Reporting reproducible imaging protocols.

A reproducible imaging protocol should include four main detailed sections. The first should describe the sample preparation and include details about the tissue and/or cell culture preparation, the staining procedure, the optical grade of the coverslip, and the type of mounting media used to mount the sample. The second section should describe the configuration and components of the microscope and include the type of stand, stage, illumination, and detector, as well as the emission (EM) and excitation (EX) filters, objective, and immersion medium specifications. Specialized microscopes may have other important components in the optical path to include. The third section should describe the settings used to acquire an image like the exposure and/or dwell time, final magnification and optical resolution, the pixel and field of view (FOV) sizes, time intervals for any time lapse, total power at the objective (i.e., directed at your sample) and number of planes and step size used to collect a 3-dimensional image, and order of operations used in multi-dimensional image acquisitions. The final section should include details about the image analysis workflow such as the image processing steps, segmentation and measurement methods used to extract information from the image, data size, and necessary computing hardware and networking requirements if data sets are >1 GB, as well as citations and versions for the software and code used to perform any of these steps. Every effort should be made to make an example dataset with accurate metadata available online. Finally, specifics about the type of replicates included in the experiment and details about the statistical analysis conducted are also necessary.

Reopening During the Unprecedented: The Association of Biomolecular Resource Facilities Community Coronavirus Disease 2019 Pandemic Response. Part 2: Efforts to Effectively Ramp Up Core Facility Activities.

Shared research resources, also known as core facilities, serve a crucial role in supporting research, training, and other needs for their respective institutions. In response to the coronavirus disease (COVID-19) pandemic, all but the most critical laboratory research was halted in many institutions around the world. The Association of Biomolecular Resource Facilities conducted 2 surveys to understand and document institutional responses to the COVID-19 pandemic from core facility perspectives. The first survey was focused on initial pandemic response and efforts to sustainably ramp down core facility operations. The second survey, which is the subject of this study, focused on understanding the approaches taken to ramp up core facility operations after these ramp-down procedures. The survey results revealed that many cores remained active during the ramp-down, performing essential COVID-19 research, and had a more coordinated institutional response for ramping up research as a whole. The lessons gained from this survey will be indexed to serve as a resource for the core facility community to understand, plan, and mitigate risk and disruptions in the event of future disasters.

Melanopsin expression in the cornea.

A unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA in a subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor.

Concurrent Imaging of Receptor Trafficking and Calcium Dynamics by Spinning Disk Confocal Microscopy.

Synaptic activity is modulated by the activation of neuromodulator receptors present in dendrites of neurons. The majority of neuromodulator receptors are G protein coupled receptors (GPCRs), in which membrane trafficking regulates their activities. Membrane trafficking of neuromodulator receptors and their signaling occurs on a rapid time scale and emerging studies indicate that neuromodulator receptors function not just from the plasma membrane but also from the endocytic compartments. Here, we describe a live cell imaging approach using spinning disk confocal microscopy to investigate the effect of neuromodulator receptor activation on synaptic activity by measuring calcium dynamics in primary rat striatal neurons. The advantages of spinning disk confocal microscopy and recent improvements in the genetically encoded calcium sensor, GCaMP6, provide an imaging approach to image both the receptor membrane trafficking to endocytic compartments, and calcium dynamics at a high spatial and temporal resolution. We believe this approach of imaging both the neuromodulator receptor membrane trafficking and synaptic activity using GCaMP6 is a powerful tool to address many questions regarding possible roles of membrane trafficking of neuromodulator receptors in synaptic activity.

Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus.

To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.

An S-opsin knock-in mouse (F81Y) reveals a role for the native ligand 11-cis-retinal in cone opsin biosynthesis.

In absence of their natural ligand, 11-cis-retinal, cone opsin G-protein-coupled receptors fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knock-in mutation respond to light with maximal sensitivity red-shifted from 360 to 420 nm, consistent with an altered interaction between the apoprotein and ligand, 11-cis-retinal. However, cones expressing F81Y S-opsin showed an ∼3-fold reduced absolute sensitivity that was associated with a corresponding reduction in S-opsin protein expression. The reduced S-opsin expression did not arise from decreased S-opsin mRNA or cone degeneration, but rather from enhanced endoplasmic reticulum (ER)-associated degradation of the nascent protein. Exogenously increased 11-cis-retinal restored F81Y S-opsin protein expression to normal levels, suggesting that ligand binding in the ER facilitates proper folding. Immunohistochemistry and electron microscopy of normal retinas showed that Mueller cells, which synthesize a precursor of 11-cis-retinal, are closely adjoined to the cone ER, so they could deliver the ligand to the site of opsin synthesis. Together, these results suggest that the binding of 11-cis-retinal in the ER is important for normal folding during cone opsin biosynthesis.

What are the Effects of Severe Visual Impairment on the Cortical Organization and Connectivity of Primary Visual Cortex?

The organization and connections of the primary visual area (V1) were examined in mice that lacked functional rods (Gnat-/-), but had normal cone function. Because mice are nocturnal and rely almost exclusively on rod vision for normal behaviors, the Gnat-/- mice used in the present study are considered functionally blind. Our goal was to determine if visual cortex is reorganized in these mice, and to examine the neuroanatomical connections that may subserve reorganization. We found that most neurons in V1 responded to auditory, or some combination of auditory, somatosensory, and/or visual stimulation. We also determined that cortical connections of V1 in Gnat-/- mice were similar to those in normal animals, but even in normal animals, there is sparse input from auditory cortex (AC) to V1. An important observation was that most of the subcortical inputs to V1 were from thalamic nuclei that normally project to V1 such as the lateral geniculate (LG), lateral posterior (LP), and lateral dorsal (LD) nuclei. However, V1 also received some abnormal subcortical inputs from the anterior thalamic nuclei, the ventral posterior, the ventral lateral and the posterior nuclei. While the vision generated from the small number of cones appears to be sufficient to maintain most of the patterns of normal connectivity, the sparse abnormal thalamic inputs to VI, existing inputs from AC, and possibly abnormal inputs to LG and LP may be responsible for generating the alterations in the functional organization of V1.

Genetic and epigenetic contributions to the cortical phenotype in mammals.

One aspect of cortical organization, cortical field size, is variable both within and across species. The observed variability arises from a variety of sources, including genes intrinsic to the neocortex and a number of extrinsic and epigenetic factors. Genes intrinsic to the cortex are directly involved in the development and specification of cortical fields and are regulated from both signaling centers located outside of the neocortex, which secrete diffusible molecules, and the expression of transcription factors within the neocortex. In addition, extrinsic factors such as the type, location and density of sensory receptor arrays and how these receptor arrays are utilized, are also strongly related to cortical field size. Epigenetic factors including the relative activity patterns generated by the different types of physical stimuli in a given environment also contribute to differences in cortical organization, including cortical field size. Since both genetic and epigenetic factors contribute to cortical organization, some aspects of the cortical phenotype evolve, while other aspects of the cortical phenotype persist only if the environment in which an individual develops is relatively stable.

Retrograde tracing with recombinant rabies virus reveals correlations between projection targets and dendritic architecture in layer 5 of mouse barrel cortex.

A recombinant rabies virus was used as a retrograde tracer to allow complete filling of the axonal and dendritic arbors of identified projection neurons in layer 5 of mouse primary somatosensory cortex (S1) in vivo. Previous studies have distinguished three types of layer 5 pyramids in S1: tall-tufted, tall-simple, and short. Layer 5 pyramidal neurons were retrogradely labeled from several known targets: contralateral S1, superior colliculus, and thalamus. The complete dendritic arbors of labeled cells were reconstructed to allow for unambiguous classification of cell type. We confirmed that the tall-tufted pyramids project to the superior colliculus and thalamus and that short layer 5 pyramidal neurons project to contralateral cortex, as previously described. We found that tall-simple pyramidal neurons contribute to corticocortical connections. Axonal reconstructions show that corticocortical projection neurons have a large superficial axonal arborization locally, while the subcortically projecting neurons limit axonal arbors to the deep layers. Furthermore, reconstructions of local axons suggest that tall-simple cell axons have extensive lateral spread while those of the short pyramids are more columnar. These differences were revealed by the ability to completely label dendritic and axonal arbors in vivo and have not been apparent in previous studies using labeling in brain slices.

Development of layer-specific axonal arborizations in mouse primary somatosensory cortex.

In the developing neocortex, pyramidal neurons use molecular cues to form axonal arbors selectively in the correct layers. Despite the utility of mice for molecular and genetic studies, little work has been done on the development of layer-specific axonal arborizations of pyramidal neurons in mice. We intracellularly labeled and reconstructed the axons of layer 2/3 and layer 5 pyramidal neurons in slices of primary somatosensory cortex from C57Bl6 mice on postnatal days 7-21. For all neurons studied, the development of the axonal arborizations in mice follows a pattern similar to that seen in other species; laminar specificity of the earliest axonal branches is similar to that of mature animals. At P7, pyramidal neurons are very simple, having only a main descending axon and few primary branches. Between P7 and P10, there is a large increase in the total number of axonal branches, and axons continue to increase in complexity and total length from P10 to P21. Unlike observations in ferrets, cats, and monkeys, two types of layer 2/3 pyramidal neurons are present in both mature and developing mice; cells in superficial layer 2/3 lack axonal arbors in layer 4, and cells close to the layer 4 border have substantial axonal arbors within layer 4. We also describe axonal and dendritic arborization patterns of three pyramidal cell types in layer 5. The axons of tall-tufted layer 5 pyramidal neurons arborize almost exclusively within deep layers while tall-simple, and short layer 5 pyramidal neurons also project axons to superficial layers.

RORalpha coordinates reciprocal signaling in cerebellar development through sonic hedgehog and calcium-dependent pathways.

The cerebellum provides an excellent system for understanding how afferent and target neurons coordinate sequential intercellular signals and cell-autonomous genetic programs in development. Mutations in the orphan nuclear receptor RORalpha block Purkinje cell differentiation with a secondary loss of afferent granule cells. We show that early transcriptional targets of RORalpha include both mitogenic signals for afferent progenitors and signal transduction genes required to process their subsequent synaptic input. RORalpha acts through recruitment of gene-specific sets of transcriptional cofactors, including beta-catenin, p300, and Tip60, but appears independent of CBP. One target promoter is Sonic hedgehog, and recombinant Sonic hedgehog restores granule precursor proliferation in RORalpha-deficient cerebellum. Our results suggest a link between RORalpha and beta-catenin pathways, confirm that a nuclear receptor employs distinct coactivator complexes at different target genes, and provide a logic for early RORalpha expression in coordinating expression of genes required for reciprocal signals in cerebellar development.