Ludise Malkova - US grants

DESCRIPTION (provided by applicant): The goal of this proposal is to understand how early brain insults influence the short-term and long-term development of cognitive and socioemotional functions. This understanding is vital for the approach to neurodevelopmental disorders such as autism, and at the same time is relevant to an entire spectrum of behavioral disorders that emerge as a result of a host of neurological disturbances in children (epilepsy, cerebral palsy, cortical dysgenesis, etc.). The analysis of human cases and experiments in animals suggest the hypothesis that dysfunction in medial temporal lobes, and the amygdala in particular, is an etiological factor in autism. Therefore the goal of our proposal is to investigate the role of the amygdala and its specific subdivisions for socioemotional behavior and to identify the critical developmental periods and neural triggers for developmental abnormalities in an animal model of autism. The first Specific Aim concentrates on the effects of pharmacologically-induced imbalances in neurotransmission in the amygdala on social interactions in infant animals. Drugs known to either block or enhance GABA-ergic or glutamatergic transmission will be focally infused into specific subdivisions of the amygdala and social interactions in the experimental animals will be observed. The second Specific Aim is to compare the effects of drugs (as obtained in Aim I) with the effects of discrete lesions of subregions of the amygdala, when damaged by axon-sparing excitotoxic lesions, in infant monkeys. The third aim will evaluate the effects of early prolonged seizures, known to disrupt the function of the amygdala and its projection network, on the observed behavioral categories. This aim is directed to the understanding why certain seizure disorders in infants (e.g., infantile spasms) give rise to autism. An important facet of these studies will be the analysis of the extent to which socioemotional disturbances produced by various anatomically site-specific insults are accompanied by impairment in cognitive functions. Well standardized procedures will be used to evaluate various components of socioemotional interactions of infant and juvenile animals in dyads and to assess their emotional reactions to positive and negative stimuli. Cognitive tasks will include concurrent visual objects discrimination and auditory-visual crossmodal associations and memory. The results of the proposed studies will identify specific amygdaloid nuclei critical for regulation of social and emotional interactions and determine the critical stage(s) in development during which amygdala dysfunction (by lesions, drugs or seizures) can lead to long-term socioemotional abnormalities. This information will provide a rationale basis for the design of therapeutic interventions directed at correcting the underlying biological dysfunctions that give rise to autism and related socioemotional disorders.

[unreadable] DESCRIPTION (provided by applicant): The research and career development plans proposed in this application will expand the candidate's expertise in neuropharmacology, normal and abnormal socioemotional development, epileptology, and magnetic resonance imaging (MRI) as applied to neurodevelopmental disorders such as autism, mental retardation, and cerebral palsy. The Research Plan proposed will take advantage of the recent evidence that dysfunction in medial temporal lobes, and the amygdala in particular, may be an etiological factor in autism. The goal of the application is to investigate the role of amygdala and its specific subdivisions for socioemotional behavior, and to identify the critical periods and neural triggers for developmental abnormalities in an animal model of autism. Specific Aim 1 concentrates on the effects of pharmacologically induced imbalances in neurotransmission in amygdala on social interactions in infant animals. Specific Aim 2 will compare the effects of drugs (as obtained in Aim 1) with the effects of discrete lesions of subregions of amygdala, damaged by axon-sparing lesions. Specific Aim 3 will evaluate the effects of early prolonged seizures, known to disrupt the function of amygdala and its projection network, on the neurodevelopmental behavioral outcomes. The experiments designed to pursue these aims will provide an opportunity for the candidate to gain both theoretical and practical expertise in the combined use of pharmacological, physiological, and MRI approaches to the study of the animal models. The candidate's background in behavioral research will be applied to the analyses of various components of socioemotional interactions of infant and juvenile animals in dyads and the assessment of their cognitive functions. The career development plan will facilitate a substantial shift in the candidate's research capabilities and scope, so that it will become possible for her to build an independent research program devoted to the identification of neural mechanisms underlying autism and other developmental disorders. The ability to evaluate the multifactorial impact of pharmacological interventions in animal models will prepare the candidate to pursue research on animal models of neurodevelopmental disorders and place her research skills in the context of pharmacology, epileptology, and physiology. Structured activities and short courses will guide the development of expertise in these areas necessary for the proposed studies as well as for the long-term career advancement of the candidate as a versatile neuroscientist. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A large proportion of subjects with autism exhibit seizure disorders and epilepsy. This co morbidity has been reported to be in the range of 30-40%. Epilepsy and abnormal EEG patterns occur at a significantly higher rate in individuals in the more impaired range of the autism spectrum. Seizures in the autistic population often include complex partial seizures involving the temporal lobe. The limbic network supporting these seizures is composed of the amygdala, hippocampus, medio-dorsal thalamus, piriform, rhinal, and orbitofrontal cortices. Recurrent and/or prolonged complex partial seizures alter the functional connectivity of this network in a manner that can impact both cognitive function and socio-emotional regulation. Importantly, this is the same network implicated in autistic pathophysiology and therefore these seizures may pose the greatest risk for adverse psychiatric outcomes in this population. Dysfunction (often in the absence of structural abnormalities) in the network anchored in the amygdala, and interconnected with the orbital frontal cortex, has been found in many cases of autism. This dysfunction is likely to be exacerbated by the aberrant plasticity induced in response to repeated seizure discharge. The goal of this application is to determine if a history of repeated seizure activity changes the responsiveness of this network and whether it predisposes this network to amygdala-mediated behavioral disturbances. Our recent findings showed that reversible manipulations of the GABAA receptors within the basolateral amygdala (BLA) by focal intracerebral infusions of GABAA receptor agonists or antagonists resulted in profound changes in social interactions and reward evaluation in nonhuman primates. The Specific Aims will determine whether a history of complex partial seizures, focally-evoked from the piriform cortex in one hemisphere, result in an increased vulnerability to disinhibition within BLA in nonhuman primates. This shift in sensitivity will be probed by evaluating specific behavioral responses to focal manipulations of GABAA transmission within BLA: Social interactions (Aim 1), reinforcer devaluation (Aim 2), and emotional conditioning (Aim 3). The studies will address a recognized comorbidity, not yet studied in pre-clinical animal models, for which clinical studies cannot sort out the extent to which seizures may exacerbate the autistic symptomatology. The combination of co-investigators provides a unique blend of expertise in experimental epilepsy models, nonhuman primate models of socio-emotional disturbances, and neural substrates of human disorders of affect and psychopathy. The team is ideally suited to approach the analysis of comorbidity of seizures and autistic-like symptoms evoked from amygdala and to permit a translationally meaningful analysis of the animal data. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The proposal aims to identify the neural substrates in the brain that are responsible for abnormal socio-emotional behavior. This understanding is vital for developing both diagnostic and therapeutic approaches to identify and treat the abnormal brain circuitry in neuropsychiatric conditions such as affective and anxiety disorders. The field of social behavior research still lacks appropriate animal models for social pathology that leads to inappropriate aggression, social anxiety/phobias, social withdrawal/detachment, impulsivity, or defensiveness. The study of the neurobiology of social behavior in non-rodent models is especially promising since the impairment in social interactions and emotional processing generates symptomatology that resembles psychopathology observed in human patients. Moreover these studies will permit an analysis of social interactions at a level not feasible in rodents. Therefore, this work promises to substantially advance our understanding of the neural substrates of human social interactions in the norm and pathology with a special relevance to anxiety and depression. The proposed research seeks to investigate the novel components of the amygdala-based network that regulates socioemotional responses in order to account for imbalances that can give rise to psychopathology in the absence of structural lesions. The effects of the DLSC manipulations on the vulnerability to behavioral abnormalities evoked by disinhibition within nuclei of the amygdala will be analyzed to determine the role of DLSC in mediating and/or modulating the influence of the amygdala circuitry. An understanding of the functional relationship between the amygdala-derived and colliculus- derived regulation of defensive and aggressive emotional tone is expected to reveal novel targets for both etiology and therapeutic intervention for affective and anxiety disorders.

DESCRIPTION (provided by applicant): The proposal aims to identify the neural substrates in the brain that are responsible for abnormal socio-emotional behavior. This understanding is vital for developing both diagnostic and therapeutic approaches to identify and treat the abnormal brain circuitry in neuropsychiatric conditions such as affective and anxiety disorders. The field of social behavior research still lacks appropriate animal models for social pathology that leads to inappropriate aggression, social anxiety/phobias, social withdrawal/detachment, impulsivity, or defensiveness. The study of the neurobiology of social behavior in non-rodent models is especially promising since the impairment in social interactions and emotional processing generates symptomatology that resembles psychopathology observed in human patients. Moreover these studies will permit an analysis of social interactions at a level not feasible in rodents. Therefore, this work promises to substantially advance our understanding of the neural substrates of human social interactions in the norm and pathology with a special relevance to anxiety and depression. The proposed research seeks to investigate the novel components of the amygdala-based network that regulates socioemotional responses in order to account for imbalances that can give rise to psychopathology in the absence of structural lesions. The effects of the DLSC manipulations on the vulnerability to behavioral abnormalities evoked by disinhibition within nuclei of the amygdala will be analyzed to determine the role of DLSC in mediating and/or modulating the influence of the amygdala circuitry. An understanding of the functional relationship between the amygdala-derived and colliculus- derived regulation of defensive and aggressive emotional tone is expected to reveal novel targets for both etiology and therapeutic intervention for affective and anxiety disorders.

DESCRIPTION (provided by applicant): The proposal aims to identify the neural substrates in the primate brain that are responsible for abnormal socio-emotional behavior. This understanding is vital for developing both diagnostic and therapeutic approaches to identify and treat the abnormal brain circuitry in neuropsychiatric conditions such as affective and anxiety disorders. The field of social behavior research still lacks appropriate animal models for social pathology that leads to inappropriate aggression, social anxiety/phobias, social withdrawal/detachment, impulsivity, or defensiveness. The study of the neurobiology of social behavior in the nonhuman primate is especially promising since the impairment in social interactions and emotional processing generates symptomatology that resembles psychopathology observed in human patients. The proposed research seeks to investigate the novel components of the amygdala-based network that regulate socioemotional responses in nonhuman primates in order to account for imbalances that can give rise to psychopathology in the absence of structural lesions. We have determined that reversible pharmacological manipulations of specific sites within the amygdala result in profound changes in social interactions and emotional behavior in macaque monkeys. Further, we have discovered that the activation of the intermediate and deep layers of the superior colliculus (DLSC; a midbrain structure) by focal infusions of GABAA antagonist bicuculline, has a profound impact on emotional reactivity and defensive responses. In the proposed experiments, we will use the method of intracerebral focal drug infusions of either the GABA agonist muscimol or the GABA antagonist bicuculline aimed at various sites within either amygdala or DLSC to test further the role of these structures in socioemotional behavior. Specific Aim 1 will define specific nuclei within the amygdala responsible for GABA- mediated regulation of socioemotional responses. Aim 2 will test the hypothesis that disinhibition of DLSC will evoke aggressive behavior and emotional hyperactivity. Aim 3 will assess the role of DLSC and amygdala in fear-potentiated startle, a conditioning paradigm that is altered in human patients with PTSD. Social interactions will be assessed in dyads, each infused animal will be paired with a familiar non-infused partner; videotaped behaviors will be analyzed by two independent observers using a standardized behavioral categorization. Special attention will be paid to reciprocal social interactions, aggressive behavior, and emergence of any abnormal behaviors (e.g., stereotypies, self-directed behaviors). Emotional reactivity will be tested by presenting the animals with a standard set of stimuli (neutral, positive, and negative). An understanding of the functional relationship between the amygdala-derived and colliculus-derived regulation of social interactions and emotional tone is expected to reveal novel targets for both etiology and therapeutic intervention for affective and anxiety disorders.

DESCRIPTION (provided by applicant): The Interdisciplinary Program in Neurosciences (IPN) at Georgetown University is a broad-based, transdisciplinary, non-departmental program leading to a Ph.D. in Neuroscience. The program, established in 1994, trains students in the scholarly pursuit of research in integrative neuroscience, from the cell to the intact behaving organism. The 32 core training faculty and 20 supporting faculty are drawn from 14 clinical and basic science departments on the Main Campus and Medical Center; they span a breadth of inquiry, ranging from neurotransmitter receptors and signal transduction, to behavior and human disease. Areas of research strengths include 1) neural injury, degeneration, and plasticity; 2) synaptic modulation and signal transduction; 4) neural substrates of autism, epilepsy, schizophrenia, dementias, and addiction; and 5) telencephalic neural networks subserving sensory processing, memory and language. Students gain training in a range of approaches, including molecular, genetic, neurophysiological, cognitive testing, computational and imaging techniques. Training Grant funds support prethesis training (8 slots during the first 2 years); research grants and individual fellowships support thesis research. The program enrolls 40-50 thesis and prethesis students. Aggressive recruitment of underrepresented racial and ethnic applicants continues to be a top priority. The training environment fosters interactive, pandisciplinary research of both faculty and trainees. Over 40% of the core training faculty are in close proximity in the Research Building, with state-of-the-art core facilities and custom designed laboratory and office space. Faculty are highly collaborative; students are encouraged to seek co-mentorship between faculty with interfacing interests and complementary approaches. All core training faculty have research grant support and fully equipped facilities for training pre-and postdoctoral students. The recent recruitment of several neuroscience faculty into the Departments of Pharmacology, Psychology and Neuroscience, has expanded the equipment, facilities and faculty expertise available to the training program. The training program includes broad-based didactic coursework, as well as rotations in laboratories of the training faculty. The trainees participate in a seminar series, national professional meetings, journal clubs, intensive laboratory research, and training in several essential professional skills (writing and reviewing manuscripts, grantsmanship, mentorship, teaching, conflict resolution, career choices, oral presentations) and their ethical dimensions. Opportunities for gaining practical teaching experience at the undergraduate and secondary school levels are abundant and encouraged.

The Interdisciplinary Program in Neurosciences (IPN) at Georgetown University is a broad-based, non- departmental program that leads to a PhD in Neuroscience. The IPN was established in 1994 and supported by the NIH from 2001 to 2015. The primary goal of this training program is to develop ?Stewards of the Discipline? by training students in the scholarly pursuit of research in integrative neuroscience. The 25 core training faculty and 19 supporting faculty in this application are drawn from nine clinical and basic science departments on the Main Campus and Medical Center, contiguous campuses in Washington, DC. The IPN faculty span a breadth of inquiry; the core faculty are split evenly between cellular/molecular and cognitive sciences, with several researchers collaborating across these disciplines. IPN students are very successful, publishing an average of 4.7 papers from their thesis training, with 2.3 as first author. Students average 5.3 years to defense, with a median time of 5 years. Training Grant funds are requested to support prethesis training (4 slots each for first and second years), while Georgetown University supports additional students in prethesis training (4 slots each for first and second years). The support was greatly increased over the last two years in the absence of training grant funding. Institutional support also covers recruitment, a seminar series, a program retreat, and bridge funds for thesis students. Two NIH training grants provide additional thesis research training. The IPN consists of 40-50 thesis and prethesis students. Aggressive recruitment of applicants from underrepresented racial and ethnic groups have been successful (currently 32% of students), and continues to be a top priority. IPN students benefit from the collaborative nature of our faculty: over 70% of student publications have more than one IPN faculty member. The recent recruitments of neuroscience faculty into the Departments of Pharmacology & Physiology, Psychology, Neurology, and Biology have expanded the equipment, facilities and faculty expertise available to the training program. The training program includes broad-based didactic coursework with increased emphasis on quantitative aspects of inquiry and the rigor of scientific approaches. The trainees participate in a seminar series, national professional meetings, journal clubs, intensive laboratory research, and training in several essential professional skills (writing and reviewing manuscripts, grantsmanship, mentorship, teaching, conflict resolution, career choices, oral presentations) and their ethical dimensions. Students are also very active in governance of the IPN. Opportunities to explore career options are developed throughout the graduate career, particularly with the establishment of a new graduate Office of Career Strategy and Professional Development. Upon graduation, 60-70% of IPN students pursue post-doctoral research training; 11 of them (since 2005) have earned tenure track faculty positions (with more progressing quickly on research active tracks). Approximately 15% go into careers related to science policy and administration, based largely on our location in the nation's capital and the proximity of the NIH.

Exaggerated emotional reactivity, impaired social function, aberrant regulation of defense behaviors, and autonomic dysregulation are a constellation of debilitating symptoms that are present in a range of anxiety disorders. Anxiety disorders, as a group, impact about 20% of the US population and treatments for anxiety disorders are only partially effective and often associated with side effects. While most attention has focused on fronto-limbic circuitry, a current gap in knowledge is the contribution of hindbrain circuits. A second major gap is how hindbrain and forebrain sites interact. Moreover, the vast majority of circuit-level characterization has occurred in rodent models, which leads to the third major gap in knowledge: the functional organization of these circuits in non-human primates. Indeed, as evidenced by findings in our lab and by others, the primate brain is organized in often surprisingly different manners than the rodent brain. Thus, understanding the organization of these circuits in the primate brain is essential to understanding the organization of the human brain. We have previously found that acute disinhibition of the deep layers of the superior colliculus (DLSC), a midbrain structure, by focal infusions of the GABAA antagonist, bicuculline, precipitated a state of exaggerated defensive and emotional reactivity (DER). Concurrent inhibition of the basolateral amygdala (BLA) reduced some but not all of the defense responses, suggesting differential circuitry underlying individual components of the defensive response. In this application, we propose to determine the circuit architecture by which hindbrain (DLSC, PAG) and forebrain (BLA, central nucleus of the amygdala, pulvinar) regions interact to produce defensive emotional reactions, unconditioned fear, dysregulation of social behavior, and autonomic arousal. In the two proposed specific aims, we will test the hypotheses that induced inhibition of the limbic components will attenuate the DER evoked from the midbrain structures and that induced inhibition of midbrain structures will attenuate the DER evoked from the forebrain. Using MRI-guided intracerebral microinfusions, we will transiently activate and inactivate components of this network and determine the resulting impact on anxiety- relevant behavioral responses. Following these experiments, we will employ anatomical tracer techniques to characterize projection pathways of interest. We will also perform validation experiments using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), which have grown in use in rodents, but remain rarely used in primates, to help move this translational technology forward. We expect that our data will have implications for understanding the pathology of anxiety disorders.