Visualizing Synaptic Specificity with GRASP

A maxim of modern neuroscience holds that the structures of neural circuits dictate their function. Circuit assembly requires each neuron to exercise remarkable precision as it selects a unique ensemble of synaptic partners from a vast array of potential targets. Visualization of individual synapses in the central nervous system is difficult in some contexts and impossible in others, and the ability to rapidly monitor connectivity in living animals would greatly facilitate deeper understanding of synaptic specificity. In this thesis, I describe the development of a method, GFP reconstitution across synaptic partners (GRASP), that allows visualization of defined synapses in vivo, and apply this method at one set of synapses in the brain of the nematode C. elegans. The principle of GRASP relies on bimolecular assembly of two GFP fragments expressed on two cells at a synapse. To this end, I appended fragments of green fluorescent protein (GFP) to the extracellular portions of transmembrane carrier proteins in apposing cells. When complementary CD4-tethered GFP fragments were brought into proximity at sites of cell contact, GFP fluorescence was observed both in vitro and in vivo. Split GFP fragments fused to the presynaptic phosphatase PTP-3A labeled synapses when expressed in connected neurons. This method detected known mutations that alter synaptic connectivity, such as syg-1 and syg-2, which affect development of synapses between HSN neurons and their postsynaptic nerve and muscle partners. These observations suggest that GRASP could aid efforts to trace behavioral circuits and investigation of the mechanisms of synaptic specificity. Additional tools based on Cre recombinase were developed to confine labeling to single cells and synapses of interest. The ability of GRASP to detect known specificity mutants prompted an investigation of synapse formation in the central nervous system of C. elegans. I generated a transgenic strain, kyIs501, in which GRASP labels synapses formed by the ASH sensory neuron onto the AVA interneuron. A genetic screen in kyIs501 identified one promising mutation, ky957, that causes loss of GRASP labeling. However, subsequent analyses revealed that ky957 is not a bona fide specificity mutant, and appears instead to be associated with alterations in the integrated kyIs501 transgene. Potential solutions to the problems raised by transgene-based approaches as well as further refinements of GRASP are discussed

Global 'worming'

A report on the 16th International Caenorhabditis elegans Meeting, Los Angeles, USA, 27 June-1 July 2007

Orientation columns in the mouse superior colliculus

More than twenty types of retinal ganglion cells conduct visual information from the eye to the rest of the brain. Each retinal ganglion cell type tessellates the retina in a regular mosaic, so that every point in visual space is processed for visual primitives such as contrast and motion. This information flows to two principal brain centres: the visual cortex and the superior colliculus. The superior colliculus plays an evolutionarily conserved role in visual behaviours, but its functional architecture is poorly understood. Here we report on population recordings of visual responses from neurons in the mouse superior colliculus. Many neurons respond preferentially to lines of a certain orientation or movement axis. We show that cells with similar orientation preferences form large patches that span the vertical thickness of the retinorecipient layers. This organization is strikingly different from the randomly interspersed orientation preferences in the mouse’s visual cortex; instead, it resembles the orientation columns observed in the visual cortices of large mammals. Notably, adjacent superior colliculus orientation columns have only limited receptive field overlap. This is in contrast to the organization of visual cortex, where each point in the visual field activates neurons with all preferred orientations. Instead, the superior colliculus favours specific contour orientations within ~30° regions of the visual field, a finding with implications for behavioural responses mediated by this brain centre

Growing up with cancer: Accommodating the effects of cancer into young people’s social lives.

Adolescence and young adulthood are transitional periods of rapid and dramatic personal change. Few events can cause as unpredictable and challenging alterations to this process as the onset of a serious illness, such as cancer. Although we know much about the physical and psychological consequences of having cancer at this time, we know little about the effect of cancer on young people’s relationships. We conducted interviews with 15 women and 12 men aged between 16 and 29 years, who had survived cancer. Our findings demonstrate that the experience of cancer and how it affects relationships is complex. It arrests young people’s development by increasing their dependence on parents, giving them life experiences unavailable to peers, and complicating the process of establishing new relationships. However, it also accelerates development by facilitating closer and more mature relationships with parents and giving young people wisdom and insight not shared by peers. Cancer profoundly shapes how young people conduct their relationships. These changes require ongoing accommodation by young people with cancer, their parents, peers, and new acquaintances

The Habitable Exoplanet Observatory (HabEx) Mission Concept Study Final Report

The Habitable Exoplanet Observatory, or HabEx, has been designed to be the Great Observatory of the 2030s. For the first time in human history, technologies have matured sufficiently to enable an affordable space-based telescope mission capable of discovering and characterizing Earthlike planets orbiting nearby bright sunlike stars in order to search for signs of habitability and biosignatures. Such a mission can also be equipped with instrumentation that will enable broad and exciting general astrophysics and planetary science not possible from current or planned facilities. HabEx is a space telescope with unique imaging and multi-object spectroscopic capabilities at wavelengths ranging from ultraviolet (UV) to near-IR. These capabilities allow for a broad suite of compelling science that cuts across the entire NASA astrophysics portfolio. HabEx has three primary science goals: (1) Seek out nearby worlds and explore their habitability; (2) Map out nearby planetary systems and understand the diversity of the worlds they contain; (3) Enable new explorations of astrophysical systems from our own solar system to external galaxies by extending our reach in the UV through near-IR. This Great Observatory science will be selected through a competed GO program, and will account for about 50% of the HabEx primary mission. The preferred HabEx architecture is a 4m, monolithic, off-axis telescope that is diffraction-limited at 0.4 microns and is in an L2 orbit. HabEx employs two starlight suppression systems: a coronagraph and a starshade, each with their own dedicated instrument

The Habitable Exoplanet Observatory (HabEx) Mission Concept Study Final Report

The Habitable Exoplanet Observatory, or HabEx, has been designed to be the Great Observatory of the 2030s. For the first time in human history, technologies have matured sufficiently to enable an affordable space-based telescope mission capable of discovering and characterizing Earthlike planets orbiting nearby bright sunlike stars in order to search for signs of habitability and biosignatures. Such a mission can also be equipped with instrumentation that will enable broad and exciting general astrophysics and planetary science not possible from current or planned facilities. HabEx is a space telescope with unique imaging and multi-object spectroscopic capabilities at wavelengths ranging from ultraviolet (UV) to near-IR. These capabilities allow for a broad suite of compelling science that cuts across the entire NASA astrophysics portfolio. HabEx has three primary science goals: (1) Seek out nearby worlds and explore their habitability; (2) Map out nearby planetary systems and understand the diversity of the worlds they contain; (3) Enable new explorations of astrophysical systems from our own solar system to external galaxies by extending our reach in the UV through near-IR. This Great Observatory science will be selected through a competed GO program, and will account for about 50% of the HabEx primary mission. The preferred HabEx architecture is a 4m, monolithic, off-axis telescope that is diffraction-limited at 0.4 microns and is in an L2 orbit. HabEx employs two starlight suppression systems: a coronagraph and a starshade, each with their own dedicated instrument.Comment: Full report: 498 pages. Executive Summary: 14 pages. More information about HabEx can be found here: https://www.jpl.nasa.gov/habex

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Transcriptional profiling of mouse projection neurons with VECTORseq.

Existing techniques for transcriptional profiling of projection neurons could be applied to only one neuronal population per experiment. To increase throughput, we developed VECTORseq, which repurposes retrogradely infecting viruses to deliver multiplexable RNA barcodes, enabling projection anatomy to be read out in single-cell datasets. In this protocol, we describe the delivery of viral barcodes to mouse brain to label different projection neurons. We then detail single-cell or nuclei isolation for sequencing, followed by the analysis of single-cell sequencing data. For complete details on the use and execution of this protocol, please refer to Cheung et al. (2021)

LETTERS A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans

Innate social behaviours emerge from neuronal circuits that interpret sensory information on the basis of an individual's own genotype, sex and experience. The regulated aggregation behaviour of the nematode Caenorhabditis elegans, a simple animal with only 302 neurons, is an attractive system to analyse these circuits. Wild social strains of C. elegans aggregate in the presence of specific sensory cues, but solitary strains do not Many naturally isolated social strains of C. elegans aggregate into feeding groups with dozens of animals, although other strains, including the laboratory strain N2, are solitary How NPR-1 acts to regulate behaviour is not well understood. A previous report using a genomic npr-1 fragment identified the oxygensensing URX neuron as a site of npr-1 action, but behavioural rescue was incomplete, with rescue of aggregation, partial rescue of bordering, and no rescue of rapid movement We next addressed whether RMG expression of npr-1 is sufficient to suppress aggregation. No RMG-specific promoter is known, so an intersectional strategy was developed to drive npr-1 expression only in cells that express both flp-21 and ncs-1, using Cre-mediated recombination between loxP sites that flanked transcriptional stop sequences. When ncs-1::nCre and flp-21::LoxStopLox::GFP strains were crossed together, the intersection between ncs-1 and flp-21 allowed strong and consistent GFP expression only in RMG and M2 pharyngeal neurons Mammalian neuropeptide Y receptors generally inhibit neurotransmitter release Inspection of the C. elegans wiring diagram 13 revealed that RMG is the hub of a gap-junction network connecting seven classes of neurons, including the oxygen-sensitive URX neurons and the nociceptive ASH and ADL neurons previously implicated in aggregation behaviou