Linda E. Wilbrecht - US grants

DESCRIPTION (provided by applicant): How does the brain change with experience? Once the brain is mature, can it be rewired by growth of new synapses and loss of older ones? Classic studies measuring spine densities or synapse numbers have relied on post-mortem techniques which do not allow direct observation of spine growth and loss. Recently, 2-photon microscopy in fluorescent protein transgenic animals has made it possible to follow dendrites and spines over weeks to months of an animal's life. The aim of the project will be to describe the long-term effect of sensory experience changes on dendrite and spine persistence in layer V and layer I I/I 11 neurons of the barrel cortex. Receptive field plasticity has been observed in deprived barrel columns where responses to deprived whiskers become depressed within days and responses to adjacent spared whiskers are enhanced 1-3 weeks later. It is not known if either process involves the formation or loss of synapses. Using long-term in vivo imaging and electron microscopy in wild-type and alphaCamKIIT286A (receptive field plasticity impaired) mice, we will document the loss and formation of synapses that occur normally and 1- 3 weeks after chessboard whisker deprivation.

DESCRIPTION (provided by applicant): Genetic variation that alters activity-dependent circuit formation may contribute to an experience-dependent imbalance in circuit development. Brain derived neurotrophic factor (BDNF) is a neurotrophic factor involved in developmental maturation of neural processes and cell survival. 20-30% of Caucasians carry a polymorphism in the BDNF gene, where codon 66 is altered from a valine to methionine (Shimizu et al., 2004), a change which has been shown to alter activity regulated release of BDNF (Chen et al., 2006). The BDNF val66met polymorphism has been linked to anxiety and depression (although not without controversy), with stronger correlation in persons who have had the greatest amount of early life stress (Gatt et al., 2009). We hypothesize that individuals possessing the met66 allele of BDNF have weakened parahippocampal inputs to the cingulated and that early life stress enhances amygdala inputs to the cingulated. We hypothesize an imbalance in projections to the frontal cortex biases emotional experience and behaviors. In our animal model of this process, underdevelopment of the (parahippocampal) perirhinal (PRH) cingulated afferents enable enhanced, competitive development of the basolateral amygdala (BLA) cingulated afferents leading to abnormal dominance of harm avoidance information in frontal circuits involved in action selection and cognitive control. We propose a PRH:BLA cingulated projection imbalance, with diverse causes, may underlie a range of disorders in the spectrum of anxiety and novelty seeking. We will test the hypothesis that BDNF met66 variant mice which show enhance anxiety (Chen et al. 2006) also show unbalanced development of PRH and BLA inputs to the cingulated cortex. To pursue this putative endophenotype of anxiety, we will use state of the art longitudinal in vivo imaging of synapse formation (Aim 1) and optogenetic tools (Aim 2) to probe the adolescent development of long range afferents from the PRH and BLA to the cingulated cortex in BDNF val66met knocking mice (Chen et al., 2006). Specific measures will include in vivo cingulated spine and PRH and BLA bouton turnover and density (Aim 1), the ratio of excitatory and inhibitory currents driven by the BLA and PRH afferents in the cingulated, and the average PRH- and BLA-cingulated AMPA:NMDA ratio (Aim 2). We will also investigate the effects adolescent stress and cognitive training on plasticity (Aim 1) and connectivity (Aim 2) of PRH and BLA afferents. We predict that cognitive training in tasks that engage the PRH and the cingulated together will protect against the development of PRH:BLA imbalance and reduce anxiety behavior. Our experiments will inform understanding of the developmental etiology of disorders of anxiety and harm avoidance, provide an endophenotype that may be transferred to human studies, and test forms of cognitive therapy to rebalance circuit development. 7. Project Narrative Excess anxiety or its opposite, low harm avoidance, can profoundly disrupt human lives. In our model, abnormal juvenile development leads to a lack of balance in inputs that relay information about safety and danger to the frontal cortex adversely affecting decision making. We will test this model to illuminate the developmental causes of disorders of anxiety and harm avoidance, provide a biological measure that can be transferred to human studies, and test forms of cognitive therapy to repair an imbalance in these circuits.

DESCRIPTION (provided by applicant): Adolescent exposure to stimulants, such as cocaine, may permanently affect the coordinated development of the frontal cortex at the synaptic, circuit and behavioral level. The development of the frontal cortex takes place during late childhood and adolescence, a critical moment for the development for substance use (Paus et al., 2007;Chambers et al., 2003;Spear, 2000). Classic histological analysis and recent longitudinal anatomical structural imaging studies have shown that human frontal cortical development is highly dynamic during adolescence (Lewis et al.,1997;2008;Paus et al., 2008). Volatility of this period may create vulnerability to the development of addiction and serious mental health issues. Repeated stimulant exposure consistently enhances spine density in the apical dendrites of the medial prefrontal cortex in adult rodents (Robinson and Kolb, 2004). It is unclear if this effect is due to fewer synapses lost or more gained in the dynamic process of spine turnover which continues in the cortex through adulthood (Holtmaat et al., 2005). It is also unclear if these extra spines represent greater connectivity from the amygdala, the thalamus, or other regions that innervate frontal dendrites. Further work needs to be done to understand how stimulant exposure affects spine plasticity and synapse properties specifically during the volatile period of adolescent maturation. Our understanding also needs to be refined, so that we better understand the mechanisms of these synaptic changes and specificity to particular circuits. We are using multi-photon imaging technology to determine the effect of early and late adolescent binge cocaine exposure on spine structural dynamics in vivo (spine growth and loss, Aim1 ) and optogenetic technology to measure the balance of input from specific, isolated, long-range afferents that drive frontal cortex (Aim 2). We supplement these anatomical and functional studies of synapses with behavioral analysis to assess the function of the frontal cortex in mice exposed to cocaine at early and late stages of adolescence and saline controls (Aim 3). We will compare the short and long term effects of cocaine exposure on spine dynamics, synapses and behavior in both adolescent and adult mice. Our studies will illuminate the developmental synaptic and circuit mechanisms that make adolescence a high risk period for the development of substance use problems and will inform clinicians and stimulant users of possible negative impacts of use on specific frontal circuit synapses at different stages of development. By identifying specific circuits, synapses, and synaptic plasticity mechanisms that are disrupted by stimulant exposure, our data will also serve as a guide for selection and testing of future drug and cognitive therapies to ameliorate the negative effects of adolescent stimulant exposure on specific neural circuits in adult brains. PUBLIC HEALTH RELEVANCE: Adolescent exposure to stimulants, such as cocaine, may permanently affect the coordinated development of the frontal cortex at the synaptic, circuit and behavioral level. We will measure the effect of cocaine exposure on in vivo spine dynamics, synapses and behavior in both adolescent and adult mouse models. By identifying specific circuits, synapses, and synaptic plasticity mechanisms that are disrupted, our data will also serve as a guide for selection and testing of future drug and cognitive therapies to repair the negative effects of adolescent stimulant exposure on adult neural circuits.

DESCRIPTION (provided by applicant): Adolescent exposure to stimulants, such as cocaine, may permanently affect the coordinated development of the frontal cortex at the synaptic, circuit and behavioral level. The development of the frontal cortex takes place during late childhood and adolescence, a critical moment for the development for substance use (Paus et al., 2007; Chambers et al., 2003; Spear, 2000). Classic histological analysis and recent longitudinal anatomical structural imaging studies have shown that human frontal cortical development is highly dynamic during adolescence (Lewis et al.,1997;2008; Paus et al., 2008). Volatility of this period may create vulnerability to the development of addiction and serious mental health issues. Repeated stimulant exposure consistently enhances spine density in the apical dendrites of the medial prefrontal cortex in adult rodents (Robinson and Kolb, 2004). It is unclear if this effect is due to fewer synapses lost or more gained in the dynamic process of spine turnover which continues in the cortex through adulthood (Holtmaat et al., 2005). It is also unclear if these extra spines represent greater connectivity from the amygdala, the thalamus, or other regions that innervate frontal dendrites. Further work needs to be done to understand how stimulant exposure affects spine plasticity and synapse properties specifically during the volatile period of adolescent maturation. Our understanding also needs to be refined, so that we better understand the mechanisms of these synaptic changes and specificity to particular circuits. We are using multi-photon imaging technology to determine the effect of early and late adolescent binge cocaine exposure on spine structural dynamics in vivo (spine growth and loss, Aim1 ) and optogenetic technology to measure the balance of input from specific, isolated, long-range afferents that drive frontal cortex (Aim 2). We supplement these anatomical and functional studies of synapses with behavioral analysis to assess the function of the frontal cortex in mice exposed to cocaine at early and late stages of adolescence and saline controls (Aim 3). We will compare the short and long term effects of cocaine exposure on spine dynamics, synapses and behavior in both adolescent and adult mice. Our studies will illuminate the developmental synaptic and circuit mechanisms that make adolescence a high risk period for the development of substance use problems and will inform clinicians and stimulant users of possible negative impacts of use on specific frontal circuit synapses at different stages of development. By identifying specific circuits, synapses, and synaptic plasticity mechanisms that are disrupted by stimulant exposure, our data will also serve as a guide for selection and testing of future drug and cognitive therapies to ameliorate the negative effects of adolescent stimulant exposure on specific neural circuits in adult brains.

This Science of Learning Collaborative Network brings together researchers from across the University of California-Berkeley and the University of California-San Francisco, to advance scientific understanding of developmental changes that occur in the learning processes of children and adolescents. Growing evidence shows that learning processes and the underlying brain systems go through important developmental changes. These changes begin during infancy and early childhood, but they also extend much later in pubertal maturation and adolescent development. A team-science approach will be used to address these complex issues. The collaborative research network includes expertise in the developmental science of adolescence, and in the science of learning in both human and animal models. A deeper understanding of the developmental changes in specific learning processes in adolescence will inform educational methods and interventions. With greater developmental precision, it should be possible to design more effective education for specific age groups. The long-term goals are to help transform the adolescent "window of vulnerability" (when so many youth become bored and disengaged from school) into a "window of opportunity" (a natural period of curiosity, exploration, and unique learning opportunities).

This collaborative research network builds upon (and helps to integrate) four distinct areas in the science of learning: a) the developmental science of adolescence; b) animal models of brain development in adolescence; c) animal models of learning, and d) computational modeling of learning in humans and animals. The network members will work together to develop new methods, tasks, and analyses that better isolate specific learning variables under transition at adolescence. By tracking pubertal measures as well as age, the work is expected to illuminate the role of puberty onset in developmental transitions in learning, independent from age. The use of mouse models will enable experiments that delineate the role of specific aspects (and timing) of puberty in relation to these specific changes in learning. The integration of human and animal models in parallel experiments will establish a bridge between the fields of developmental science, computational neuroscience, cognitive neuroscience, and systems neurobiology. Scientists and trainees will participate in 'cross-training' opportunities through network meetings, contributing to building a stronger interdisciplinary culture of interaction and collaboration. Undergraduate trainees from underserved backgrounds will also participate in the network.

The award is from the Science of Learning-Collaborative Networks (SL-CN) Program, with funding from the SBE Division of Behavioral and Cognitive Sciences (BCS), the SBE Office of Multidisciplinary Activities (SMA), and the CISE Division of Computer and Network Systems (CNS).

PROJECT SUMMARY Dopamine, norepinephrine, and serotonin are major modulatory neurotransmitters that are implicated in a wide variety of psychiatric and neurological disorders, including addiction. Our available methods to quantify the dynamics of these neurotransmitters in extracellular space are not as fast, sensitive, direct or as clean as we would like. Here we propose to leverage recent progress in nanoscience to solve this problem by moving new near infrared (nIR) nanosensor technology from the nanoscientists' bench into the neurobiologists' rig. For ease of calibration and strong relevance to addiction, we will start with a dopamine nanosensor. We will integrate new nIR nanosensor tools with existing imaging and recording methods in brain slices and intact mice to enable greater understanding of the biology of modulatory neurotransmission. In Specific Aim 1, we plan to develop and calibrate the use of dopamine sensitive nanosensors for ex vivo detection of evoked dopamine release in striatal and frontal cortex brain slices. In Specific Aim 2 we will test the feasibility of using these sensors over long time scales in vivo. In Specific Aim 3, we will image nanosensor response to evoked dopamine release in vivo in intact and potentially awake behaving mice. Our goal is to set the stage to optically monitor dopamine and other neurotransmitter levels in vivo, in response to cues and rewards in conditions which induce reward prediction error in mouse models of health and disease. Development and dissemination of the dopamine nanosensor alone will greatly inform our understanding of substances with abuse potential and the effects of a broad variety of pharmacological agents. Experimental data collected using the dopamine nanosensor will then be applied to facilitate development of the norepinephrine and serotonin sensors. Our ultimate goal is to develop methods for measuring dopamine, norepinephrine, and serotonin in the cortex in vivo simultaneously. New infrared nanosensors have the potential to greatly advance our understanding of the brain, granting us new eyes to see modulatory neurotransmission in real time.

Abstract In the United States, 20% of children live in food insecure or marginally secure conditions, defined by limited or uncertain access to food (1, 2). Even at the lowest levels of severity, food insecurity is paradoxically associated with increased body mass index (BMI) (3) and populations that experience food insecurity are at greater risk for substance abuse issues (4-8). Further progress in delineating the role of food insecurity on substance use and obesity in humans is handicapped by co-occurrence of other aspects of adversity such as neglect, abuse and maternal mental illness. Here we propose to further develop our mouse model of food insecurity in which meals are delivered to mice in an irregular pattern for a brief developmental window. We compare these mice to stably food restricted and ad libitum fed controls. Because dopamine neurons fire phasically in response to unpredicted events (reward prediction error), we hypothesize that uncertainty may be an important variable independent from scarcity. We posit that cumulative experience of unpredictable food delivery will alter maturation of dopamine neurons and downstream circuits, altering reward and decision- making behavior. Our initial experiments show that adult male mice that experienced food insecurity P21-40 are less flexible in a reversal task compared to mice that experienced a history of regular food restriction or ad libitum fed controls. Females show no effect of treatment on flexibility in reversal but a treatment related increase in adult ad lib weight. Here we propose to test if there is a sensitive period for exposure to food insecurity by moving exposure to different ages (Aim 1) and to evaluate the impact of treatment at different ages on adult reward integration (Aim 1). We will also use pre-pubertal ovariectomy to test the role of pubertal hormones in the observed sex difference (Aim 1). We will next (Aim 2) determine if food insecurity enhances vulnerability in the context of substance use by measuring ethanol consumption in an intermittent access two bottle choice paradigm (which can elicit binge drinking) and aversion resistant ethanol consumption in which the only available ethanol is adulterated with quinine (a model of compulsive drinking). Here we will also test both sexes to investigate sex differences. We will also investigate if inflexibility in a cognitive test of reversal will predict later insensitivity to quinine adulteration of ethanol. Our major goals for the end of the grant period are: 1) To have established when it matters most to the developing brain to experience food insecurity, a question highly relevant to policy and public health. 2) To have tested the hypothesis that early food insecurity predisposes individuals to show greater binge or compulsive substance use in the context of ethanol, a causal link that is impossible to isolate without large confounds in humans. A future R01 will investigate neurobiological changes in the dopaminergic system and its cortical-striatal targets that may mediate observed changes in behavior.