Albert M. Galaburda - US grants

The purpose of this research is to specify further the anatomical characteristics of the brain in developmental dyslexia. Three brains have been examined in our lab, and all have shown anatomical anomalies that can be traced to the fetal period. Although all 3 brains shared in common the presence of neuronal ectopias and architectonic dysplasias, the locaton and severity of these lesions are variable. Additional brains will be examined in an attempt to correlate the severity and location of the anatomical findings to dyslexic syndromes. A search will also be made for additional pathologies, "negative cases", and developmental anomalies in unaffected family members and unrelated controls. Brains have been and will continue to be received through a brain bank established with the Orton Dyslexia Society. The specimens will be processed in whole-brain celloidin-embedded serial sections stained for cell bodies, myelinated fibers and additional strains as needed to interpret the pathological findings. Attention will be paid to anomalies of developmental origin and to cytoarchitectonic structure and asymmetry. The second, and related, portion of the grant will aim at characterizing the cortical anomalies that have been discovered in the brains of autoimmune New Zealand mice. The relevance for further study in this animal is that: 1) immune-related disorders have been shown to be increased in frequency among left-handers and learning disabled individuals and their families; and 2) the cortical anomalies in the New Zealand mice bear a strong resemblance to the anomalies described in the dyslexics. The major effort in this portion of this study will be to specify the architectonic and histological characteristics of the abnormalities in this animal, and to relate these to age, sex, laterality, and, preliminarily, to behavior. The results will be used in future experiments aimed at disclosing intrauterine influences in the formation of brain anomalies. The New Zealand mouse may then become a powerful animal model for the study of anatomical mechanisms in dyslexia.

neuroanatomy; model design /development; biological models; reading disorder; brain disorders;

Immune-defective mice show developmental brain anomalies that are similar to those seen in dyslexics and exhibit abnormal learning behaviors. Dyslexics and their families may have a greater incidence of some immune disorders. In the first period of the Program Project, we documented brain, behavioral and immunological abnormalities in strains of immune- defective mice and searched for causes of the brain abnormalities. We also developed a second model of induced minor developmental brain anomalies, which share many features of the spontaneous anomalies and offer experimental advantages. We carried out preliminary research that suggests a special role of the uterine environment for some immunological and behavioral characteristics. The purpose of the continuation research is to pursue lines of evidence that have been productive and convergent. One is the further study of minor developmental brain malformations. A detailed analysis of the onset and evolution of the spontaneous and induced brain abnormalities will be carried out in the Anatomy 1 Research Project, which will also look at the effect of timing and severity of the insult and the effect of neuroprotective substances on the resultant brain lesion. Changes in cellular and connectional architecture and behavior will be described in standard and manipulated lesions by the Anatomy 2 and Behavior research projects. Behavior research will also look at effects of early life experience and other ways of facilitating learning, including the use of drugs. The Embryo Transfer Research Project will assess the effects of changing the uterine and neonatal environments on anatomy, immunology, and behavior and at the effects of modulating autoimmunity in mothers. A Breeding Core will be established to insure uniform supply of difficult-to- obtain animals. A Neuroanatomy Core will screen brains arising in the research components for anomalies. A Data Registry and Processing Core will keep and analyze databases arising from the components of the Program Project. It is expected that the proposed work will contribute to the understanding of the onset, course, consequences, and causes of minor cortical malformations with an aim to improving prevention and treatment of learning disorders in children and adults.

The goal of the proposed research is the understanding of mechanisms underlying structural asymmetry of the brain and, ultimately, functional lateralization. Lateralization of neural structure and function is seen throughout the animal kingdom. In humans, the left hemisphere of most individuals is functionally specialized for linguistic tasks and the right for nonlinguistic tasks. The planum temporale, a language-related neuroanatomical area, is larger on the left in the majority of individuals. There is, however, significant individual variability in lateralization, especially among left-handers and nonright handers, who are more likely to show absence of asymmetry. Variability in brain asymmetry is also seen in nonhuman species, and, through the use of the rat as an experimental model, we intend to investigate histological, connectional, and developmental factors characterizing symmetry and asymmetry in the brain. The combined volume of homologous brain substrates in the two hemispheres decreases with increasing degree of asymmetry, and the asynmmetrical cases have fewer neurons on one side, rather than more neurons on the other. The question to be addressed in the first set of experiments is whether certain immunocytochemically identified neuronal subtypes are more involved than others in the production of this volume asymmetry. Thus, neurons that are immunocytochemically-reactive to each of four antibodies in specific architectonic areas of the cerebral cortex will be counted, their laminar distributions noted, and the findings will be related to volumetric asymmetry. The pattern and density of connections, in pilot studies, differ in brains with symmetrical and asymmetrical areas - symmetrical brains having a denser and more complex pattern of callosal terminations. The second set of experiments we will seek to replicate and extend these findings and to investigate whether this principle holds for thalamocortical connections, or whether the opposite is found since the two types of connections tend to be complementary. Specifically, the corpus callosum will be sectioned and the patterns of axonal termination in architectonic areas of the neocortex will be analyzed with respect to cerebral asymmetry and laminar location. In addition, the laminar and areal pattern of thalamocortical axonal terminals in the visual cortex of the rat will be examined after transneuronal transport of radioactive anterogade tracers injected into the eyes. In the third set of experiments, the ontogenetic processes underlying structural asymmetry will be examined. Pilot data do not point to asymmetry in rates of cell proliferation. Expanding on this study neocortical neurons labelled with [3H]Thymidine after injection during gestation will be counted in animals sacrificed at several postnatal ages. Analysis of these neuronal counts with respect to hemispheric asymmetry will help to determine whether neuronal count differences in asymmetrical and symmetrical brain areas relate to (1) the number of neurons in the original germinal zone or (2) developmental neuronal disappearance.

Immune-defective mice and a perinatal surgical rodent model developed in our laboratories, both of which exhibit abnormal learning behaviors, show developmental brain anomalies that are similar to those seen in dyslexics. In the first period of the Program Project, we documented brain, behavioral and immunological abnormalities in an animal model. We carried out preliminary research that suggested a special role of the uterine environment for some immunological and behavioral characteristics. During the second period, we characterized further cellular, connectional, behavioral (including early life experience), and developmental characteristics of the anomalies, as well as genetic influences on their origin and effects of pharmacological manipulations. We also demonstrated the lack of substantial interaction among malformations, immunological parameters, and intrauterine environment. Related research in humans and animals demonstrated that the distinction between defects in low-level sensory processing and those in higher-level cognition is pivotal in determining the pathogenesis of dyslexia. The purpose of the continuation of this program project is to pursue lines of evidence that have been productive and convergent between our line of research and that of our colleagues. Four research projects and 4 core functions will comprise the Program Project. Two anatomy projects will look at the developmental anatomical consequences of minor cortical malformations, either spontaneous or induced, on connectionally related cortical and subcortical regions, in an attempt to explain functional abnormalities at both high and low levels of processing. Amelioration of anatomic effects will be attempted through environmental enrichment. A neurophysiology project will examine the synaptic characteristics of these connections, which are likely to be part of the mechanism by which cross-level developmental influences act. A neurobehavioral project will investigate behaviors at multiple levels of processing and the effects of early environmental manipulation. The 2 anatomical cores will support these projects and an especially designed neuroimaging core will be instituted to allow for longitudinal research in living animals and to optimize methods for best demonstrating minor cortical malformations in vivo . A data processing Core will serve all projects.

The specific objectives of the Neurohistology Core are to provide neuroanatomic and neuropathologic support for several aspects of the research comprising the Program Project and other activities of the laboratory. This arrangement has worked very well during the past 5 years. This includes the morphologic analysis of some of the mouse and rat brains arising from 1) the neuroanatomy projects, 2) Neurobehavior Research Project , 3) and 4) Neurophysiology Research Project. Some of the additional activities of the laboratory include the processing of human brains of individuals with learning disorders and control specimens, which are used to describe neuroanatomic findings and generate neuroanatomic hypotheses that can be tested in animal models such as those in the present Program Project. The Neurohistology Core will be responsible for insuring proper collection, registration, randomization, and processing of all neuroanatomic materials generated by the component projects. This core will determine the presence, location, and severity of cortical malformations in mice from the neuroanatomic, behavioral, and neurogenetic experiments. The Neurohistology Core will play the most involved role in the Neurogenetics Component. All offspring generated by this component will be screened by the core for both macro- and microscopic structural abnormalities. Our primary interest is in nervous system abnormalities, however, the entire animal will be grossly examined to look for any obvious defects in external structure (e.g. polydactyly) and in internal organs (e.g. situs inversus) that may correlate with brain changes. Additional markers of brain involvement, e.g., hippocampal anomalies, will be screened as well. Additional neuroanatomic studies, which include surgery or other specialized manipulations, morphometric studies, special processing such as immunofluorescence, autoradiography, tracer methods, immunohistochemistry, in situ hybridization will be carried out within the specific neuroanatomic projects themselves. The Neurohistology Core will maintain all the neuroanatomic materials, processed and unprocessed.

DESCRIPTION (provided by applicant): "Language, brain, and cognitive development," an open international meeting in Paris in May 2001, is designed to foster interactions amongst the specialists of three disciplines: adult psycholinguistics, infant cognitive development, and cognitive neuroscience. By exposing students and young researchers to top-level research in these fields, the meeting will play a pedagogical role and will encourage further research that cuts across those disciplines. Special emphasis will be put on the contribution of new findings in neuropsychology and brain imaging to the interaction between cognitive developmental psychology and biology. The meeting will consist of twenty half-hour lectures and three round-table discussions, in which experts in the field and participants will discuss the links between language and thought and between maturation and learning, as well as theories of cognitive and linguistic development and the complementarity between biological (neuropsychology and brain imaging) and cognitive psychological research.

DESCRIPTION (provided by applicant): The dyslexic brain displays focal microgyria and related cortical neuronal migration anomalies. Freezing damage to the cortical plate of the rat results in the formation of a malformation resembling human microgyria. Although seemingly focal in nature, rodent research from our and other laboratories has shown that microgyria is accompanied by widespread disruption of cerebral organization, demonstrable at the anatomic, physiologic, and behavioral levels. For example, one of the most robust findings of the past four years of research is the suppression of a system involved in rapid general sound processing in the rodent by the induction of microgyria. This sound-processing anomaly is comparable to that seen in many human dyslexics. We have found that, in relation to the induction of microgyria, correlated abnormalities can be demonstrated anatomically (including architectonics, histochemistry, histometry, and connectivity), physiologically (including field potentials and single cell recordings), and behaviorally (including operant conditioning, gap detection and oddball startle reduction paradigms). A striking sex difference has emerged from the research: microgyria induction does not induce a sound-processing anomaly in female rats. We are struck by this gender difference in developmental plasticity-one adaptive, one maladaptive-which then becomes the focus of the proposed research. In this project, our overall goal is to deepen and broaden our investigations, while concentrating on issues of developmental plasticity and sex differences. We are particularly interested in comparing cellular and molecular mechanisms affecting the development of the aforementioned cortico-cortical and cortico-thalamic circuits. Each specific aim of the current project is designed, therefore, to directly assess differences between microgyric and nonmicrogyric subjects at a number of different stages of development, as well as evaluating differences between the sexes, which we believe will shed light on the more adaptive female response. The Specific Aims are: 1) To describe the changes in types, locations, and numbers of cortical and thalamic neurons and receptors following early injury to the cortical plate;2) To describe the changes in neuron growth and survival associated with early injury to the cortical plate;3) To describe the changes in the outgrowth and survival of connections in the forebrain following early injury to the cortical plate;and 4) To describe the effects of pharmacologic manipulation of these molecular changes on the anatomy, behavior, and physiology of the forebrain following early injury to the cortical plate.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This application requests funds for establishing a state of the art training program in Cognitive Neurology at Beth Israel Deaconess Medical Center, Youville Rehabilitation Hospital, and Harvard University with a focus on research. The arrival of the baby boom generation into late middle age and improved survival after stroke, head injury, and infection, together with a higher life expectancy in general, is resulting in growing numbers of individuals with cognitive impairment. Consistently productive work in cognitive psychology and cognitive neuroscience has led to improved methodologies and better models of normal cognitive, emotional, and behavioral function. These advances are beginning to be applied to the study of human cognitive disorders. However, the marked complexity of the neurocognitive models and methodologies requires formal training for mastery of concepts and the appropriate use of tools. The main motivation for this training grant application is to prepare young neurologists for carrying out up-to-date research in human neurobehavioral disorders at the systems level, which we hope will ultimately result in improved rehabilitation. We propose to train two neurocognitive fellows per year for a period of three years selected from applicants with M.D. or M.D., Ph.D. degrees who have completed a neurology residency. Clinical and research training takes place at three linked sites: the Division of Cognitive Neurology, the Department of Psychology, and the Neurorehabilitation Center, linked to two Neuroimaging Centers . Twenty-four interactive faculty members (9 core, and 15 ancillary) with expertise in cognitive neurology, cognitive neuroscience, neuroimaging, aging, and neurorehabilitation, will work under the guidance of a Program Director and Executive Committee. Principal areas of training include perception, motor control, language, memory, attention, visual cognition, executive functions, and development, as they pertain to normal subjects and to neurocognitively impaired patients. [unreadable] [unreadable]

The Administrative and Research Core units will serve each of the Projects of the Program. In addition, these units will be a resource for grant supported research projects and protocols related to the overall goals of the participating laboratories. Dr. Galaburda, as Director of the Administrative Core (Core A), is the origin and clearing house of ideas that link the projects together, suggests coordinated changes, represents the Program to other institutions and programs, and motivates plans for future research and external collaborations. Dr. Galaburda will also review experimental design and data management, appropriate neurobehavioral instruments, ethical issues concerning animals, and budgetary effects on the Core units. The Executive Committee, which is composed of Drs. Galaburda, LoTurco, Fitch, and Rosen, will also review and advise on publications and meeting participation. The use of the Core budgetary resources will be at the discretion of the Core Director. Any redistribution of funds will require the approval of the Program Director. The use of the Cores is summarized in Table 1. The In Utero Electroporation Core (Core B) is located at the University of Connecticut and directed by Dr. Joseph J. LoTurco. This Core will be responsible for providing Research Projects with embryonicallytransfected live animals (Projects I and III) or perfused brains (Projects I and II). The Neurohistology, Morphometry, and Data Processing Core (Core C) is located at the applicant institution (Beth Israel Deaconess Medical Center) and is directed by Dr. Glenn D. Rosen. The Core will histologically process animals arising in the various research components, will morphometrically assess these brains, and will maintain a database of all animals being used in the Program Project.

DESCRIPTION (provided by applicant): This proposed program project is to study a unique rat model of developmental learning disability that uses methods of developmental neurobiology, structural anatomy, and behavior to analyze the functions of three candidate dyslexia susceptibility genes (CDSGs). Neuropathologic studies in human dyslexic brains and previous animal models have underscored the importance of focal neuronal migration defects and developmental plasticity for some of the dyslexic deficits. The discovery of CDSGs challenges us to analyze the effects of this genetic variation on brain development, structure, and behavior with respect to learning disability. Using an in utero electroporation method developed in their laboratories, the investigators will transfect into young neurons in the ventricular zone short hairpin RNAs or overexpression constructs targeted against homologs in the rat of CDSG Dyx1c1, Kiaa0319, or Dcdc2. They have already seen that this procedure leads to abnormal neuronal migration, alters neuronal morphology, and causes secondary effects in untouched neighboring neurons, thus producing a picture reminiscent of dyslexic brains. Interesting behavioral alterations are also seen. Project I will analyze Dyx1c1's interaction with genes with known molecular pathways involved in process extension, nuclear movement, and cell adhesion, the domains on the Dyx1c1 critical to function. Project II will characterize anatomic changes (cortical architecture, cell identity, morphology, and connectivity) associated with knockdown or overexpression of CDSGs. Project III will uncover behavioral consequences of CDSG disruption (auditory processing and learning), and will attempt to ameliorate the effects of these genetic manipulations by behavioral interventions. The three interactive projects will be supported by an Administrative Core, an in utero Electroporation Core, and a Neurohistology, Morphometry, and Data Processing Core. A better understanding of the functions of CDSGs will shed a broader light on mechanisms of normal brain development and on the abnormalities seen in developmental dyslexia, but also offering the possibility of earlier detection, biologically-based subtyping, and improved treatment. RELEVANCE: Animal models of human disorders have traditionally been helpful for moving the field forward and leading to better diagnostic and treatment approaches. There are few animal models for learning disorders in general, and only one for dyslexia. Results from the proposed work are apt to help us understand human dyslexia more fully, diagnose it more accurately, and define better treatment modalities.

Recent evidence indicates that candidate dyslexia susceptibility genes (CDSGs) have roles in the development of the cerebral cortex, especially in neuronal migration and maturation. In Project II, we will investigate postnatal anatomic consequences of neuronal migration disorders induced by embryonic transfection with small hairpin RNAs (shRNA) targeted against CDSG homologs Dyxld, Kiaa0319, or Dcdc2 in the rat cerebral cortex. Based on preliminary results, and because it is not yet known in humans whether all of these gene variants result in loss of function, we will also investigate the effects of CDSG overexpression. Since all CDSGs share among them an association with dyslexia, in Aim 1 we will address anatomical RNAi and overexpression phenotypes that appear to be shared among the genes?namely a bimodal distribution of transfected cells that either undermigrate or migrate past their expected laminar locations. We will use molecular and birthdate markers to assess the phenotypes of these mismigrated neurons, whether or not layer appropriate. In addition, we will co-transfect gain and loss of function neurons with a wheat germ agglutinin transgene that will allow precise determination of the connectivity of transfected neurons in both control and experimental cases. We will compare the intra- and inter-hemispheric, cortico-cortical, cortico-thalamic, and thalamo-cortical connections in rats transfected with different CDSG shRNAs, as well as between experimentals and controls. In the expectation that this work can guide research on dyslexia subtyping, Aim 2 will focus on systematic differences that are seen in the brains of rats embryonically transfected with shRNA or overexpression plasmids for each of the CDSG homologs. Following completed work in embryos, we will use in situ hybridization and immunohistochemistry to compare the genes' temporal and spatial expression patterns in the postnatal rat. We will also assess the neuronal morphology of transfected neurons and their processes. Aim 3 examines widespread changes in anatomic organization, which are hypothesized to arise directly from local transfections of shRNA or overexpression constructs and as a result of secondary plasticity-related effects. We will use efficient and accurate stereologic probes to estimate neuron number, neuron size, and regional volume throughout the neocortex and thalamus. An accurate description of the forebrain anatomy that results from either knockdown or overexpression of rat homologs of CDSGs, both cell autonomous and secondary effects, and the course of their development, serve as a good bridge between genetics and behavior and will help to shed a broader light on the neurobiological substrates underlying developmental dyslexia in humans. We will link results from this project down to developmental and molecular mechanisms studied in Project I and up to behavioral changes to be characterized in Project III.

DESCRIPTION (provided by applicant): The aim of this award is to create an opportunity for selected residents in the neurology residency training programs of the Beth Israel Deaconess Medical Center (BIDMC) and the Children's Hospital Boston (CHB) to participate for 9 to 24 months in an intensive, mentored, research educational experience during the third year of residency and subsequent fellowship years. This training will be designed to prepare participating residents for successful competition for independent mentored research awards, and will facilitate the transition from resident/fellow to clinician-scientist. Each participant will work with one of 37 mentors, who have been recruited from the faculties of CHB, BIDMC, and Harvard Medical School (HMS). All have active NIH funding and a history of training clinician-scientists. The proposed mentors cover all major areas of the clinical and basic neurosciences, and include 20 investigators from CHB, 16 investigators from BIDMC, and one investigator from HMS (Dr. Michael Greenberg, former leader of the Neurobiology Program at CHB, now Chair of Neurobiology at HMS). Seventeen of these investigators are engaged in clinical/translational neuroscience research, twelve conduct basic neuroscience research, and eight are involved in both basic and clinical/translational research. Mentors have been drawn not only from the Departments of Neurology/Neurobiology, but also from the CHB Division of Neuropathology, the CHB Division of Neuroradiology, the CHB Department of Neurosurgery, the BIDMC Division of Neuropathology, and the Pain Research Program of the BIDMC Department of Anesthesia. Selected resident participants will learn state-of- the-art laboratory skills and will acquire the critical expertise necessary for the conduct of responsible research. Data collected and analyzed will serve for publications as well as for future NIH proposals. The program will be governed by a Steering Committee consisting of the PD/PIs, the Department Chairs, and the Residency Directors of the participating residency programs. This Committee will work together to recruit and select trainees, to monitor their progress, and to evaluate the effectiveness of the training experience. PUBLIC HEALTH RELEVANCE: Illnesses affecting the nervous system remain a great challenge to medical research. The discovery of the causes of these illnesses, leading to earlier diagnosis and better treatments, requires research that relies increasingly on conceptual complexities and sophisticated methodologies. The present R25 proposal is aimed at developing researchers that can combine the clinical expertise obtained during residency with a well developed and focused investigative research program. Neurology residents are keenly aware of the problems that need to be solved and are especially motivated and energized to solve them. The CHB/BIDMC residency program combines both adult and child neurology within an environment that has great depth in basic and clinical neuroscience. Mentors chosen for the R25 include leading NIH funded basic cellular and molecular neuroscientists, as well as successful physician scientists actively engaged in clinical and basic neuroscience and experienced in, and eager to train physician scientists. The main focus of the proposed program will be to prepare physicians for independent and competitive research.