All-optical assay to study biological neural networks

This work was supported by the Cunningham Trust Ph.D. Studentship, the RS MacDonald Charitable Trust Neurophotonics Grant and EPSRC programme Grant EP/P030017/1.We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetics construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (jRCamp1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. We also developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity.Publisher PDFPeer reviewe

Wide-Field Multiphoton Imaging Through Scattering Media Without Correction

Funding: This work is supported by the UK Engineering and Physical Sciences Research Council for funding through grants EP/P030017/1 and EP/M000869/1, and has received funding from the European Union’s Horizon 2020 Programme through the project Advanced BiomEdical OPTICAL Imaging and Data Analysis (BE-OPTICAL) under grant agreement no. 675512, The Cunningham Trust and The RS MacDonald Charitable Trust. KD acknowledges the financial support of Elizabeth Killick and Susan Gurney.Optical approaches to fluorescent, spectroscopic, and morphological imaging have made exceptional advances in the last decade. Super-resolution imaging and wide-field multiphoton imaging are now underpinning major advances across the biomedical sciences. While the advances have been startling, the key unmet challenge to date in all forms of optical imaging is to penetrate deeper. A number of schemes implement aberration correction or the use of complex photonics to address this need. In contrast, we approach this challenge by implementing a scheme that requires no a priori information about the medium nor its properties. Exploiting temporal focusing and single-pixel detection in our innovative scheme, we obtain wide-field two-photon images through various turbid media including a scattering phantom and tissue reaching a depth of up to seven scattering mean free path lengths. Our results show that it competes favorably with standard point-scanning two-photon imaging, with up to a fivefold improvement in signal-to-background ratio while showing significantly lower photobleaching.Publisher PDFPeer reviewe

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and their differentiation into neural lineages is a revolutionary experimental system for studying neurological disorders, including intellectual and developmental disabilities (IDDs). However, issues related to variability and reproducibility have hindered translating preclinical findings into drug discovery. Here, we identify areas for improvement by conducting a comprehensive review of 58 research articles that utilized iPSC-derived neural cells to investigate genetically defined IDDs. Based upon these findings, we propose recommendations for best practices that can be adopted by research scientists as well as journal editors

Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts

Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. Here, we identify five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons that recapitulate the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibit TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we show that the technique can reveal novel aspects of human disease phenotypes in vitro

All-optical assay to study biological neural networks

As life span increases, neurodegenerative diseases such as dementia, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis become an emerging problem in modern society. In particular Alzheimer’s disease (AD), characterized by a progressive cognitive impairment and memory loss, is the dominant cause of disability in people aged over 60. Due to the lack of accurate models, understanding the disease mechanisms and developing a cure for AD remains challenging. However, a novel approach based on human induced pluripotent stem cell (iPSC) technology may offer an opportunity to overcome the limitations of the current models. These cells obtained by reprogramming patient’s somatic cells such as fibroblasts can be differentiated in vitro into various types of neural cells which further develop complex networks. To explore these heterogeneous neural networks, it is often critical to understand the activity of multiple neurons and how they communicate with each other. The work presented in this thesis focuses on the development of the first molecular optogenetic tool called OptoCaMP used in an all-optical assay enabling simultaneous stimulation and calcium imaging of a large population of neurons with a single-cell readout. This assay was further adapted to study the spread of excitation in a network thus allowing the quantification of its connectivity. The application of this assay in conditions where the neuronal connectivity was enhanced or decreased successfully demonstrated its sensitivity to changes in connectivity. This assay together with the iPSC technology bring the promise to greatly improve disease models studies and drug screening platforms."This work was supported by the Cunningham Trust PhD studentship and the Wellcome Trust Institutional Strategic Support Fund (ISSF), the RS MacDonald Charitable Trust Neurophotonics Grant, the EPSRC programme Grant EP/P030017/1 and the generous financial support of the University of St Andrews for my attendance at multiple conferences." -- Fundin

All-optical assay to study biological neural networks

We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetics construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (jRCamp1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. We also developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity

Deep tissue, wide-field multiphoton imaging using TEMPPIX

We demonstrate a new approach, temporal focusing microscopy with single-pixel detection (TEMPPIX), for wide-field multiphoton imaging through scattering media without any a priori knowledge or requirement to determine the properties of the media.</p

Image_1_All-Optical Assay to Study Biological Neural Networks.PDF

<p>We introduce a novel all-optical assay for functional studies of biological neural networks in vitro. We created a novel optogenetic construct named OptoCaMP which is a combination of a channelrhodopsin variant (CheRiff) and a red genetically encoded calcium indicator (GECI) (jRCaMP1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. Additionally, we have developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity.</p