Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide

Gut-seeded α-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice

Parkinson’s disease is a synucleinopathy that is characterized by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (α-Syn) aggregates. Evidence suggests that α-Syn aggregation can originate in peripheral tissues and progress to the brain via autonomic fibers. We tested this by inoculating the duodenal wall of mice with α-Syn preformed fibrils. Following inoculation, we observed gastrointestinal deficits and physiological changes to the enteric nervous system. Using the AAV-PHP.S capsid to target the lysosomal enzyme glucocerebrosidase for peripheral gene transfer, we found that α-Syn pathology is reduced due to the increased expression of this protein. Lastly, inoculation of α-Syn fibrils in aged mice, but not younger mice, resulted in progression of α-Syn histopathology to the midbrain and subsequent motor defects. Our results characterize peripheral synucleinopathy in prodromal Parkinson’s disease and explore cellular mechanisms for the gut-to-brain progression of α-Syn pathology

Top-Down Control of Serotonergic Systems in Socioaffective Choices and Depression-Like Behaviors

Regulation of social behaviors is necessary to achieve social inclusion, establish relationships and sustain those relationships through adversity. Impairments in socio-emotional function and competence are prominent and debilitating features of major depression, yet are not traditionally recognized as cardinal symptoms of the disease. However, these deficits often persist in patients whose other mood symptoms have remitted and can predict risk of relapse, indicating an important role as a vulnerability factor. Understanding the neurobiology of socioaffective dysfunction in depression is thus important for determining the pathology of the disorder and developing effective treatments. Human imaging studies of depressive patients have consistently reported abnormal activity in the ventromedial prefrontal cortex (vmPFC), an area important for emotional processing and social cognition. Tracing studies in animals and tractography in humans have shown that the dorsal raphe nucleus (DRN) is a major projection target of the vmPFC. The DRN contains the most serotonin (5-HT) producing neurons in the brain and its output has been shown to regulate behaviors along an affiliative-agonistic axis, however it is neuronally heterogeneous. This thesis investigated the cytoarchitecture of the vmPFC-DRN microcircuit and its relevance to socioaffective behaviors using genetic mapping, whole cell electrophysiology and optogenetics. I showed that GABAergic neurons, which are the primary non-serotonergic neuronal population in the DRN, mediated top-down projections from the vmPFC onto mood-regulating 5-HT neurons and demonstrated the relevance of this pathway in mediating socioaffective decisions using the chronic social defeat stress (CSDS) paradigm. In addition, I used deep brain stimulation of the vmPFC as an antidepressant model to show that therapeutic response may rely on restoring the excitatory/inhibitory balance of inputs to 5-HT neurons. Together, these results will provide a better understanding of socioaffective circuitry and could lead to the development of more effective and efficient strategies to treat mood disorders

Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide

Changing practice in dementia care in the community: developing and testing evidence-based interventions, from timely diagnosis to end of life (EVIDEM)

Background Dementia has an enormous impact on the lives of individuals and families, and on health and social services, and this will increase as the population ages. The needs of people with dementia and their carers for information and support are inadequately addressed at all key points in the illness trajectory. Methods The Unit is working specifically on an evaluation of the impact of the Mental Capacity Act 2005, and will develop practice guidance to enhance concordance with the Act. Phase One of the study has involved baseline interviews with practitioners across a wide range of services to establish knowledge and expectations of the Act, and to consider change processes when new policy and legislation are implemented. Findings Phase 1, involving baseline interviews with 115 practitioners, identified variable knowledge and understanding about the principles of the Act. Phase 2 is exploring everyday decision-making by people with memory problems and their carers

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

Background. Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods. A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results. The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences. The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

Background. Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods. A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results. The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences. The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry

Systemic AAV vectors for widespread and targeted gene delivery in rodents

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing

Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family

Spiders produce multiple silks with different physical properties that allow them to occupy a diverse range of ecological niches, including the underwater environment. Despite this functional diversity, past molecular analyses show a high degree of amino acid sequence similarity between C-terminal regions of silk genes that appear to be independent of the physical properties of the resulting silks; instead, this domain is crucial to the formation of silk fibres. Here we present an analysis of the C-terminal domain of all known types of spider silk and include silk sequences from the spider Argyroneta aquatica, which spins the majority of its silk underwater. Our work indicates that spiders have retained a highly conserved mechanism of silk assembly, despite the extraordinary diversification of species, silk types and applications of silk over 350 million years. Sequence analysis of the silk C-terminal domain across the entire gene family shows the conservation of two uncommon amino acids that are implicated in the formation of a salt bridge, a functional bond essential to protein assembly. This conservation extends to the novel sequences isolated from A. aquatica. This finding is relevant to research regarding the artificial synthesis of spider silk, suggesting that synthesis of all silk types will be possible using a single process