. We will also identify specific alterations in population dynamics of pyramidal neurons and different subtypes of inhibitory interneurons that are responsible for deficits in sensory discrimination in Fmr1 knockout mice. Building on our recently published study in Nature Neuroscience (Goel et al., 2018), we will address the following important questions: 1. Does distraction worsen performance in a sensory discrimination task in Fmr1 knockout mice and in adult subjects with FXS? (Aim 1)? 2. Is the firing of parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing interneurons disrupted in Fmr1 knockout mice during the sensory discrimination task, especially in the presence of sensory distractors? (Aim 2A)? 3. Can silencing VIP interneurons (or exciting PV neurons) with DREADDs rescue behavioral performance in Fmr1 knockout mice? (Aim 2B)? 4. Do mice and humans with FXS share similar deficits in neural oscillations (Aim 3)? The mouse studies will be performed in the laboratory of established FXS investigator Carlos Portera-Cailliau (PI) at UCLA. Craig Erickson (co-I), who runs the world?s 3rd largest FXS clinic at the University of Cincinnati, will conduct the human studies. The experimental design exploits cutting edge in vivo imaging techniques (e.g., chemogenetics, in vivo two-photon calcium imaging, Cre-Lox genetics, silicon probe recordings, phase- amplitude coupling analysis of EEG) and seeks to address important knowledge gaps in ASD pathogenesis.