Takao K. Hensch - US grants

PROJECT SUMMARY (See instructions): The Harvard University Conte Center Administrative Core will be charged with the overall management and administration of the center and its objectives. The Administrative core will facilitate communications between the projects and cores, as well as with the University and outside programs/entities that will benefit from the research and progress made at the Conte Center. A fulltime Center Administrator will be responsible for overseeing and managing the center's budget, events (including but not limited to Public Outreach, Training, and Advisory Board events), website development, and planning programs for training, public outreach and the Conte Center Summer Program, as well as day-to-day operations. To foster education about mental illness and integration across Conte Center laboratories, a Journal Club and website will be established. In addition to disseminating knowledge, the Conte Center will also reach out to under-represented minority students more specifically by online/mobile Cyber Labs in conjunction with summer program students and teachers. Advisory board members each year will be asked to give a major public lecture at one of the Harvard University lecture halls to be advertised broadly in conjunction with the Harvard Center on the Developing Child.

Brain plasticity at the right time and place is of paramount importance during development with lasting consequence throughout life. Just as sensory input in early life shapes primary sensory cortex, emotional experience may shape the limbic-cortical relay system. Fibers from the BLA actively sprout within the mPFC during the post-weanling period, forming increased contacts with GABAergic interneurons as late as the early adult period. This connectivity requires further evaluation ultrastructurally (with Lichtman), and the identity of the receptors at these appositions merits investigation (with Zhuang). Here, we focus on the shift of neural circuit mechanisms from amygdala to mPFC underlying fear extinction across development. Using these stages as a milestone, we will measure developing synaptic inputs onto individual PV-cells of the mPFC by whole-cell recording techniques in slices from PV-EGFP mice crossed to various mouse models. The maturational state of afferents from the BLA in particular, will be examined by optogenetic tagging of this projection. Ultimately, we aim to determine whether dynamic epigenetic regulation (such as by imprinted genes identified by Dulac) dictates the functional maturation of these pivotal cortical inhibitory circuits. Identifying cell-specific mechanisms that underlie epigenetic regulation of critical periods may provide valuable insight into potential circuit-based therapies for pathologies arising from aberrant environment-gene interactions.

DESCRIPTION (provided by applicant): Brain function is shaped by genes and environment during critical periods of neuronal circuit development. Mental illness may arise when the complex convergence of these factors results in aberrant wiring. Here, we propose to meet this challenge by sophisticated, whole genome and neural circuit analyses at single-cell resolution in developing systems. We unite recent insights by the PIs regarding the true magnitude of genomic imprinting, which may underlie parent-of-origin effects in a variety of disorders; the identification of specific cell-types that trigger the re-wiring of circuits in response to early life experience; and innovative technologies to visualize and reconstruct all synaptic inputs and outputs of an individual neuron in the mammalian cortex. Taking advantage of vastly improved computational power and methods, our goal in this project is to use a suite of new neuronal circuit analysis tools to attain a rather simple, but heretofore unattainable goal: the complete connectional diagram and imprinted gene expression profile of a pivotal cell type implicated in multiple cognitive developmental disorders. To begin, we focus strategically on the parvalbumin (PV)-positive GABA neuron in medial prefrontal cortex (mPFC). This inhibitory cell type plays a critical role in timing normal brain development and processing, and is particularly vulnerable to a broad spectrum of genetic and environmental stressors, as are imprinted genes. Shared features of neural circuit dysregulation across animal models are likely to inform the human disorder being modeled. The pipeline to obtain such data will then be very similar for other cases, so that once it is established for one cell-type, age, sex, or mutant, it will be straight forward to repeat for others. Our collective goal is to establish a paradigm for the systematic dissection of developmental 'connectopathies,' which should inspire novel circuit-based therapies for mental illness.

DESCRIPTION (provided by applicant): The human process of attention is vital to basic survival and higher learning, yet its basic neurological mechanisms remain poorly understood. During certain stages of sleep and types of epilepsy, the brain is unresponsive to sensory input. The brain center responsible for focusing the neural searchlight of attention and for generation spindles during sleep is the thalamic reticular nucleus (TRN), where electrical synapses are a major source of connectivity between neurons in the nucleus. Neurons in the TRN spike in two behaviorally distinct modes - burst and tonic firing - and are densely connected by electrical synapses formed by gap junctions. Gap junctions are a unique type of inter-neuronal connection that physically link membranes of neighboring neurons with small pores, allowing charged ions and small molecules to pass between neurons. These synapses are expressed widely throughout the mammalian brain and are thought to synchronize neuronal activity among coupled neighboring neurons. The strength of electrical synapses, in general, directly affects the synchrony or coherence of connected neurons, and in particular, it modulates the afferent output of the TRN. However, the effect of neuronal activity on electrical synaptic strength has been largely unexplored. We propose two aims that address the fundamental question of how the TRN gates attention by evaluating the role of specific neuronal activity patterns in modifying the strength of electrical synapses in the TRN. In Aim 1, we will record and induce pairings of naturalistic TRN activity patterns - burst and tonic firing - in coupled neurons to determine whether coordinated spiking activity induces changes in electrical synapses. In Aim 2, we will begin to dissect the mechanisms underlying electrical synaptic plasticity by testing whether sodium-based spiking is necessary to induce electrical synaptic modification and by examining the contributions from the prominent low-threshold calcium current in these neurons. Because electrical synapses are widespread throughout the brain, the results of this research will open a promising new field of study and a new perspective on the dynamics of networks that include electrical synapses.

DESCRIPTION (provided by applicant): Epilepsy and intellectual disability (ID), including autism are often comorbid with one another in early life. Early postnatal development is characterized by a critical period (CP) of enhanced synaptic plasticity and learning. If epilepsy and seizures occur in the setting of rapid synaptic development, as is the case during the CP, there is the potential for excessive induction of activity dependent synaptic modification (plasticity) as well as disruption of the normal excitatory:inhibitory balance unique to this age window, and this in turn could affect brain development and neurobehavior. The central hypothesis of this proposal is that early life seizures can alter synaptogenesis and network plasticity, thereby disrupting aspects of the subsequent CP. To date, the limited evidence for an effect of seizure on CP events has been at the level of cellular and molecular changes, but has not been addressed quantitatively in vivo at a systems level. We will assess auditory cortical CP resulting from in vivo tone rearing in animals exposed to early life seizures (Aim 1). Next, we wil examine how the maturation of inhibition (Aim 2) and excitation (Aim 3) in specific auditory cortical networks contribute to the CP and how this is altered by early life seizures. Finally, we will perform pilot proof-of-principle experiments to test how seizure induced disruption of auditory CP correlates with seizure-induced neurobehavioral deficits, as well as whether seizure control in 2 mouse models of autism syndromes. If successful, these experiments will reveal new therapeutic targets for the treatment of ID and autism that accompany early life seizures.

Administrative Core Reducing the stigma of mental illness through state-of-the-art neuroscience is the primary mission of the Conte Center at Harvard. The administrative core has a threefold purpose: 1) to facilitate planning, synergy and evaluation of the multidisciplinary science, 2) to support facilities and service resources streamlining joint research efforts, and 3) to promote public outreach and training of the next generation of mental health advocate. During our first phase, the exceptional commitment of a dedicated Outreach Director and science writer, Dr Parizad Bilimoria, working alongside the Center Director established a robust infrastructure and network of partners upon which the current core will build. First, management of the scientific enterprise will include a monthly journal club and progress report in rotation through the four labs, a Center Retreat including summer student interns, and a strong Scientific Advisory Board (SAB) to convene annually. Our original SAB comprised of Drs Hyman, Sudhof, Yuste, Greenberg and Ferguson-Smith, will be reconfigured to fit the new Center direction to include female expertise in stem cell and marmoset neurobiology. Second, access to information technology (formerly a core of the Center) as well as standardized mouse behavioral phenotyping at the Neurodevelopmental Behavioral Core facility in Boston Children's Hospital will be managed on a fee-for- service basis through the administrative core. This will prioritize our intense sequencing and connectomic demands at the Harvard University Research Computing facility, and provide consistent cohorts of well- characterized animals for subsequent anatomical and physiological analyses. By serving multiple laboratories in the Center, the administrative core will foster synergy and increase efficiency. Third, our Outreach program will be strengthened and expanded. Already dubbed ?a model for how all Conte Centers (and even research universities generally) should operate? by our NIH site visit, our highly visible presence on the Harvard campus and on-line (conte.harvard.edu) will be maintained. Innovative programs such as the Wintersession on mental health careers for undergraduates, summer High School Teacher Training Workshop, public lecture series at the Boston Museum of Science and minority summer internships in Center laboratories will be expanded. Monthly public Colloquium series lectures, ?Team Conte? participation in several walks for mental illness, Brain Awareness Week events and Middle School visits will be continued. Our activities to enhance science literacy are supported by a rich network of collaborating institutions including the Department of Molecular Cellular Biology Life Sciences Outreach, the Harvard Center on the Developing Child, Boston Museum of Science and the National Alliance on Mental Illness (NAMI). One initiative which we aim to spearhead in the second phase is a national gathering of Conte Centers to share their science, ethical concerns and outreach experiences. Our trainees will thus form a pipeline of future investigators at the cutting edge of neuroscience research, who are able to address a broad audience about the implications of their work with empathy and care.

Overall abstract Most mental illnesses emerge during vulnerable windows of brain development to impact cognitive function in humans. While hundreds of genes and/or environmental factors have been linked to mental illnesses, even neighboring point mutations on a single gene (eg. Shank3) can lead to `early' disorders such as autism or `late' schizophrenia. This poses a double challenge to understanding their etiology and potential treatment ? how to track the trajectory of circuit derailment and the relevance of such studies in animals like mice whose cognitive skills may be less well-characterized. Our proposed Conte Center renewal will tackle these problems directly by uniting four pioneering neurobiologists focused on the formation and refinement of neuronal circuits in two stages of development, fetal and pre-adolescent critical periods. Our central hypothesis is that whatever the predisposing environmental factors or genetic bases, the proximate cause of aberrant behavior in cognitive disorders will be found in distinct patterns of altered neuronal connectivity. First, to examine how excitatory- inhibitory balance is established in fetal life, Arlotta combines cutting edge stem cell, genomic, imaging and physiological recording technology for the longitudinal study of human brain organoids carrying specific gene mutation. Second, key conceptual insights from Hensch in the first phase of our Center identified the pivotal role of parvalbumin (PV+) cells in determining postnatal critical period timing. Because of their high metabolic activity, PV+ cells are vulnerable to oxidative stress in mental illness, as are the gamma oscillations which they generate (in association with cognition). Manipulations altering PV+ cell maturational profiles powerfully shift plastic windows in sensory cortex, indicating that malleability of critical periods themselves may contribute to cognitive disorders as well. Third, Hensch and Feng confirmed an impairment of multisensory integration in the insular cortex of mice carrying autism risk mutations in Shank3 and Mecp2. Notably, these lie on opposite ends of PV+ circuit hypo- or hyper-maturation. Here, we will take advantage of reversible and conditional genetic mutations in these genes to map critical periods for other higher functions of relevance: attention, cognitive flexibility, and social preference? all established in the Hensch lab. Moreover, for direct comparison to his mice, Feng will produce marmosets carrying Cre recombinase in PV+ cells or Shank3 deletion using CRISPR technology. His unique infrastructure will enable manipulation and analysis of the same circuits in this primate with better evolved frontal cortex and behaviors. Fourth, we capitalize on a sophisticated platform for complete 3D electron microscopic circuit reconstruction established by Lichtman during the first phase of our Center to compare and contrast the emergence of `connectopathies' from human organoids to mice and marmosets. Ultimately, reducing the stigma of mental illness through state-of-the-art neuroscience to train the next generation and conveying this knowledge through our strong, active Outreach program is the primary mission of the Conte Center at Harvard.

Abstract Mice ? with their short gestation time, lifespan, large litter size, and above all, genetic tractability ?are powerful experimental tools with which to explore mammalian brain development. One striking example is the reversible disruption of autism risk genes, like Mecp2 or Shank3. Switching on these genes after the emergence of fullblown phenotypes rescues several motor- related symptoms, including inertia, abnormal gait, weight, irregular breathing, repetitive grooming or hind-limb clasping. Instead, other neurological aspects are uncorrected in adulthood, such as anxiety or motor coordination. Of central relevance to human patients, it remains unknown to what extent higher cognition is impaired in Shank3 or Mecp2 mutant mice and whether recovery would also be limited to a critical period. The Hensch project will directly address sensitive periods and reversibility of dorsomedial prefrontal cortical (dmPFC) functions using novel behavioral tests of attention, cognitive flexibility and acoustic preference along with associated physiological measures from relevant areas. Pioneering work in the first phase of the Conte Center established the pivotal role of particular inhibitory neurons underlying critical period timing in mouse sensory systems. Parvalbumin (PV+) GABA circuit maturation dictates both the onset and closure of these windows of circuit refinement. Manipulations of psychiatric risk factors, such as circadian Clock gene disruption or redox dysregulation, can delay or extend developmental trajectories by upsetting the vulnerable PV+ component of local circuit excitatory-inhibitory balance. Recently, Hensch and Feng extended this principle to higher-order multi-sensory integration (MSI, commonly impaired in patients with autism) in the insular cortex. Shared MSI impairments in mice lacking either Shank3 (weak PV+) or Mecp2 (excessive PV+) suggest an optimal range of PV+ network function enables proper pruning of connections in the insula. Here, we will examine circuit physiology and anatomy before/after restoration of Shank3 or Mecp2 in mice, ultimately by full 3D EM circuit reconstruction with Lichtman also in marmosets carrying the same Shank3 deletion (from Feng). Further, our touchscreen two- choice visual attention assay, a multiple-choice foraging task to assess flexible rule learning, and preference for acoustic stimuli (music, ultrasonic calls) experienced early in life will probe dmPFC function in mutant and rescued mice. Electrophysiological recording and two-photon Calcium / Chloride imaging from the dmPFC in vivo will focus on PV+ networks in these areas across development, starting with comparison to Arlotta?s human organoids. Based on these many insights from mice, the impact of silencing/activating PV+ circuits in corresponding frontal cortical regions of PV-Cre marmosets (by Feng) using focal injections of viral DREADD constructs can be tested on analogous primate tasks of attention, cognitive flexibility and preference behavior at MIT. Our collective work will determine whether biological determinants of critical periods in sensory systems play a similar role in cognition, the corresponding circuit changes which are corrected when Shank3/Mecp2 symptoms are reversed and how much the mouse and primate brain differ.