Neuroscience 2020

Neurodegenerative disease research can be considerably accelerated by using fibrillar or oligomeric constructs of prion-like proteins in in vitro or in vivo studies. These exist in a variety of forms, and are able to generate different outcomes depending on how they are prepared. The most popular constructs are pre-formed fibrils (PFFs) of certain proteins, including Alpha Synuclein and Tau, relevant in Parkinson’s and Alzheimer’s Diseases.  However, despite being popular in this regard, their preparation – requiring sonication – has not been extensively studied from a phase perspective. In this discussion we examine current best practices and provide direction of why sonication is important and how it affect pre-formed fibrils.

The accumulation of neurotoxic amyloid beta peptides and/or neurofibrillary tangle formation are key pathological hallmarks of neurodegenerative diseases including but not limited to Alzheimer’s disease, frontotemporal dementia and multi-system atrophy. The brain has been considered as an immune-privileged organ, however, increasing evidence from translational, genetic, and pathological studies suggests that activation of distinct innate immune pathways are a third important disease hallmark which, once initiated, actively contributes to progression and chronicity of neurodegenerative disease.

Microglia play a pivotal role in this immune response and are activated by binding of aggregated proteins or aberrant nucleic acids to pattern recognition receptors. This immune activation leads to the release of inflammatory mediators, but also distracts microglia cells from their physiological functions and tasks, including debris clearance and trophic factor support. NLRP3 inflammasome activation and release of ASC specks contribute to spreading of pathology and impair microglia clearance mechanisms, and together contribute to neuronal spine loss, neuronal degeneration, and ultimately to spatial memory deficits. In keeping with this immune hypothesis of neurodegeneration, inhibition of this and other immune pathways protect from neurodegeneration in cellular and murine models of neurodegenerative disease. Modulation of the microglia driven innate immune response at key signaling steps might therefore be protective and alter disease progression. However, the microglia are not a stable population, but have continuous turn over, most likely resulting in more than one generation of microglia being involved in disease progression.  Moreover their turnover is increased in response to neurodegeneration. Along with the regional diversity of microglia, these phenomena need to be understood in more detail prior to targeting innate immune mechanisms for therapeutic purposes.

The US Brain Research through Advancing Innovative Neurotechnologies is a research program focused on building fundamental knowledge of how brain circuits process information to enable human behavior. Research began in 2014 and considerable progress has occurred to open up extraordinary research possibilities for understanding the circuits underlying animal and human behavior. These pose new opportunities for dissecting the circuits that underlie neuro/mental/substance abuse disorders. BRAIN Initiative research has already included advances in brain recording and stimulation for neuro/mental health disorders such as closed-loop simulation for Parkinson's, Essential Tremor and Epilepsy. However, it's major impact will come from the application of technologies to patients that are now in animal studies. These will include new brain activity monitoring techniques to serve as diagnostic readouts of neural circuits. New therapies will come from genomic -enabled precision modulation of specific brain cell types by chemicals, electromagnetic, ultrasound energy or other controlling technologies. In addition to the US program complimentary programs in Europe, the Human Brain Project, Japan (BRAIN/MINDS), Korea, Australia and soon China will add to this scientific revolution. In the past Neurology has been built off of neuropathology and neuroanatomy with the patient's symptoms/deficits presumed due to interruption of these static features. The BRAIN Initiatives will bring the previously invisible dynamic features of brain circuits into Neurology, Psychiatry and NeuroSurgery.

Learning Objectives:

1. Understand the potential for new Neurotechnologies to enable powerful abilities to monitor, modulate, and map brain circuits.

2. Understand the promise for new Neurotechnologies to more effectively diagnose and treat neuro/mental/substance abuse disorders.

3. Become aware of ethical questions that are raised by the new Neurotechnologies.

Autosomal dominant missense mutations that hyperactivate the LRRK2 protein kinase are a common cause of inherited Parkinson’s disease and therapeutic efficacy of LRRK2 inhibitors is being tested in clinical trials. In my presentation I will we discuss the nuts and bolts of our current research that has revealed that LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with a new set of effectors such as RILPL1/2.  I will discuss how mutations in other components linked to Parkinson’s such as  Rab29 and VPS35 activate LRRK2 and promote Rab protein phosphorylation. I will discuss the identification and characterization of a novel PPM family phosphatase member that counteracts LRRK2 signalling by dephosphorylating Rab proteins. I will present data that indicates that the LRRK2 parlog termed LRRK1 also functions as a Rab kinase.  Finally, I will discuss what is known about how LRRK2 regulates ciliogenesis, the endosomal-lysosome system, and immune responses and how this might be linked to Parkinson’s disease.

Learning Objectives:

1. Define the nuts and bolts of the LRRK2 signaling pathway and how it is linked to Parkinson's disease

2. Showcases new approaches that could be exploited to better diagnose and Parkinson's disease

Miltenyi Biotec offers a variety of neuroscience and immunology workflows that cover all steps from sample preparation and cell isolation to cell culture and flow cytometry.  Discover the breadth of products, detailed scientific data and protocols Miltenyi  Biotec has to support your neuroimmunology research.

Learning Objectives:
1. Highlight the importance of efficient and reproducible sample preparation of your starting materials.
2. Describe the fundamentals of MACS technology and magnetic cell separation for the isolation and cultivation of neurons and immune cells.
3. Cover important considerations for reliable and interpretable analysis of downstream applications.

The study of biological function in intact organisms and the development of targeted cellular therapeutics necessitate methods to image and control cellular function in vivo. Technologies such as optogenetics serve this purpose in small, translucent specimens or surgically accessed organs, but are limited by the poor penetration of light into deeper tissues. In contrast, non-invasive techniques such as ultrasound – while based on energy forms that penetrate tissue effectively – are not as effectively coupled to cellular function. Our work attempts to bridge this gap by engineering biomolecules with the appropriate physical properties to interact with sound waves, and by enhancing the transport of engineered biomolecules into tissues such as the brain. In this talk, I will describe our work on Acoustically Targeted Chemogenetics (ATAC) – a technology that couples ultrasound with molecular engineering to enable non-invasive control of specific neurons. In this approach, we use focused ultrasound to transiently open the blood–brain barrier and transduce neurons at specific locations in the brain with virally encoded engineered G-protein-coupled receptors. The engineered neurons subsequently respond to systemically administered designer compounds to activate or inhibit their activity. In a mouse model of memory formation, we showed that this approach can modify and subsequently activate or inhibit excitatory neurons within the hippocampus (1). Now we are adapting and improving this technology for application in larger species.

(1) Szablowski JO, Lue B, Lee-Gosselin A, Malounda D, Shapiro MG. Acoustically targeted chemogenetics for the non-invasive control of neural circuits. Nature Biomedical Engineering 2, 475-484 (2018).

Learning Objectives:

1. Introduce the technology of acoustically targeted chemogenetics.

2. Describe the use of ultrasound in modulating neural circuits.

Open Science has changed research by making data accessible and shareable, contributing to replicability to accelerate and disseminate knowledge. However, for rodent cognitive studies the availability of tools to share and disseminate data is scarce. Automated touchscreen-based tests enable systematic cognitive assessment with easily standardized outputs that can facilitate data dissemination. Here we present an integration of touchscreen cognitive testing  with an open-access database public repository (mousebytes.ca), as well as a Web platform for knowledge dissemination (https://touchscreencognition.org). We complement these resources with the largest dataset of age-dependent high-level cognitive assessment of mouse models of Alzheimer’s disease, expanding knowledge of affected cognitive domains from male and female mice of three mouse strains. We envision that these new platforms will enhance sharing of protocols, data availability and transparency, allowing meta-analysis and reuse of mouse cognitive data to increase the replicability/reproducibility of datasets.

Learning Objectives:

1. Understand the pitfalls and advantages of cognitive testing in mouse models of disease
2. Learn about new initiatives in Open Science and data sharing for behavioral neuroscience

Misfolded and accumulated neurodegenerative disease associated proteins (NDAPs, such as tau and alpha-synuclein) represent the major pathological hallmark in Alzheimer’s and Parkinson’s disease patient brain cells. Compelling evidence indicate that spreading of NDAPs within the patient brain occurs via interconnected neurons. While progress in deciphering the mechanisms of neuronal uptake of NDAPs and NDAP-caused protein aggregation has been made, our knowledge about other cellular processes, such as axonal uptake and transport of NDAPs as well as neuron-to-neuron transfer of NDAPs, is still limited. In the presentation, I will present Cellectricon’s approach to decipher individual cellular processes that are occurring in progressive proteopathies in cortical neuronal circuits. We apply an experimental in vitro paradigm to generate compartmentalized neuronal cells by culturing primary mouse cortical neurons in high capacity microfluidic co-culture plates. By using fluorescently labelled, seed competent NDAPs and high-content imaging, we can assess NDAP axonal uptake, axonal transport and protein aggregation in cortical neurons. By combining our neuronal compartmentalisation approach with neuronal labelling techniques, we aim to decipher the spreading mechanisms of neuropathologies in neuronal circuits. We aim to provide insights into specific disease-relevant neuronal processes, which can pave the way for novel intervention approaches for modulation of progressive proteopathies.

Learning Objectives:

1. Introduce the audience the principles of progressive neurodegenerative diseases

2. Present a concept that enables the assessment of specific cellular processes involved in progressive neurodegenerative diseases

3. Present data obtained from Cellectricon´s high-capacity microfluidics platform showing alpha-synuclein axonal uptake, transport and caused protein aggregation in a sub-population of primary cortical neurons

Recent technological advancements in neuroprosthetics allow for wireless recording and stimulation of brain activity in freely moving human participants. At the same time, advancements in virtual and augmented reality (VR/AR) and other wearable technologies are occurring at an unprecedented rate. I will discuss our recently developed platform that allows for wireless and programmable recording and stimulation of deep brain activity in freely moving human participants integrated with wearable sensors (e.g., heart rate, skin conductance, respiration, eye tracking, and scalp EEG) and VR/AR technologies. This research platform allows for a naturalistic and ecologically valid environment for elucidating the neural mechanisms underlying freely moving human behaviors in the real world. As a proof-of-concept, I will present data captured from this platform that provides insight into deep brain oscillatory dynamics that underlie human spatial navigation and episodic memory in real and virtual environments.

Learning Objectives:

1. To learn about new opportunities that enable the study of the human brain in naturalistic settings
2. To gain understanding of the brain mechanisms that support navigation and memory in humans

Early phase clinical trials investigating novel applications of neural devices, such as deep brain stimulation (DBS) devices, pose ethical challenge during the recruitment of human subjects and in supporting ongoing informed consent over the course of research participation. As these clinical trials end, research participants face complicated decisions about the surgical removal or post-trial use of investigational brain implants. Drawing from qualitative research into the experiences of research participants who have exited from early phase clinical trials in deep brain stimulation, this session will highlight ethical considerations related to participant motivations for enrolling in research, understanding of study procedures at the time of initial informed consent, the process of exiting from research, decisions about device removal or post-trial device use, and concerns about access to long-term follow-up care. Ethical safeguards and approaches to involving ethicists in the earliest stages of clinical research will be proposed. This presentation will also reflect upon the development of evidence-based guidelines to support best practices in informed consent and robust standards for the ethical design and conduct of early phase clinical trials in DBS and other implanted brain devices.

Learning Objectives:
1. Enhance awareness and understanding of ethical challenges that arise in early phase brain device research.
2.Describe practical and ethical challenges in obtaining initial and ongoing informed consent to early phase research participation.
3.Reflect on ethical safeguards that should be in place and the role of ethicists in supporting best practices in brain device research.

I describe a framework for improving the targeting and precision of transcranial magnetic stimulation (TMS), a noninvasive brain stimulation technique used for research and clinical applications. The framework designs coils that double the precision of spatial targeting (focality) of existing TMS coils. This is the first significant advancement in the depth-focality trade-off of TMS coils since the introduction of the standard figure-of-eight coil three decades ago, and likely represents the fundamental physical limit. Moreover, the framework quantifies uncertainty in TMS induced electric fields due to system setup and patient variability, and it identifies key parameters that affect targeting precision. Results show that coil position is a key contributor to TMS variability, supporting the need for more precise neuro-navigation devices.  Finally, I show how this framework can also be used for determining coil placements that optimally target specific brain regions. By improving the accuracy and precision of TMS targeting this frameworks enables the development of more effective clinical and research TMS protocols.

Learning Objectives:

1. Learn about the physics behind transcranial magnetic stimulation, and the spatial characteristics of the TMS coil stimulating fields.
2. Learn about the specific TMS session parameters that contribute most to variation in the TMS induced stimulating field. Furthermore, they should learn about different approaches to coil placement and their precision in terms of targeting.

Autism constitutes a spectrum of clinical symptoms that are caused by a constellation of genetic and environmental causes; thus, to improve early diagnosis of the disorders and provide possible routes to therapy, it has become important to identify common pathophysiologies among autism cases. Recently, attention has turned to an area of the brain that is commonly malformed among autism patients, the cerebellum. This talk will review evidence that cerebellar disruption is sufficient to cause autism symptoms, that cerebellar dysfunction is common in preclinical models of autism, and how the cerebellum might be involved in the development of autism symptoms. This talk will also explore some unanswered questions about the cerebellum and autism and touch on how this subject can be studied at a small, primarily undergraduate institution.

Learning Objectives:

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress‐inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co‐chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in‐depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co‐chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co‐chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age‐dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age‐dependent hippocampal neurodegeneration in mice.

Learning Objectives:

1. Introduce the advantages of using a hypomorphic allele mouse model to study the chaperone machinery in mice.

2. Learn about a protein that is a potential key player in neurodegeneration and aging.

In Andalman et al, 2018, we explored the activity of over 10,000 neurons across more than 15 regions imaged simultaneously in larval zebrafish in a novel behavioral challenge paradigm. Complementing the experimental findings, we developed a computational model of the activity in the habenula and raphe, identifying a significant and specific change in intra-habenular connectivity and in raphe-to-habenula projection strengths as a result of behavioral challenge. These results were consistent with both the key experimental findings of Andalman et al as well as with converging evidence from other studies. Building on this paper, supported by the NIH BRAIN Initiative, for the first time, we consider complex multi-dimensional interactions at the whole brain level, while also relating them causally to observed behavior of the intact organism. We present a novel, more robust, and scalable class of circuit models – multi-region neural network models. These models are constrained at the outset by experimental data at two levels simultaneously: (a) large-scale neural dynamics – cellular resolution activity imaged from all 15 larval zebrafish brain regions – and (b) the behavior of the organism – tail movements tracked realtime in a range of continuous behavioral and experiential states. We develop an innovative method for analyzing and visualizing essential features extracted from our multi-region network models fit directly to these data, and derive from them the measurable signatures of brain-wide fluctuations that are causally predictive of transitions between behavioral states of the fish.

Our findings demonstrate that theories and mechanistic models constructed in tight conjunction with data, as in this test case, will have far reaching applications for other studies using a range of collection methods and across nervous systems, even those sampled with differing levels of granularity.

Learning Objectives:

1. Exemplify the role of principled theory and computational modeling in neuroscience

2. Present new class of multi-region recurrent network (RNN) models constrained, from the outset, by experimental data

3. Demonstrate that reverse engineering data-inspired RNN models can uncover key mechanisms of brain-wide inter-area communication that are inaccessible from measurements alone

Brain machine interfaces or neural prosthetics have the potential to restore movement to people with paralysis or amputation, bridging gaps in the nervous system with an artificial device. Microelectrode arrays can record from hundreds of individual neurons in motor cortex, and machine learning can be used to generate useful control signals from this neural activity. Performance can already surpass the current state of the art in assistive technology in terms of controlling the endpoint of computer cursors or prosthetic hands. The natural next step in this progression is to control more complex movements at the level of individual fingers. Our lab has approached this problem in three different ways. For people with upper limb amputation, we acquire signals from individual peripheral nerve branches using small muscle grafts to amplify the signal. After a successful study in animals, human study participants have recently been able to control individual fingers online using indwelling EMG electrodes within these grafts. For spinal cord injury, where no peripheral signals are available, we implant Utah arrays into finger areas of motor cortex, and have successfully decoded flexion and extension in multiple fingers. Decoding “spiking band” activity at much lower sampling rates, we recently showed that power consumption of an implantable device could be reduced by an order of magnitude compared to existing broadband approaches, and fit within the specification of existing systems for upper limb functional electrical stimulation. Finally, finger control is ultimately limited by the number of independent electrodes that can be placed within cortex or the nerves, and this is in turn limited by the extent of glial scarring surrounding an electrode. Therefore, we developed an electrode array based on 8 um carbon fibers, no bigger than the neurons themselves to enable chronic recording of single units with minimal scarring. The long-term goal of this work is to make neural interfaces for the restoration of hand movement a clinical reality for everyone who has lost the use of their hands.

Learning Objectives:
1. Explain the recording limitations of conventional electrodes
2. Describe the new capabilities of carbon fiber electrodes compared to the state of the art.

Striatal neuromodulation through G-protein-coupled receptors (GPCRs) regulates complex voluntary motor actions, involving decision-making, learning, and action selection. The dorsal striatum integrates information from neuromodulatory inputs from the thalamus, midbrain, and various parts of cortex, as well as intrinsic neuromodulators within in its microcircuity. The integration of these signals initiates the communication of information through the basal ganglia to control action learning and performance. Many of these inputs form synapses onto indirect and direct-pathway projection medium spiny neurons (MSN). Both subpopulation of MSNs contains GPCRs that can oppositely regulate the cAMP-PKA signaling pathway. This signaling pathway is critical in the modulation of synaptic transmission, long-term synaptic plasticity and learning and memory. In the striatum, the dynamic interplay between dopamine and cAMP-PKA signaling is important for the induction of long-term depression. However, the spatial and temporal dynamics of these intracellular effector molecules in real-time during the induction of synaptic plasticity and behavior remain unclear. Using newly optimized intensity- and FRET-based genetically encoded cAMP biosensors together with optical techniques, we can now measure activity-induced changes in cAMP accumulation in real-time in live brain slices and in freely moving animals. Simultaneous recordings of activity-induced cAMP transients detected by cAMP Difference Detector in situ (cADDis) intensity-based sensor and dopamine transients were performed using slice photometry and fast-scan cyclic voltammetry, respectively. Single pulse electrical stimulation generates increased cAMP in D1 dopamine receptor-expressing MSNs and decreased cAMP in D2 receptor expressing MSNs. These signaling changes coincide with increased extracellular dopamine. We are currently examining the kinetics of the dopamine and cAMP signals. Both transients can be altered by changes in stimulation duration and frequency, and are blocked by tetrodotoxin, indicating that they are driven by neuronal activation. Our findings indicate the feasibility of using optical biosensors combined with traditional methods to investigate precise temporal dopamine-cAMP dynamics in real-time in the striatum.

Learning Objectives:

1. Learn about newly developed and optimized genetically encoded fluorescent sensors to measure second messenger effector molecules

2. Explain optical imaging tools to measure these sensors in vitro (live brain slices) and in vivo during freely moving behaviors

The dopamine D4 receptor (D4R) is enriched in the prefrontal cortex where it plays important roles in cognition, attention, decision making and executive function. Novel D4R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer’s disease and substance use disorders (SUD). To identify new D4R-selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A-412997 (2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide). Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cAMP inhibition assays, and molecular dynamic computational modeling. We identified several novel D4R-selective (Ki ≤ 4.3 nM and >100-fold vs. other D2-like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. These compounds highlight receptor-ligand interactions that control efficacy at D2-like receptors and may provide insights to targeted drug discovery leading to a better understanding of the role of D4Rs in neuropsychiatric disorders such as SUD.

Learning Objectives:

1.  Identify various challenges in designing medication in treating Substance Use Disorders

2.  Understanding the role of dopamine receptors in developing therapies for the treatment of  Substance Use Disorders

3. Understanding of receptor-ligand interactions that control efficacy.

Normal behavior in any moving animal, including humans, relies on communication between motor systems that control movements, and the sensory systems we use to guide these actions. A critical task for the brain is distinguishing between sensations created by our own actions from those caused by external sources; disruptions in this process cause significant impairments in normal behavior. Yet the neural circuits that aid in this task are generally not well understood. To understand these processes at the cellular, circuit and behavioral level, I study the small, tractable brain of the fruit fly Drosophila melanogaster. In this presentation, I will describe current research focusing on the neural circuits that control movements and mechanosensation in the antennae of the fruit fly. Through this work, I aim to discover fundamental principles underlying the brain’s ability to make sense of its own movements through the world.

Learning Objectives:

1. Define active sensing

2. Introduce the audience to tools and advantages of studying neural circuit function in the fruit fly (Drosophila melanogaster)

3. Explain current strategies to develop the fruit fly as a model for active sensing