Attila Losonczy - US grants

DESCRIPTION (provided by applicant): Maladaptive fear responses due to brain circuit pathology have been widely implicated in several psychiatric disorders such as post-traumatic stress disorder (PTSD), anxiety, and panic disorders. Clinical solutions to these disorders thus require a greater understanding of the basic structure and function of neural circuits implementing these memory functions. Here we propose to apply new technologies - cellular-resolution functional neural imaging and cell type-specific manipulations of genetically identified hippocampal circuits in behaving mice - for studying contextual fear learning and storage. Our result will inform our basic understanding of how we learn and remember, and suggest specific targets for treatment and rehabilitation of maladaptive fear responses in disorders of fear, anxiety, and memory.

? DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is the most common epilepsy in adults. TLE is often refractory to current anti- epileptic drugs, and systemic treatments are frequently accompanied by significant negative side effects. However, the cellular and circuit mechanisms underlying TLE are not yet understood, which poses a challenge for the development of novel treatment strategies. Recently, we discovered that closed-loop optogenetic intervention (COI) can significantly curtail on-going electrographic and behavioral seizures in chronic experimental TLE with high spatial, temporal, and cell-type specificity, and that it can be used as a powerful, versatile research tool for hypothesis testing o understand TLE mechanisms. Here we propose to test the hypothesis that COI achieves long-term seizure control, as well as the amelioration of cognitive comorbidities and pathological functional network properties, during both the chronic and latent phases of TLE. The hypothesis will be tested in experimental mouse models of TLE, and the assessment will be carried out with electrophysiological and behavioral techniques as well as large-scale in vivo functional imaging methods in the CA1 region of the mouse hippocampus. It is anticipated that defining the functional consequences of COI in TLE will have a significant impact by advancing our understanding of the role of activity-dependent pathological processes in chronic epilepsy and epileptogenesis, and aid the future development of novel anti-epileptic treatment strategies.

Sharp wave ripples (SPW-Rs) are self-organized, synchronized population events in which hippocampal circuit excitability is markedly and transiently enhanced. The temporal distribution of SPW-Rs is highly irregular and strongly influenced by behavioral and brain states, indicating the existence of control mechanisms for the initiation of these population events and the memory replay which they support. There is accumulating evidence that suggests that SPW-Rs occurrence is controlled by both extra-hippocampal and intra-hippocampal influences. However, the precise distal and local circuit mechanisms by which these extra- and intra-hippocampal influences control SPW-R initiation are unknown. In Project 1, we will perform a comprehensive set of studies which will provide new information on the precise temporal relationship (through observational experiments using integrated high-resolution methods) and perturbational influence (using optogenetic interventions) of key suspected extra- and intra-hippocampal controllers of SPW-R initiation.

Although neuroscience has provided a great deal of information about how neurons work, the fundamental question of how neurons function together in a network to produce cognition has been difficult to address. Our group has been at the forefront of developing methods that allow large scale monitoring of identified neurons, monitoring of voltage signals by optical means and elucidation of subcellular events in dendrites, all of which can now be done in awake behaving animals. We propose to use these methods to provide a deep understanding of how the neurons of the hippocampal region generate the sharp-wave ripple (SPW- R). This remarkable signal has been shown to depend on prior learning and to produce high-speed replay of memory sequences (e.g. a path along a track). The function of this signal is memory consolidation; disruption of SPW-Rs results in strong deficits in memory-guided behavior. Because much is known about the hippocampal cell types involved and their network connections, understanding the SPW-R is a tractable target for the first major effort to elucidate the cellular/network mechanism of a mammalian brain signal at an analytical level comparable to that achieved in the study of simple invertebrate systems. Project 1 is aimed at understanding the external and intra-hippocampal pathways that control the initiation of SPW-Rs. Project 2 deals with the events that occur during the SPW-R, including the timing of activity in identified cell types and understanding the fundamental network architecture by which memory sequences are produced. Project 3 deals with how the information that is replayed during the SPW-R is encoded. We will attempt to create an artificial memory and then determine whether the memory is replayed during a SPW-R; we will also interfere with molecular mechanisms of memory storage to determine whether we can erase the memories that are replayed during the SPW-R. Project 4 builds upon recent work indicating that differentially projecting CA1 pyramidal cells have distinct properties and will test the possibility that SPW- Rs in distinct output channels may carry different information and affect different behaviors. In Project 5 we will develop the first non-reduced computational model of the hippocampus, incorporating information about cell types and connections. This will be a major new resource for our group and the research community that will permit unprecedentedly close interplay between experiment and computation. To the extent that the model can account for the experimental observations, we can use it to understand underlying network principles and design interventional experiments to validate this understanding. To the extent that the model cannot explain results, it will help point us to aspects of network function that require further elucidation. Taken together, Projects 1-5 provide a tractable path to a major breakthrough in understanding how a cognitively important brain signal is generated.

Schizophrenia is a debilitating psychiatric disorder that effects 1% of the population, with an additional 2-3% developing a schizoaffective disorder. SCZ patients exhibit a spectrum of cognitive deficits including defective episodic memory, present prior to the onset of psychosis and frequently expressed in relatives of affected individuals. Episodic memory formation is dictated in part by spatially tuned (place cell) activity of principal cells in the hippocampus. The biological mechanisms driving this learning capacity in the healthy hippocampus remain largely unknown, let alone their disruption in schizophrenia, leaving large gaps in our knowledge that need to be addressed. Using in vivo functional imaging in mouse dorsal hippocampal area CA1 during head-fixed during learning behaviors, we recently uncovered specific alterations in in vivo physiological properties of CA1 pyramidal cells in the Df(16)A+/? transgenic mouse model of 22q11.2 deletion syndrome, the largest known genetic risk to develop SCZ. Df(16)A+/? CA1 place cells exhibit reduced long-term stability, impaired context- related and lack of reward-related reorganization. A novel form of synaptic plasticity, termed behavioral time- scale synaptic plasticity (BTSP), has been found to drive rapid formation of spatially selective firing fields in CA1 pyramidal cells; notably, our preliminary studies suggest that this form of plasticity is dysregulated in Df(16)A+/? mice. We thus hypothesize that BTSP, a major form of plasticity that drives place cell-recruitment during learning, is disrupted by SCZ risk mutations. These findings at the neuronal population level provide entry points for dissecting the underlying cellular, molecular and microcircuit dysfunctions caused by schizophrenia risk mutations. To gain these mechanistic insights we will unite the complementary expertise of the Losonczy lab and the Gogos lab in etiologically valid genetic mouse models of neuropsychiatric disorders to carry out multiscale dissection of microcircuit, cellular and molecular pathophysiology of schizophrenia-related memory deficits in the adult mouse hippocampal CA1 circuitry. Aim 1 is aimed at assessing altered synaptic plasticity in CA1 pyramidal cells during episodic learning in Df(16)A+/? mice. Aim 2 deals with dissecting inhibitory microcircuit dynamics during episodic learning, while Aim 3 is focused at dissecting altered excitatory and neuromodulatory input dynamics to CA1 during episodic learning in Df(16)A+/? mice. Taken together, Aims 1-3 provide a tractable path to a deeper, mechanistic understanding of hippocampus-related cognitive memory deficits in schizophrenia.

PROJECT SUMMARY / ABSTRACT Understanding how cognitively-relevant behavioral functions emerge from activity patterns of identified cell- types is predicated on the ability to record large-scale ensemble dynamics from genetically-identified and longitudinally-tracked neuronal populations across multiple brain regions and layers with high spatial and temporal resolution over behaviorally-relevant time-scales. Two-photon scanning microscopy in combination with genetically-encoded calcium (Ca2+) indicators is currently the most essential tool for in vivo optical recording of neuronal activity, its application to deep brain regions. However, currently the commercially available 2pM systems are limited in their applications due to constraints related to the obtainable imaging depth, volumetric field-of-view (VFOV), and temporal resolution at which neuronal population dynamics can be effectively captured. We have recently developed and demonstrated the proof of principle of a new high-speed volumetric Ca2+-imaging platform termed Hybrid Multiplexed Sculpted Light (HyMS) Microscopy that combines 2pM with three-photon microscopy (3pM). HyMS allows for volumetric recording of neuroactivity at single-cell resolution within volumes up to ~1 × 1 × 1.22 mm at up to 17 Hz in cortical as well as sub-cortical regions of awake behaving mice. The impact of this tool will depend on a successful optimization, neurobiological application and dissemination strategy within the neuroscience community. While we will provide open source access for technically skilled labs, given the technical complexity and costs of such a system, the most effective strategy is through partnership with industry and through commercialization of the system. Here we propose a roadmap towards this objective. Building on our current existing system, we will implement a number of technical refinements and optimizations. Leveraging the ongoing collaboration with the Losonczy Lab at the Columbia University, we will use our optimized HyMS system to perform high-speed multiphoton volumetric Ca2+ imaging of functional circuitry across the entire depth of the mouse dorsal hippocampus (HPC), encompassing all major regions of the HPC trisynaptic circuitry. This application will provide us valuable feedback for further optimization and refinement and development of our HyMS prototype system. In parallel, we will develop together with our industrial partner a first prototype of the HyMS system (?-HyMS) This prototype will be again used and tested by the Losonczy Lab. The obtained insights and user feedback from their application will drive the development of a beta prototype (?-HyMS) which will be used to engage broader local users as beta testers. 9 user labs, mainly from the NYC area, with a broad range of biological questions and applications, will participate as beta testers and provide us with iterative user feedback which will ultimately drive and be incorporated both into the into the commercialization of HyMS as well its open source model of the access to this technology.

Temporal lobe epilepsy (TLE) is the most common epilepsy in adults, and it is frequently refractory to current anti-epileptic drugs, with treatments often exerting a variety of debilitating side effects. A major barrier for the development of novel treatment strategies is our insufficient understanding of the precise cellular and circuit mechanisms underlying TLE. A centrally important but unresolved question in TLE concerns the mechanisms underlying the excessive, dysregulated production of action potentials at the axon initial segment (AIS) of excitatory principal cells (PCs). Synaptic control of AIS is provided by a unique, evolutionarily conserved, GABAergic cell-type, the axo-axonic cells (AACs). AACs form synaptic contacts exclusively with the AIS of PCs, placing AACs in a strategic position to control action potential generation. However, due to technical limitations, our knowledge about the in vivo function and regulation of AACs in the normal and epileptic hippocampus has been extremely limited. Here we propose to employ a combination of recent technical breakthroughs to test hypotheses about the in vivo functional effects, activity dynamics and efficacy of AAC- mediated control of AIS in mouse models of chronic TLE. The planned project will also determine if it is possible to mitigate epilepsy-related pathologically hyperactive circuits and cognitive deficits through interventions selectively directed at the AAC-dependent, endogenous GABAergic processes regulating AIS in chronic epilepsy. The proposed project aims to fill a major knowledge gap and address long-standing controversies concerning the interneuronal regulation of AIS in epilepsy by leveraging expertise in novel large- scale, high-resolution in vivo functional imaging techniques in combination with advanced electrophysiological, behavioral, optogenetic and computational modeling techniques in the CA1 region of the mouse hippocampus. It is anticipated that defining the function, regulation and therapeutic potential of AACs in TLE will have a significant impact by advancing our understanding of key circuit control mechanisms in chronic epilepsy and aid the future development of novel anti-epileptic treatment strategies.

Although neuroscience has recently provided a great deal of information about how neurons represent and encode behaviorally relevant information at the population level, the fundamental question of how individual neurons are selected and recruited to memory coding ensembles has been difficult to address. Our group has been at the forefront of developing experimental methods that allow high-resolution monitoring of identified neurons, monitoring subcellular events in dendrites and axons, all of which can now be done in awake behaving animals. We propose to use these experimental methods in combination with circuit modeling to provide a deep understanding of how the neurons in the mouse hippocampus are recruited to neural ensembles during contextual memory encoding. Because much is known about the excitatory and inhibitory cell types involved and their network connections at the main CA1 output node of the rodent hippocampus, this circuit represents a tractable target for the first major effort to elucidate the microcircuit/cellular/subcellular mechanisms of cell- selection at a mechanistic level comparable to that achieved in the study of simple invertebrate systems. Aim 1 is aimed at characterizing collective inhibitory dynamics in CA1 during contextual learning. Aim 2 deals with the events that occur in cell bodies and dendrites of CA1 pyramidal cells during contextual leaning, including targeted manipulation in identified inhibitory cells types and understanding the fundamental network architecture by which cellular activity patterns conducive to memory encoding are regulated. Aim 3 deals with how the information that is encoded during contextual learning converges onto individual CA1 pyramidal cells during contextual learning. Finally, Aim 4 builds upon recent work indicating that CA1 pyramidal cells can be reliably recruited to memory coding ensembles through a plasticity mechanism that requires dendritic spikes and somatic bursting activity. We will use optogenetic means to create artificial firing fields in neurons and determine whether these cells can encode context-related and reinforcement related signals; we will also interfere with local circuit inhibition to determine whether cell selection through plasticity is regulated by inhibition. Throughout the proposal we will leverage unprecedentedly close interplay between experiment and computation by using a biophysically detailed model of the hippocampal CA1 microcircuit. To the extent that the model can account for the experimental observations, we can use it to understand underlying network principles and design interventional experiments to validate this understanding. To the extent that the model cannot explain results, it will help point us to aspects of network function that require further elucidation. Taken together, Aims 1-4 provide a tractable path to a major breakthrough in understanding how cognitively important neural activity dynamics are generated at the microcircuit-, cellular- and subcellular-levels.