A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all organisms

The Impact of Mild Traumatic Brain injury on Neuronal Networks and Neurobehavior

Despite its enormous incidence, mild traumatic brain injury is not well understood. One aspect that needs more definition is how the mechanical energy during injury affects neural circuit function. Recent developments in cellular imaging probes provide an opportunity to assess the dynamic state of neural networks with single-cell resolution. In this dissertation, we developed imaging methods to assess the state of dissociated cortical networks exposed to mild injury. We probed the microarchitecture of an injured cortical circuit subject to two different injury levels, mild stretch (10% peak) and mild/moderate (35%). We found that mild injury produced a transient increase in calcium activity that dissipated within 1 h after injury. Alternatively, mild/moderate mechanical injury produced immediate disruption in network synchrony, loss in excitatory tone, and increased modular topology, suggesting a threshold for repair and degradation. The more significant changes in network behavior at moderate stretch are influenced by NMDA receptor activation and subsequent proteolytic changes in the neuronal populations. With the ability to analyze individual neurons in a circuit before and after injury, we identified several biomarkers that confer increased risk or protection from mechanical injury. We found that pre-injury connectivity and NMDA receptor subtype composition (NR2A and NR2B content) are important predictors of node loss and remodeling. Mechanistically, stretch injury caused a reduction in voltage-dependent Mg2+ block of the NR2B-cotaning NMDA receptors, resulting in increased uncorrelated activity both at the single channel and network level. The reduced coincidence detection of the NMDA receptor and overactivation of these receptors further impaired network function and plasticity. Given the demonstrated link between NR2B-NMDARs and mitochondrial dysfunction, we discovered that neuronal de-integration from the network is mediated through mitochondrial signaling. Finally, we bridged these network level studies with an investigation of changes in neurobehavior following blast-induced traumatic brain injury (bTBI), a form of mild TBI. We first developed and validated an open-source toolbox for automating the scoring of several common behavior tasks to study the deficits that occur following bTBI. We then specifically evaluated the role of neuronal transcription factor Elk-1 in mediating deficits following blast by exposing Elk-1 knockout mouse to equivalent blast pressure loading. Our systems-level behavior analysis showed that bTBI creates a complex change in behavior, with an increase in anxiety and loss of habituation in object recognition. Moreover, we found these behavioral deficits were eliminated in Elk-1 knockout animals exposed to blast loading. Together, we merged information from different perspectives (in silico, in vitro, and in vivo) and length scales (single channels, single-cells, networks, and animals) to study the impact of mild traumatic brain injury on neuronal networks and neurobehavior

Stobe Photography Mapping of Cell Membrane Potential with Nanosecond Resolution

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all organisms

Novel Tools to Investigate Cortical Activity in Paroxysmal Disorders

This PhD project is at the interface between academic research and industry, and is jointly sponsored by the BBSRC and the industrial partner– Scientifica UK. The goal of this research is the development of new instruments and approaches to monitor and manipulate neuronal network activity in disease states. Firstly, (I) I collaborated with Scientifica to develop and utilise the newly developed Laser Applied Stimulation and Uncaging (LASU) system. The combined usage of the LASU system, alongside novel spatially-targeted channelrhodopsin variants, has al- lowed me to test the limits of single-photon optogenetic stimulation in achieving specific activation of targeted neurons. The presented findings demonstrate that, al- though high-resolution stimulation is achievable in the rodent cortex, single-photon stimulation is insufficient to achieve single-cell resolution stimulation. Secondly, (II) I have combined the high temporal resolution of novel, transparent 16-channel epicortical graphene solution-gated field effect transistor (gSGFET) arrays with the large spatial coverage of bilateral widefield Ca2+ fluorescence imaging; to per- form investigations of the relationship between spreading depolarisation (SD) and cortical seizures in awake head-fixed mouse models of epilepsy. To analyse these complex datasets, I developed a bespoke, semi-automated analysis pipeline to pro- cess the data and probe the seizure-SD relationship. I present the advantages of this dual-modality approach by demonstrating the strengths and weaknesses of each recording method, and how a synergistic approach overcomes the limitations of each technique alone. I utilise widefield imaging to perform systematic classification of SD and seizures both temporally and spatially. Detailed electrophysiological anal- ysis of gSGFET data is then performed on extracted time periods of interest. This work demonstrates the complex interaction between seizures and SD, and proposes several mechanisms describing these interactions. The technological and analytical tools presented here lay the groundwork for insightful and flexible experimental paradigms; altogether, able to probe paroxysmal activity in profound detail

Neural Coding and Organization Principles in the Drosophila Olfactory System

Sensory systems receive and process external stimuli to allow an organism to perceive and react to the environment. How is sensory information subsequently represented, transformed, and interpreted in the neural system? In this dissertation, I have investigated this fundamental question using the fruit fly (Drosophila melanogaster) olfactory system.Chemical cues are transduced into neural signals in the insect antenna by the olfactory receptor neurons (ORNs). The ORNs send their axons to the antennal lobe (AL), with each ORN type innervating a specific neuropil (glomerulus), where they synapse onto excitatory and inhibitory projection neurons (ePNs and iPNs). The ePNs project their axons to the 3rd order stages, the calyx (CL) and lateral horn (LH). On the other hand, the iPNs only innervate the LH. In this dissertation, I first examined how well the peripheral neural activities evoked by an odorant could predict the final behavioral output. As the stimulus intensity increases, a fly’s preference for some odorants switch from attraction to aversion. Behavior assay suggested this phenomenon may help the fly evade harmful environment. Our results indicate that at the level of ORNs, increases in stimulus intensity could result in oscillatory extracellular field potentials that arise entirely due to abrupt changes in cell excitability. Notably, combining the activity of a few ORNs was sufficient to predict intensity-dependent preference changes with odor intensity. How is the sensory input organized in the downstream neural circuit, the insect antennal lobe? Odor-evoked signals from sensory neurons (ORNs) triggered neural responses that were patterned over space and time in cholinergic ePNs and GABAergic iPNs within the antennal lobe. The dendritic-axonal (I/O) response mapping was complex and diverse, and the axonal organization was region-specific (mushroom body vs. lateral horn). In the lateral horn, feed-forward excitatory and inhibitory axonal projections matched ‘odor tuning’ in a stereotyped, dorsal-lateral locus, but mismatched in most other locations. In the temporal dimension, ORN, ePN, and iPN odor-evoked responses had similar encoding features, such as information refinement over time and divergent ON and OFF responses. Notably, analogous spatial and temporal coding principles were observed in all flies, and the latter emerged from idiosyncratic neural processing approaches. In sum, these results provide key insights necessary for understanding how sensory information is organized along spatial and temporal dimensions

The Role of Dentate Granule Cell Age and Morphology in Seizure-induced Plasticity.

Temporal lobe epilepsy (TLE) is a common type of medically intractable epilepsy among adults. In TLE, pathological changes within the hippocampus are hypothesized to play a critical role in epileptogenesis. However, the relationship between observed neuropathology and the development of seizure activity is not well understood. The dentate gyrus is a region of particular interest for epilepsy-related plasticity because of its position as a gate for much of the incoming excitatory input to the hippocampus. In addition, it is capable of a unique type of neuronal plasticity, due to ongoing neurogenesis. DGCs born after an epileptogenic insult in rodent models are much more likely to display aberrant, pro-excitatory morphology than those that were mature at the time of insult. We hypothesized that these adult-born DGCs with aberrant morphology are also the most likely to display pro-excitatory physiological features. Hilar ectopic DGCs are common in tissue from TLE patients and animal models, but rare in healthy controls. We recorded from hilar ectopic and normotopic DGCs from both rat and human TLE tissue and found increased synaptic and intrinsic excitability in rat DGCs, but decreased intrinsic excitability in human DGCs. These data present a conflicting view of the role of ectopic DGCs in hyperexcitability, but they also highlight important discrepancies between the human disease and the rodent disease model. We also hypothesized that adult-born DGCs would contribute more than neonatal-born DGCs to hyper-connectivity of DGCs. We birthdated populations of DGCs using a retrovirus carrying a fluorescent-tagged synaptophysin to study mossy fiber axonal reorganization in the rat pilocarpine TLE model. Interestingly, we found no major differences, either qualitative or quantitative, in axonal plasticity between the two populations. Thus, axonal reorganization does not require adult-neurogenesis in the rat TLE model. The work presented in this dissertation provides new insight into the role of DGC birthdate and morphology for excitability in epilepsy. It is more complex than was previously suggested. We have shown that both neonatal- and adult-born, can exhibit features consistent with increased excitability in TLE, but not all adult-born DGCs appear aberrant.PhDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111345/1/althausa_1.pd

Development of high-speed imaging techniques for C. elegans nervous system studies

We report high-speed imaging techniques for C. elegans nervous systems studies. We introduce C. elegans, the main model organism in this dissertation, and neuroscientific and biomedical studies using C. elegans involving calcium imaging, nerve regeneration, and drug screening. We review technologies including confocal microscopy and microfluidic devices used in the neuroscientific and biomedical studies We discuss development of a high-speed laser scanning confocal microscope capable of flexible control of imaging conditions, fast imaging speed, and large field-of-view. We provides the design principles used in the development of the confocal microscope including the optical, electrical, and software implementation, and the details of the confocal microscope we built based on the design principles. We present the performance characterization of the confocal microscope, then a few sample images obtained with the confocal microscope. We present development of time-lapse volumetric confocal imaging of whole animal C. elegans Ca²⁺ dynamics. We provide the design of the time-lapse volumetric confocal imaging system including a microfluidic device to accommodate the whole animal within the field-of-view of the imaging system. We examine the feasibility of the volumetric confocal imaging of a whole animal, and demonstrate imaging of the whole animal C. elegans neurons’ response to NaCl within a 630 × 150 × 25 μm³ volume at 2 Hz rate. We report a high-throughput automated imaging platform for C. elegans nerve regeneration study. We describe the design of the automated imaging platform and the automation flow, and characterizes the performance of the platform. The imaging platform can obtain high-resolution 3D confocal images of 20 animals in 10 minutes. We show sample images of C. elegans anterior lateral microtubule nerve regeneration examples acquired via the automated imaging platform. We demonstrate a planar laser activated neuronal scanning platform (PLANS), a high-throughput animal examination system for drug screening. We explain the construction of PLANS involving the optics, the microfluidic device, and the electronics. The PLANS system can scan an animal in less than 5 ms with a spatial sampling resolution of 3 μm FWHM. We show sample scanning results of a Huntington’s disease model of C. elegans. We summarize the studies discussed in this dissertation, and suggest relevant future research to follow up on the studies.Electrical and Computer Engineerin