Stefan Leutgeb, research assistant - US grants

Complex cognitive functions such as spatial cognition, language development, and episodic memory require associations between multiple sensory experiences, such as images and sound, and require that associations are remembered in sequence. The rodent hippocampus has been one of the leading models for understanding neuronal mechanisms for remembering a sequence of spatial locations. Yet, it remains unknown whether the mechanisms that are used for encoding a path through space are the same as those that are used for encoding sequences that contain multiple modalities. To address this question, the auditory modality is of particular interest since it provides a mean to present a sequence of stimuli with high temporal precision and thus a mean to investigate time constraints for sequence learning. The proposed research investigates auditory sequence learning in a rodent species that is a hearing-specialist and tests whether the hippocampus of Mongolian gerbils can encode sequences of auditory stimuli with mechanisms similar to those used for spatial sequences.

It will be tested whether place fields and theta phase precession exist in Mongolian gerbils, whether complex sound stimuli are encoded in the gerbil hippocampus in a spatially-independent manner or in association with the location of the animal, and whether sound sequences are encoded with network mechanisms related to those that are used for encoding spatial sequences. These questions will be addressed with single-unit recording from hippocampal principal cell populations of behaving animals, and experiments will be conducted in a virtual reality setup that allows for the precise delivery of auditory stimuli. Performing these experiments in a rodent species with an auditory specialization might result in important advances in addressing how the hippocampus encodes multimodal sequences. This research can provide important insight into neural network mechanisms for sequence coding and can lead to a better understanding of the contribution of the hippocampus to language development in humans.

This project is jointly funded by Collaborative Research in Computational Neuroscience and the Office of International Science and Engineering. A companion project is being funded by the German Ministry of Education and Research (BMBF).

DESCRIPTION (provided by applicant): High-level neural functions, including perception, cognition, and memory, rely on the coordinated activity of at least thousands of cortical neurons. The synchronized neuronal activity of large cortical cell assemblies results in electrical oscillations in electroencephalographic records and in local field potentials. Psychiatric, neurological, and neurodegenerative diseases, including schizophrenia, epilepsy, and Alzheimer's disease are characterized by a change in oscillatory timing and patterns. However, it is currently not known which therapeutic interventions could restore temporal control of neural circuits to achieve improvements in cognitive function. It has been suggested that the hippocampal theta rhythm provides temporal control for neuronal firing patterns that support memory processes, but this can only be tested if theta oscillations can be controlled with high temporal precision. We therefore propose to use the temporal precision of optogenetic inactivation techniques in neural circuits for theta generation to test the hypothesis that theta oscillations provide temporal coordination for hippocampal neurons during memory encoding and retrieval. We will test this hypothesis in two aims. The first aim is to attain precise tempora control of the theta rhythm and the second aim is to use temporally precise disruption to determine whether the coordination of hippocampal spike timing by theta oscillations is required during memory acquisition, retention, or retrieval. For both aims, we will infuse AAV9-Arch3.0-EYFP or AAV9-ArchT3.0-EYFP into medial septum and place recording electrodes in the hippocampus and/or the medial entorhinal cortex of rats. AAV9 was selected as a viral vector because it provides widespread infection of brain tissue, and Arch3.0 and ArchT3.0 were selected as opsins because they are effective light-induced inhibitors of neuronal activity. After recovery from surgery, rats will, for aim 1, be trained to continuously run on a track or randomly forage. Behaviorally relevant illumination protocols will be used to determine the effects of oscillating and discrete light pulses in the septal area on hippocampal theta oscillations. For all protocols, the analysis will focus on changes in the amplitude and frequency of theta oscillations and also on the effect of theta/gamma coupling. For aim 2, rats will be trained on a figure-eight delayed spatial alternation task with distinct encoding, retrieval, and retention phases. On separate days, light pulses will be delivered during one of these phases to determine when theta oscillations are required. For light- stimulation protocols that disrupt behavioral performance, we will examine the changes in the spike timing of hippocampal cells during decreased memory performance. By using temporally precise inactivation of a pacemaker for oscillations, the proposed aims will determine which phases of a memory task require oscillatory neural activity and to what extent the precisely timed neural activity in hippocampus is required for memory processes. Because electrical stimulation paradigms can also be effective by inhibiting ongoing neuronal activity, these experiments will provide new insight for the therapeutic use of deep brain stimulation.

DESCRIPTION (provided by applicant): Synaptic loss or dysfunction is believed to be one of the major factors responsible for the memory and cognitive deficits seen in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. According to the amyloid cascade hypothesis, the gradual accumulation in brain of amyloid ¿-peptide (A¿), derived from the amyloid precursor protein (APP), is hypothesized to trigger the cascade of events that lead to AD. The mechanisms by which A¿ may initiate these events, which include synapse loss or synaptic dysfunction, are unclear. Recent studies suggested that both amyloid deposition in extracellular space and intracellular neurofibrillary degeneration, the two hallmarks of AD, may progress in a trans-synaptic or anterograde fashion. That is, the spread of AD pathology in brain, as must occur as the disease develops, expands in a manner that is suggestive of neuron-to-neuron progression. If true, this suggests that A¿-induced synaptic injury should be initiated by the presynaptic neuron to alter function of the postsynaptic neuron. Indeed, we have recently obtained preliminary data that support this concept. Specifically, impairment of synaptic plasticity is present only when A¿ is derived from the presynaptic neuron but not in the reverse situation. These novel observations were obtained from transgenic mice that restrict APP expression preferentially to CA3 or CA1 neurons of the hippocampus. These transgenic mice therefore provide the unique opportunity to ask key questions related to neuronal function or dysfunction caused by local production and release of A¿ in brain. These questions cannot be addressed with existing transgenic mice where there is pan-neuronal expression of APP at high levels or the recently developed mice with expression restricted to entorhinal cortex. This application will examine the degree to which injury to synaptic function or neuronal circuits develops with respect to the neuronal population where A¿ is produced. Specifically, we will utilize transgenic mice with spatial and temporal control of APP expression directed to neurons in CA1, CA3, or dentate gyrus by using transgenic mouse lines that express Cre recombinase in CA1, CA3, or dentate gyrus granule cells, respectively. In addition, we will test the reversibility of synaptic and circuit dysfunction in these mouse lines as well as in the original tTA/tet-APP line. Two Aims are proposed: 1) we will explore whether behavior, biochemical, and morphological changes accompany the impairment in synaptic plasticity initiated by A¿ released from pre- vs. postsynaptic neurons and whether these functional changes become irreversible with age and 2) assess neuronal dysfunction in these mice by measuring field potentials and place cell firing patterns. Collectively, results from these studies using selective and reversible APP expression in subregions of the hippocampus will provide fresh insights into A¿-induced neuronal dysfunction in vivo.

DESCRIPTION (provided by applicant): Neuron loss and the reorganization of neural circuits in the medial temporal lobe are hallmarks of traumatic brain injury, temporal lobe epilepsy, brain ischemia, and Alzheimer's disease. Various degrees of memory impairments are among the troubling symptoms of each of these diseases, but the exact pattern of histopathology varies between diseases. The memory loss that is common to the diseases is thought to emerge from entorhino-hippocampal dysfunction. The entorhinal cortex and hippocampus function as a feedback loop and a loss of function could thus emerge by disrupting neuronal processing when damaging any part of the circuit. Alternatively, each subregion within the circuit may be able to independently perform its characteristic function, but different pattern of neuronal injury within the medial temporal lobe might nonetheless manifest in a common way because the entorhinal cortex and hippocampus can only incompletely compensate for each other's function. Although questions about the mechanisms of neural dysfunction can be studied in animal models that are specific for a neurological disease, an understanding of the sources for memory problems can also be obtained from investigating different patterns of injury within the medial temporal lobe. Because many cell types for spatial processing have been described in the medial entorhinal cortex (MEC) and hippocampus, we propose to initially focus on these brain regions. We have begun to investigate the extent of spatial memory impairments after lesions to the rat hippocampus and/or MEC. Our preliminary data show substantial dysfunction of spatial and temporal processing in the hippocampus after MEC lesions and in the MEC after hippocampal lesions. We also find that memory impairments are less severe after lesions to individual brain regions compared to combined lesions. Based on our preliminary results, we hypothesize that spatial functions can, in part, be independently performed by the MEC and the hippocampus, but that temporal aspects of MEC and hippocampal neuronal processing require that the entire loop be intact. This hypothesis will be tested in three aims: (1) further characterize memory dysfunction after complete MEC lesions and after combined lesions of the MEC and the hippocampus with behavioral testing, (2) determine the extent of neuronal network dysfunction in hippocampus after MEC lesions with single-unit recordings during behavior, and (3) determine which neuronal firing patterns in MEC are disrupted after complete hippocampal lesions and, additionally, identify whether neuronal computations in the MEC can be restored by brain stimulation. Identifying spared functions after different patterns of damage and revealing how manipulations of the remaining circuits can compensate for lost functions will provide insight into the network mechanisms that can be strengthened or restored in neurological and neurodegenerative diseases.

Project Summary Neuron loss and the reorganization of neural circuits in the superficial layers of entorhinal cortex are hallmarks of Alzheimer?s disease and temporal lobe epilepsy, and memory impairments are among the troubling symptoms of these diseases. Despite the knowledge that superficial entorhinal cell layers are selectively vulnerable in these diseases, the local connectivity of entorhinal circuits, how different functional cell types within these layers emerge, and how each cell type contributes to memory and spatial processing is only beginning to be revealed. The entorhinal cortex has extensive recurrent connectivity between its layers, and harbors many functional cell types such as grid cells, head-direction cells, border cells, context-selective cells, and other types of spatial and nonspatial cells. However, this functional diversity neither maps directly onto particular cell layers nor onto anatomically defined cell classes within layers. Each cell?s functional identity may therefore predominantly be determined by local microcircuits. The objective of this proposal is to examine whether functional cell identities in mEC, including grid cell firing and context-selective firing, emerge from circuit computations. We focus on local connectivity within the superficial layers and hypothesize that layer II pyramidal cells selectively contribute to rate coding in layer II stellate cells and that layer III inputs selectively contribute to spatial coding, including grid firing, in layer II stellate cells. This hypothesis will be tested in two specific aims. First, we will use viral tracing and patch clamp recordings with optical stimulation in entorhinal slices to determine the connectivity of mEC layer II pyramidal cells and layer III pyramidal cells and, for comparison, layer II stellate cells. Second, we will record from mEC cells in behaving mice while optogenetically stimulating or inhibiting entorhinal cell populations. In two separate subaims, we will examine (1) the effects of layer II pyramidal cell manipulations on the spatial and context-selective firing patterns of layer II stellate cells and layer III pyramidal cells and (2) the effects of layer III pyramidal cell manipulations on spatial and context- selective coding by layer II cells. Results from our aims will identify how local entorhinal circuits contribute to the generation of specialized entorhinal cell types, including grid cells and context-selective cells. This will not only fill gaps between theoretical models and experimental data, but also develop methods to selectively manipulate different functional cell types in mEC. Our results will therefore advance our understanding of the contribution of entorhinal cell types and cell layers to spatial and memory processing and thereby suggest strategies for restoring entorhinal circuit function and ameliorating the progression of neurodegenerative diseases.

Project Summary Healthy cognitive processing is associated with well-defined brain oscillations, which are not only an indication of accurate timing of neuronal activity within brain regions, but also of the coordination of neural activity patterns between brain regions. Consistent with this notion, some aspects of oscillatory activity in at least a subset of brain regions are disrupted in any of the major psychiatric and neurological diseases. However, evidence for a causal link between disrupted oscillations and impaired behavior has remained sparse. In particular, it has been challenging to manipulate timing within brain circuits without simultaneously disrupting other firing statistics of neuronal populations, such as their firing sparsity or firing rate. Preliminary data are presented which indicate that optogenetic rhythmic stimulation of medial septal PV neurons at frequencies that are higher than the endogenous theta frequency (? 10 Hz) alters hippocampal spike timing, but without changing other firing statistics, such as spatial firing patterns, firing rate, and theta phase precession. Furthermore, we found that stimulation at 8 Hz was without effect on memory performance while stimulation at ?10 Hz resulted in a memory impairment that was comparable in its severity to a complete hippocampal lesion. We therefore hypothesize that minor shifts in the timing of neuronal activity result in a loss of coordination between neuronal networks in the hippocampus, medial entorhinal cortex, and medial prefrontal cortex, such that these brain regions can no longer support spatial working memory. We will perform three aims to address this hypothesis. (1) Determine the minimal time shift at which memory deficits emerge and measure the coordination of hippocampal neural activity while stimulating at frequencies with and without memory deficits. (2) Determine whether manipulations of theta frequency critically alter firing patterns in medial entorhinal cortex (mEC). (3) Determine whether oscillations within the endogenous theta frequency range are necessary for the coordination between hippocampus and medial prefrontal cortex (mPFC). In each of the three aims, we will perform LFP and single unit recordings while mice perform a spatial alternation task. Two versions of the task will be compared, a delayed version which is dependent on hippocampal and prefrontal function, and a continuous version, which is included as a control. While mice are performing the task, the medial septal area will be stimulated at frequencies that do not result in memory deficits or stimulated at frequencies that result in memory deficits. These conditions will be compared to identify the critical changes in neural activity in hippocampus, mEC, and mPFC at the transition to the memory impairment. Taken together, the combined manipulations and recordings in the behavioral task will provide evidence for a causal relation between precise timing and memory function and for a role of oscillations in coordinating the exchange of information between brain regions.

PROJECT SUMMARY A combination of entorhinal, hippocampal, and prefrontal pathology has a pivotal role in most neurological and neurodegenerative diseases and in the emergence of memory impairments that are associated with these diseases. Despite the knowledge that these brain regions are particularly vulnerable, the diversity of neural computations within and across these brain regions is only beginning to be revealed. For example, a key function of the hippocampus and medial entorhinal cortex (mEC) is to bridge events that are discontinuous in time, and entorhinal and hippocampal cells that are sequentially active (?time cells?) have been proposed to be pivotal for memory retention over delay intervals of many seconds. In our previous work, we therefore investigated the firing patterns over the delay interval in a spatial working memory (WM) task. We unexpectedly found that hippocampal time cells were not a general mechanism for WM retention in hippocampus-dependent tasks. Rather, preliminary data indicate that information about past and future choices during the delay interval is evident during brief population bursts in hippocampus, while mEC cells may show memory-related activity that persists irrespective of brain state. We therefore hypothesize that memory retention does not require time-varying activity over the delay interval but is rather evident in sporadic population bursts in hippocampus and medial prefrontal cortex (mPFC) throughout the delay period and in firing patterns of entorhinal cells that provide continuity irrespective of brain state. To record activity during the delay period with varying brain states, we will ? within each animal ? use two variants of a spatial WM task, one with and one without forced running throughout the delay such that either theta or non-theta states are predominant. Aim 1 will focus on population bursts in hippocampus and determine whether they are only informative in non-theta states, as shown in our preliminary data, or a general mechanism across brain states. In addition, we will selectively interrupt hippocampal activity within the delay period to determine whether coding of future choices by population bursts and behavior are perturbed. Aim 2 will then perform recordings across deep and superficial layers and along the dorso-ventral axis of entorhinal cortex to identify how mEC contributes to WM. Finally, Aim 3 will carry out large-scale combined single-unit and LFP recordings in hippocampus and mPFC and in mEC and mPFC to reveal the coordination of mechanisms for WM retention across brain regions and brain states. Taken together, we will identify whether neuronal activity during population bursts is a general mechanism for memory retention over delay intervals irrespective of brain state, whether mEC supports WM retention with persistent activity, and how subregions of mPFC are coordinated with hippocampal and entorhinal subareas along the dorso-ventral axis. Identifying these memory computations will be the foundation for treatment approaches to restore memory functions when brain circuits deteriorate in neurological and neurodegenerative diseases.

Learning and memory are cognitive functions that are central to human behavior. It has been widely hypothesized that multiple brain regions are coordinated with hippocampus, a subcortical structure, to form the basis for learning and long-term memory. Understanding how different brain regions interact during learning can lead to better understanding of long-term memory storage in the brain. This high-risk, high-payoff project will investigate how cortex and hippocampus communicate and coordinate information transfer during learning and memory consolidation by multimodal imaging and recording experiments. However, such experiments are not currently feasible due to technical limitations. This proposal follows a transformative approach to investigate hippocampus-cortex coordination during learning and memory by combining (i) technological breakthroughs in development of novel implantable probes, (ii) carefully designed multi-modal sensing experiments, and (iii) advanced data analysis techniques. Such a capability could lead to discoveries on information processing in the brain and can help to better understand circuit dysfunctions causing memory impairment for various neurological disorders affecting a large population worldwide. Findings from this research can help with bridging critical gaps between artificial intelligence-driven models for learning and real biological learning in brain. Understanding the latter has the potential to reshape current practices in machine learning. This project will also provide opportunities for students to become engaged in cutting-edge multidisciplinary research in microfabrication, neuroscience and data analysis. The project will also provide research internship opportunities and mentoring initiatives for underrepresented minorities in engineering.

The objective of this project is to investigate how cortex and hippocampus communicate and coordinate information transfer during learning and memory consolidation by multimodal imaging and recording experiments. Wide-field calcium imaging will be used to monitor cortex-wide neural activation across large areas in awake mice. Simultaneous electrophysiological recordings from hippocampus will detect high frequency oscillations such as sharp-wave ripples and spikes from single neurons. Integration of multiple imaging and recording modalities requires development of new implantable probe technologies enabling recording from hippocampus during imaging and advanced data analysis techniques. Complementary expertise of the investigators will be leveraged to pursue; Task 1: Development of new flexible penetrating microprobes compatible with optical imaging, Task 2: Multi-modal, multi-scale experiments in awake mice generating brand new data sets synergistically combining information from calcium fluorescence, local field potentials, single units and behavior, and Task 3: Development of a novel data-driven task-aware algorithm to perform single-event analyses with multimodal calcium imaging from cortex and electrophysiological recordings from hippocampus.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

SUMMARY The basal ganglia are a group of subcortical nuclei that regulates motor and cognitive functions. Recent identification of neuronal heterogeneity in the basal ganglia suggests that functionally distinct neural circuits defined by their molecular identity and efferent projections exist even within the same nuclei. This distinction may account for a multitude of symptoms associated with basal ganglia disorders such as Parkinson's disease (PD). However, our incomplete understanding of the basal ganglia functional organization has hindered further investigation of individual circuits that may underlie distinct behavioral symptoms in different disease states. The external globus pallidus (GPe) is a central basal ganglia nucleus that can influence numerous downstream regions. While the prevailing circuit model assumes that the GPe is a homogeneous population of neurons transferring the signal in the indirect pathway of the basal ganglia, accumulating evidence suggests that neurons in the GPe are more heterogeneous than previously appreciated. Although GPe is known to be a nucleus with GABAergic neurons, we have identified novel cell types expressing VGLUT2, glutamatergic neuronal marker, at the outer layer of GPe. In our careful anatomical and molecular examination showed that VGLUT2GPe neurons project mainly to inner part of GPe, making synaptic contacts onto other neuronal populations. Recent evidence showed that the distinct cell types in GPe may have different roles in modulating basal ganglia circuitry and associated behaviors. Thus, elucidating the anatomical and functional organization of VGLUT2GPe neurons will provide novel cellular and circuit information to understand basal ganglia function. The progressive nature of behavioral deficits associated with PD is very well documented in human patients. However, what neural adaptations associated with behavioral deficits at different stages of PD are not fully understood. In this application, we try to address this with two different animal models. First, as in our preliminary results and recent reports, we will administer different doses of neurotoxin administration to induce different degrees of DA neuronal loss, which elicit the different behavioral deficits. Second, we will confirm the neurotoxin- induced PD-related behaviors in MitoPark mice which show the progressive loss of DA neurons. Examining the circuit adaptation in two animal models will provide an important information on the neural mechanisms underlying the progressive nature of PD. Therefore, using cutting-edge techniques including optogenetic, genetic and viral-mediated manipulation, in vivo multi-unit recording, and so on, we will decipher roles of VGLUT2GPe neurons in behavioral deficits in these two animal models for PD.