Peyman Golshani - US grants

[unreadable] DESCRIPTION (provided by applicant): Refractory seizures remain a major source of disability despite advances in surgical and pharmacological treatment of temporal lobe epilepsy. Status epilepticus (SE) causes chronic seizures in animal models. Transcriptional changes induced by calcium entry during SE play an important role in epileptogenesis but the mechanisms of long-term transcriptional regulation after SE have not been well characterized. As changes in DNA methylation have been shown to play an important role in activity-dependent gene expression in neurons, we wondered whether similar epigenetic mechanisms play a role in transcriptional regulation in epileptogenesis. Our preliminary results show that SE causes significant epigenetic changes to brain-derived neurotrophic factor (BDNF), a gene up-regulated by SE, and implicated in epileptogenesis. We also find that demethylation of DNA leads to dysregulation of ion channel expression, with down-regulation of the A-type potassium channel, kv4.2. Interestingly, down-regulation of dendritic kv4.2 channels after SE increases the excitability of hippocampal neurons and has been proposed to increase the probability of chronic seizures. The goal of this project is to test the hypothesis that epigenetic changes after SE are crucial for promoting epileptogenesis. The Specific Aims are to 1) test the hypothesis that dynamic changes in DNA methylation are essential for transcriptional changes after SE, and to 2) test the hypothesis that prevention of de novo DNA methylation can modulate SE-induced transcriptional changes and epileptogenesis. We will characterize genome-wide changes in DNA methylation after SE using DNA methylation microarrays. We will ascertain if DNA methylation has a direct role in changes in transcription by performing promoter luciferase expression assays in dissociated cultures. We will test whether transgenic mice with deficient de novo DNA methylation have increased down-regulation of dendritic A-type kv4.2 channels after SE. Finally we will determine if prevention of de novo methylation promotes epileptogenesis by testing the frequency and duration of chronic seizures after SE. The training offered by mentors with laboratories with specific expertise in animal models of epilepsy, epigenetic gene regulation, and advanced electrophysiological techniques will help the candidate gain expertise in multiple scientific domains during his independent career as a clinician-scientist studying epileptogenesis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Common and rare mutations in contactin-associated protein-like 2 (CNTNAP2) are strongly linked to autism, with autosomal recessive truncating mutations resulting in autism in more than two-thirds of patients. However the alterations in functional connectivity underlying CNTNAP2-associated autism are not understood. Recently our collaborators demonstrated that a knockout mouse model of CNTNAP2 shows GABAergic interneuron migration abnormalities, robust social behavioral deficits, repetitive behaviors, communication problems, and seizures, accurately modeling the human condition. Here we propose to test the hypothesis that local and long- range functional medial prefrontal cortical connectivity is altered in the CNTNAP2 model of autism and that optogenetic interventions that correct the altered connectivity will improve social behavior. Finally, as gamma- synchronization has been hypothesized to underlie the abnormal cortical function in autism, potentially serving as a biomarker for diagnosis and gauging response to treatment, we will test the hypothesis that CNTNAP2 mice show altered gamma coherence between mPFC and amygdala, leading to altered recruitment of specific interneuron types in these structures. These hypotheses will be tested using single and paired patch clamp recordings from identified pyramidal and interneurons in combination with optogenetic stimulation or silencing of specific long-range projections, both in-vitro and in-vivo. These discoveries will guide the development of circuit-specific treatments for social behavioral deficits in ASD.

DESCRIPTION (provided by applicant): A substantial proportion of individuals with epilepsy suffer from depression but the mechanisms underlying epilepsy-associated depression (EAD) are not understood. Such a level of understanding is essential for designing new modalities for the treatment and prevention of EAD. Our previous work showed that animals that develop epilepsy as result of status epilepticus (SE) develop behavioral symptoms of despair and hopelessness. These animals show decreased evoked serotonin levels in cortex and hippocampus but the cause of decreased serotonin levels in epileptic animals is still not understood. The excitatory projection from the medial prefrontal cortex (mPFC) to the RN plays an essential role in the top-down control of serotonin release, and activating this system can rescue depression-related behaviors. We therefore hypothesize that this prefrontal input is weakened in animals with epilepsy and that optogenetic methods can be used increase excitatory drive from the remaining prefrontal inputs to normalize serotonin levels and treat EAD. The main goal of this proposal is to explore optogenetic stimulation of long-range inputs into serotonergic RN as a novel therapeutic proof-of-principle approach for effective management of EAD. We will use cutting edge in-vivo optogenetic techniques, behavioral studies, and electrophysiology to find convergent evidence for the role of long-range projections in depression in epilepsy. Specifically we will test the hypothesis that EAD is characterized by the diminished excitatory drive from mPFC into the RN and that normalizing the function of the mPFC-RN pathway exerts antidepressant effects in animals with chronic epilepsy and concurrent depression.

? DESCRIPTION (provided by applicant): One of the biggest challenges in neuroscience is to understand how neural circuits in the brain process, encode, store, and retrieve information. Meeting this challenge will require methods to record the activity of intact neural networks in freely behaving animals. Spectacular advances with the development of genetically encoded indicators of neural activity and optogenetic actuators now call for methods to image and manipulate the activity of large populations of identified neurons in freely moving mice over prolonged periods of time. Multi-channel imaging is needed to unequivocally identify individual neurons based on their unique gene expression profiles or projection patterns, and a flexible platform is needed so that the scopes can be easily adapted to address diverse neuroscience questions. Optogenetic capability is needed to draw causal connections between cellular activity patterns and behavior. Finally, currently available technology is limited because it requires the mice to be tethered by wires, limiting their range of behaviors and ability to interact with other animals or their environment. In addition, commercial miniaturized scopes are extremely expensive, and cannot be altered to meet individual end-user needs. Here, we seek to remedy shortfalls in existing technology by developing truly open source next-generation two-channel optogenetics-capable, wireless, miniaturized microscopes for imaging and tracking activity patterns of large neural-cell populations in freely moving mice. We will design, manufacture, optimize and test: a two-channel microscope for imaging two fluorophores (Aim 1), an optogenetics-capable microscope for imaging and optogenetic excitation (Aim 2), a wireless miniaturized microscope (Aim 3), and a microscope with combined two-channel, optogenetics, and wireless capability (Aim 4). All throughout, we will build an online environment for sharing our microscopes with the neuroscience community, lifting barriers for others to build, modify, and implant the microscopes and analyze neural activity data with our software. Our new wearable microscopes will have a transformative impact on neuroscience by permitting for the first time the imaging and manipulation of the activity of hundreds of identified neurons and other cells such as astrocytes in freely behaving animals.

? DESCRIPTION (provided by applicant): Inhibitory neurons are key regulators of cortical operations. Their dysfunction has been implicated as a major factor in many brain disorders. While recent studies indicate physiological and functional differences between specific types of inhibitory neurons, neural circuit mechanisms that give rise to these differences in cortical regions underlying cognition and executive function are not well understood. We focus our studies of inhibitory neuron circuit organization and function in the prelimbic area of medial prefrontal cortex (mPFC). This region is highly relevant to schizophrenia, autism, attention deficit disorders and others. The guiding hypothesis for this proposal is that the distinct connectivity of each type of inhibitory neurons differentially governs computationally distinct neural signal transformations in the mPFC, and that circuit connectivity differences between these cell types can be mapped to determine their specific roles in regulation of cortical network dynamics and behavioral output. Our proposed experiments will focus on the three major, non- overlapping inhibitory cell types or groups (parvalbumin-expressing, somatostatin-expressing and vasoactive intestinal peptide-expressing interneurons). A new Cre-dependent, genetically modified rabies-based tracing system will be used to map monosynaptic global circuit connections in the intact brain to these selected inhibitory neurons. To complement the anatomical rabies tracing, physiological input characterization will be accomplished by laser scanning photostimulation and channelrhodopsin (ChR2)-assisted circuit mapping. These studies will allow mapping of both local and long-range functional inputs to identified subtypes within each targeted cell group in brain slice preparations. Building on assessing input connections, we will map local functional outputs of these major inhibitory neuronal groups. Computational and behavioral analysis of the input and output circuit connections of specific inhibitory neuron types will be applied to understand how they regulate mPFC network oscillations in vivo and how they contribute to mPFC-controlled animal learning. This will be achieved by electrophysiological recordings made in parallel with behavioral performance measures with cell-type specific genetic inactivation. Together, the proposed research will generate new maps of inhibitory neuronal circuit wiring in medial prefrontal cortex, and it will broadly illuminate how inhibitory neuronal circuits regulate normal and maladaptive behaviors linked to neuropsychiatric and neurological diseases.

? DESCRIPTION (provided by applicant): One of the biggest challenges in neuroscience is to understand how neural circuits in the brain process, encode, store, and retrieve information. Meeting this challenge will require methods to record the activity of intact neural networks in freely behaving animals. Spectacular advances with the development of genetically encoded indicators of neural activity and optogenetic actuators now call for methods to image and manipulate the activity of large populations of identified neurons in freely moving mice over prolonged periods of time. Multi-channel imaging is needed to unequivocally identify individual neurons based on their unique gene expression profiles or projection patterns, and a flexible platform is needed so that the scopes can be easily adapted to address diverse neuroscience questions. Optogenetic capability is needed to draw causal connections between cellular activity patterns and behavior. Finally, currently available technology is limited because it requires the mice to be tethered by wires, limiting their range of behaviors and ability to interact with other animals or their environment. In addition, commercial miniaturized scopes are extremely expensive, and cannot be altered to meet individual end-user needs. Here, we seek to remedy shortfalls in existing technology by developing truly open source next-generation two-channel optogenetics-capable, wireless, miniaturized microscopes for imaging and tracking activity patterns of large neural-cell populations in freely moving mice. We will design, manufacture, optimize and test: a two-channel microscope for imaging two fluorophores (Aim 1), an optogenetics-capable microscope for imaging and optogenetic excitation (Aim 2), a wireless miniaturized microscope (Aim 3), and a microscope with combined two-channel, optogenetics, and wireless capability (Aim 4). All throughout, we will build an online environment for sharing our microscopes with the neuroscience community, lifting barriers for others to build, modify, and implant the microscopes and analyze neural activity data with our software. Our new wearable microscopes will have a transformative impact on neuroscience by permitting for the first time the imaging and manipulation of the activity of hundreds of identified neurons and other cells such as astrocytes in freely behaving animals.

Project Summary: Hippocampal sharp-wave ripples are 150-250 Hz oscillations during slow-wave sleep or immobility during which large populations of hippocampal neurons sequentially replay activity patterns that occurred during exploration of the environment. Disruption of ripples during wakefulness disrupts working memory. Recent work has shown that many other extra-hippocampal brain regions are specifically activated during ripples, and this coordination of hippocampal and neocortical networks during ripples may be critical for learning and retrieval. Yet the precise identity of cell types across different neocortical regions and reliability of activation during ripples is not known. The Golshani Laboratory has recently developed a large cranial window preparation that allows us to perform systematic and unbiased calcium imaging of neurons across all brain regions extending from frontal to occipital cortex bilaterally. Here we propose to implant flexible electrode arrays developed by the Tolosa and Frank Labs as a part of the BRAIN initiative into the hippocampus in mouse implanted with the large cranial window. These flexible electrode arrays will be optimal for this purpose because they allow long lasting recordings of local field potential for >200 days; moreover, because they are flexible they can be shaped so they don't obscure the imaging window. We will first learn implantation of electrodes by visiting the Frank Lab at UCSF. We will then obtain flexible electrode arrays from the Tolosa Lab (10 arrays), and implant them into the hippocampus in Thy-1 GCAMP6s animals with the large cranial window. After assuring that we can perform low noise electrophysiological recordings and calcium imaging in head-fixed animals resting on the treadmill, we will train animals to perform a memory retrieval task. We will determine proportion of neurons in different cortical regions activated during ripples during the task and their reliability activation across days. This one year R03 will allow us to collect data showing feasibility of these experiments that we will use as preliminary data for a collaborative BRAIN initiative grant.

DESCRIPTION (provided by applicant): Effective coordination requires that the motor system predict proper movements. To make these predictions, the cerebellum integrates sensorimotor information and motor errors and, through a process of error-driven learning, build up feed-forward models of movement. Decades of cerebellar research have clarified a highly stereotyped circuit, identified roles for particular circuit elements, and suggested cellular mechanisms that might account for associative learning. However, fundamental questions remain unanswered. How do particular activity patterns in Purkinje neurons influence movement? What are the functional ramifications of the neurochemically-defined divisions in the motor map? Where within the cerebellar circuit do changes occur during cerebellum-dependent forms of motor learning? And finally, how do circuit changes alter cerebellum-dependent behavior? The following specific aims will be addressed in the project. In Specific Aim 1, we will interrogate the organization of the motor map in the simplex lobe of the mouse cerebellum using optogenetic stimuli. Preliminary data show that Purkinje neuron inhibition triggers rapid, highly stereotyped movements. Using high speed videography and motion tracking we will measure movement trajectories and speeds in response to activation or inhibition in various cerebellar neurons with patterned illumination. We will also make electrophysiological recordings from cerebellar neurons in awake mice to examine the effects of manipulating PN excitability on the circuit. In Specific Aim 2 we will test whether associative motor learning can be driven by pairing sensory stimuli with optogenetically-elicited reductions or increases in PN firing. In vivo electrophysiology will be used to determine how error signals contribute to this learning. In Specific Aim 3 we will test the hypothesis that manipulation of PN firing alters a prediction signa giving rise to feed-forward error signals. These interrelated aims make use of a novel behavioral preparation applying sophisticated optical patterning, optogenetic, electrophysiological, and behavioral methods to awake mice in order to answer fundamental questions about cerebellar physiology. Together, the proposed experiments are designed to resolve issues that have been debated for decades within the cerebellar field. We expect that our results will yield a much improved understanding of basic cerebellar physiology and resolve some long-standing mysteries regarding cerebellum-dependent learning. In addition, these findings are likely to provide conceptual insights into cerebellar dysfunction caused by inherited and sporadic forms of ataxia.

Project Summary Temporal lobe epilepsy is often associated with significant cognitive dysfunction, but the mechanisms underlying such dysfunction are not understood. In both human temporal lobe epilepsy and related models, neuronal loss occurs in selected populations of hippocampal neurons, and this cell loss could be associated with the learning and memory deficits. The effects of loss of each of the most vulnerable groups of neurons are of particular interest, and these neurons include mossy cells in the hilus of the dentate gyrus, hilar somatostatin (SOM) neurons, and SOM neurons in stratum oriens of CA1, the majority of which are oriens lacunosum-moleculare (OLM) neurons. It remains unclear how the loss of each cell type contributes to the reorganization of synaptic connections and alters the in vivo function of hippocampal circuits. The broad goal of this proposal is to determine the effects of selective ablation of each of these three groups of hippocampal neurons and associated axonal reorganization on electrophysiological and behavioral measures of cognitive function. To determine the effects of loss of each cell population, the neurons will be ablated separately through adeno-associated virus (AAV) expression of Cre-dependent diphtheria toxin A in mice with cell-type specific expression of Cre. Specific Aim 1 will test the hypothesis that selective ablation of each of the vulnerable groups of neurons will lead to unique patterns of reorganization of remaining populations of neurons. Cre-dependent transfection of eYFP in Cre-expressing mice will be used to identify changes in the axonal arborizations of remaining neurons and determine if aberrant synaptic circuits are created. Specific Aim 2 will test the hypothesis that mossy cell or hilar SOM neuron deletion, but not OLM neuron deletion, will induce desynchronization of dentate hilar neuron firing during locomotion. Silicon probe recordings of theta oscillations and multiple single-unit recordings of dentate hilar neurons will be used to determine whether mossy cell, hilar SOM interneuron, or SOM OLM deletion induces this desynchronization of dentate hilar neurons. Specific Aim 3 will test the hypothesis that OLM deletion, but not mossy cell or hilar SOM neuron deletion, will cause less precise (broadened) place related firing of CA1 pyramidal neurons. These studies will use calcium imaging of large populations of CA1 neurons in freely moving animals with custom-made miniaturized microscopes to determine which cell type is sufficient for degrading the precision of place field firing. This proposal combines the mutually complementary expertise of two laboratories to determine if loss of specific groups of neurons and related reorganization of hippocampal circuits can lead to changes in how large groups of neurons become synchronized and encode information, and thus contribute to cognitive dysfunction in epilepsy and related disorders.

To understand how the brain processes information, creates and retrieves memories, and makes decisions it is necessary to record the activity of thousands of brain cells simultaneously. New small and light-weight microscopes have been developed that can be carried on the heads of laboratory mice and rats. These microscopes take advantage of new probes that sense calcium levels and flash bright when a brain cell becomes active. The Neuronex Neurotechnology Hub has built new miniature microscopes that not only sense light but can also directly record the electrical activity of the large numbers of cells deep in the brain. This combination of electrical and optical recordings gives scientists the new ability to read out how large groups of brain cells and brain regions work together as the brain senses, learns, plans and executes actions. The Neuronex Neurotechnology Hub will also create new computer systems that can analyze these activity patterns extremely quickly (within small fractions of a second). This rapid feedback system will allow investigators to rapidly probe how the activity of specific groups of brain cells is linked to each behavior. Finally, the Hub will build and test a new miniature microscope called a "light field miniature microscope". This version of the microscope will allow investigators to make 3-D movies of brain activity, greatly improving their view of the large network of brain cells. All these technologies will be openly shared with neuroscience community through a website (miniscope.org), such that each laboratory can build each of these devices themselves at very low cost. The Hub will hold workshops to teach scientists how to build and use the various devices. Finally the hub will reach out to the broader community by holding classes for K-12 and college students, and demonstrating how these devices can give us a view of brain function.

This Neurotechnology Hub will develop and share next-generation miniaturized in vivo sensing devices that integrate optical and electrophysiological recording from hundreds or thousands of neurons in behaving animals. These devices will be coupled with energy-efficient computing hardware for real-time signal processing and closed-loop feedback capabilities. The Hub will also create light field miniaturized microscopes that will allow three dimensional optical recordings of network activity in freely behaving animals. Last, the Hub will manufacture and distribute custom made, 3 dimensional silicon microprobes for large scale electrophysiological recordings. Making these devices widely available for neuroscience research and teaching will have significant broader impacts, by accelerating discovery and broadening outreach. The devices and techniques will be distributed widely to a large community of researchers, as previously done with the open-source miniaturized microscope developed by the PIs (the website at miniscope.org already has >2500 registered users and >250 labs using our microscope), as well as with silicon microprobes (>100 devices have been shared with users). Hence, the Hub will have a broad impact upon neuroscience research, facilitating many future advances in our understanding of the neural basis for emotion, cognition, and behavior, with a high potential to catalyze major new discoveries. The PIs will establish an outreach program through partnership with the Minority Access to Research Careers program at UCLA, as well as the UCLA Center for Excellence in Engineering and Diversity (CEED), to involve highly diversified high school and undergraduate students in this research. This NeuroTechnology Hub award is funded by the Division of Emerging Frontiers within the Directorate for Biological Sciences as part of the BRAIN Initiative and NSF's Understanding the Brain activities.

Abstract A general principle of brain function is the ability to store information about the past to better predict and prepare for the future. Working memory and timing are two computational features that evolved to allow the brain to use recent information about the past to accomplish short-term goals. Working memory refers to the ability to transiently store information in a flexible manner, while timing refers to the ability to generate well timed motor responses, modulate attention in time, and predict when external events will occur. To date working memory and timing have primarily been treated as independent processes. Here we propose that because the brain seeks to use information about the past to predict the future, that working memory and timing are often multiplexed. Specifically, that the neural patterns of activity recorded during the delay period of many working memory tasks encodes both retrospective information about the past, as well as prospective predictions about the future. To test this hypothesis, we have developed novel variant of the delayed- nonmatch-to-sample task, in which the first cue predicts the duration of the delay, that is, how long an item must be held in working memory. This task will allow us to determine if network level population responses encode both retrospective information about the past and prospective information about delay duration. Preliminary results from a supervised recurrent neural network model predict that the temporal structure of the neural patterns of activity elicited by both cues will be different. This prediction will be tested using large scale Ca2+-imaging to characterize the spatiotemporal patterns of activity in brain areas associated with working memory and timing. Additionally, optogenetic perturbation experiments and longitudinal characterization of the emergence of neural patterns of activity will be performed. These experiments, will in turn, be used to ground computational studies aimed at understanding the neuronal and circuit level learning rules that underlie the emergence of patterns that encode working memory and time.

Abstract: Attachment powerfully shapes our development and remains a primary driver of health and well-being in adulthood; disruption of attachments is highly traumatic. While affiliation, defined as general positive social interactions, is shared widely among mammals, attachment, or selective affiliation as a result of a bond, is far rarer and of primary relevance to humans. While affiliation has been studied in a number of contexts, how the neural circuitry that underlies affiliation ultimately contributes to adult attachment remains largely unknown. In this proposal, we will take a comparative framework to understand how the basic circuitry and neuronal patterns that underlie non-selective affiliation are ultimately engaged and underlie selective attachment in adulthood. Specifically, we will examine how the neurobiology of affiliative behavior in mice has been elaborated to support the more complex attachments formed by monogamous prairie voles and gregarious fruit bats, representing a spectrum of social relationships. We will focus on the hippocampal CA2 region as it has been shown to play a specialized role in social behavior and receives direct inputs from oxytocin and vasopressin producing cells in the paraventricular hypothalamus. Specifically, we will test the overarching hypothesis that CA2 population activity patterns follow similar trajectories across species before and during mating, and subsequently diverge to causally drive affiliative investigation in mice (Golshani/Hong) and different forms of attachment in prairie voles (Donaldson) and bats (Yartsev). To test this hypothesis we will refine and use new generation open-source wireless miniaturized microscopes (Aharoni) that will allow prolonged recordings of large neuronal populations in freely behaving animals. Kennedy will bring computational expertise and allow a unified data analysis framework cross species. In Aim 1 we will perform in-vivo calcium imaging in mice, prairie voles and bats to test the hypothesis that mating experiences modulate CA2 neural dynamics and that CA2 activity patterns encode spatial and identity information. We hypothesize that species that form attachments to mating partners, activity patterns will differentiate preferred vs. non-preferred partners. In Aim 2 we will use chemogenetic inhibition of CA2 in all species to determine whether CA2 causally drives affiliative and attachment behaviors. In Aim 3 we will test the hypothesis that inhibition of vasopressin inputs to CA2 will reduce the dimensionality of CA2 population activity patterns after mating, diminish memory of the mate in all species, and in voles and bats, reduce the decodability of the identity of the previous mating partner. In a technology development aim, we will develop and test a ?true wireless? digital data transmitting microscope with power over distance charging capability that will allow prolonged imaging over many hours without human intervention.