Kira Poskanzer - US grants

Astrocytes are star-shaped glial cells in the brain and spinal cord. They are connective tissue cells of the nervous system that link nerve cells to blood vessels and, by wrapping round brain capillaries, help to form the blood-brain barrier. Understanding of astrocyte signaling seriously lags in comparison with that of neurons, because the astrocytes are not electrophysiologically active. It is not known what extracellular signals astrocytes respond to and how they contribute to circuit function. The research proposed by Poskanzer has the promise of elucidating these key questions. The research has high potential to gain new insight and scientific knowledge. Neural circuits involve many cell types, although the vast majority of circuit studies neglect to take into account the non-neuronal glial cells in the brain. This proposal addresses gaps in our understanding of glial cells in neural circuit function and harnesses the power of light-activatable tools to tackle them. The PI applies a suite of optochemical tools that allow spatiotemporally precise and physiologically relevant release of neurotransmitter to astrocytes and neurons in cortical brain circuits. Compounds promising for research will be made available to the scientific community, and many collaboration opportunities are available at USCF.

The PI proposes to use ruthenium-bipyridine (RuBi) cages to deliver neurotransmitters to astrocytes by photo-activation and monitoring the response from Ca++ activity in their branches by a genetically encoded Ca++ indicator using a second laser. She will test the hypothesis that astrocytes can respond to neurotransmitters and whether astrocytes can be activated in vivo. This proposal investigates the very fundamental question of how astrocytes respond to a range of neurotransmitters. Novel compounds will be developed to deliver controllable concentrations of neurotransmitters to cells. Calcium response will be monitored and astrocyte response will be studied as concentration is varied. Following initial in-vitro testing in brain slices. She will test the hypothesis that astrocytes can respond to neurotransmitters and whether astrocytes can be activated in-vivo in mice.

The PI has a unique international collaboration with the Etchenique lab in Argentina and is enthusiastic about introducing biology students to imaging techniques, as evidenced by the relevant Cellular and Molecular Neuroscience course and the position with the Woods Hole Marine Lab. Undergraduate students will be involved through the USCF summer research program, and K-12 outreach through the science and health education partnership.

A Functional Taxonomy of Cortical Astrocytes The vast majority of neural circuit studies neglect to take into account the non-neuronal cells in the brain, but in order to truly appreciate neural circuit function, we will need to monitor and manipulate activity in many cell types. Our understanding of astrocyte signaling is years behind that of neurons, because the appropriate tools have been lacking for these largely electrically silent cells. We don't know what extracellular signals astrocytes respond to, nor how they contribute to circuit function. This is due, in part, to the lack of methods that replicate the breadth of possible presynaptic activity in vivo, i.e. the release of neurotransmitter. The current proposal addresses gaps in our understanding of astrocytes in neural circuit function and harnesses the power of light-activatable tools to tackle them. We propose to apply a suite of optochemical tools that allow spatiotemporally precise and physiologically relevant release of neurotransmitter to astrocytes and neurons in cortical circuits. In Aim 1, we will test the hypothesis that astrocytes response acutely to the synaptic release of excitatory and inhibitory synaptic activity with differential and predictable activity. We will use simultaneous two-photon optochemical uncaging and calcium imaging in astrocyte branches to test their physiological response to glutamatergic and GABAergic synaptic events, and uncover the heterogeneity of molecular mechanisms that govern these responses. We will activate astrocytes in vivo using these optochemical techniques, and in Aim 2, genetically silence astrocyte-specific excitatory and inhibitory receptors during in vivo imaging and electrophysiology to determine the astrocytic signals that lead to downstream cortical state shifts. In Aim 3, we will validate novel optochemical tools to mimic the release of neuromodulators in the cortical circuit, testing their function in neurons and astrocytes. With these tools in hand, we will be able to probe the repertoire of signals to which astrocytes respond in the circuit.

PROJECT SUMMARY Motivated behaviors, included those altered in substance use disorders, are known to be regulated by monoaminergic circuits (containing neurons that release dopamine, serotonin, and norepinephrine) in multiple brain regions. Disentangling these circuits from the cell-level to the systems- and behavioral-levels has almost exclusively been limited to studying the physiology of neurons. However, recent data suggest that the activity of a non-neuronal cell type?astrocytes?may have profound effects on how monoaminergic networks function, but how astrocytes respond to monoamines and change their physiology in response to repetitive drug exposure remain almost completely unstudied. The current proposal aims to address gaps in our understanding of addiction, monoamine signaling, and multi-cell type population dynamics by uncovering fundamental cell biological signaling systems in astrocytes that are critical for the function of brain regions with monoaminergic input. In Aim 1, we will use two-photon imaging and photoactivation to interrogate the cell signaling mechanisms that prefrontal cortical astrocytes deploy to sense extracellular monoamine dynamics across cortical layers. In Aim 2, we will decode the mechanisms by which astrocytes change, both instrinsically and in their output to the neuronal population, after long-term exposure to a psychostimulant. With these aims, we will probe the roles of astrocyte physiology in baseline prefrontal cortex function and as mediators of circuit neuroplasticity in a model of addiction.

PROJECT SUMMARY/ABSTRACT Aging-associated brain disorders, including cognitive decline, are among the greatest public health challenges. But without understanding the basis of age-related brain disorders at the molecular, circuit, and systems levels, effective therapeutic strategies cannot be developed. DNA repair is emerging as a potential regulator of age- related cognitive decline and neurodegeneration. The brain may be vulnerable to genomic alterations due to its network structure, the complexity of its transcriptome, and the low or absent turnover and long lifespan of neural cell types. This suggests genome maintenance pathways are crucial for brain health: persistent or incorrectly repaired DNA double-strand breaks (DSBs) could contribute to genomic alterations, thus promoting age-related cognitive impairment and neurodegenerative disorders. The role, however, of post-developmental neuronal and astrocytic DSB repair in brain physiology and maintenance of brain function with age has not been addressed. Moreover, theories of cognitive decline have focused on potential age-related changes in neuronal function, neglecting consideration of astrocytes and the complete neuro-glio-vascular circuit. The broader implication for these fundamental gaps in knowledge is that crucial opportunities for development of therapeutics for treatment and prevention of brain disorders may be missed. This provides a strong rationale for elucidating the biology of neuronal and astrocytic DSB repair at multiple levels. Thus, our long-term goal is to determine the extent to which neural DNA double-strand break formation and repair impact brain function and disorders. This application proposes to elucidate the relationship between systems-level neural circuit function and the DNA repair machinery in neurons and astrocytes with age. The central hypothesis of the proposed project is that DNA double-strand break formation and repair in mature neurons and astrocytes impact neural physiology. To test this hypothesis and to advance toward our long-term goal, we propose the following specific aims: (1) Determine impact of classical non-homologous end-joining DNA repair on neuronal physiology; (2) Elucidate role of astrocytic DSB repair in circuit homeostasis and maintenance of neural function during aging; and, (3) Elucidate non-canonical, homology-directed DSB repair pathways in neurons. The proposed approach involves a comprehensive, multidisciplinary analysis of neuronal and astrocytic function at the genetic, organismal, and circuit level. The proposed project is significant because it will use innovative approaches to investigate emerging concepts with major implications for human brain health, age-related cognitive decline, and neurodegenerative diseases. The project is further significant because it will refine and develop new research tools and models. Insights gained from the proposed studies are also expected to inform research and knowledge in other fields related to genomic stability and aging.

Sleep and wake are critical for animal survival, but the mechanisms by which the brain coordinates switching between these brain states, from brainstem to cortex, remains unclear. Neuromodulatory signaling circuits are essential systems for brain state switching, both in sleep/wake and in other contexts. In the current proposal, we explore the role of one of these neuromodulators?histamine?in the regulation of sleep and wake in the cerebral cortex. Although histamine has long been known as a wake-promoting neuromodulator, it is relatively understudied compared to others, so our understanding of its cellular and circuit mechanisms of action is still in the early stages. Here, we take a holistic perspective of the cell types involved in sleep/wake circuits, and ask how the largest class of non-neuronal cells in the brain, astrocytes, are involved in sending and responding to histaminergic signals in the cortex. We aim to address gaps in our knowledge of sleep/wake dynamics, histaminergic signaling, and astrocytic regulation of neural circuits by uncovering critical cell biological signaling systems in astrocytes. We apply advanced optical, electrophysiological, and neural manipulation techniques to reveal how astrocytes may be integrating neuromodulatory signals in cortical circuits and coordinating populations of neurons. Our main goals include: probing the cell biological mechanisms by which histamine activates astrocytes, testing the spatiotemporal dynamics of histamine and astrocytes to determine their causal relationships, and exploring the mechanisms by which astrocytes may synchronize or desynchronize neuronal activity.