Chemoaffinity in topographic mapping revisited--is it more about fiber-fiber than fiber-target interactions?

Axonal projections between two populations of neurons, which preserve neighborhood relationships, are called topographic. They are ubiquitous in the brain. The development of the retinotectal projection, mapping the retinal output onto the roof of the midbrain, has been studied for decades as a model system. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets. In addition, however, abundant evidence has been gathered mainly in the 1970s and 80s that the mapping can adjust to variegated targets with stunning flexibility demonstrating the extraordinary robustness of the guidance process. The identification of ephrins and Eph-receptors as the underlying molecular cues has mostly been interpreted as supporting the fiber-target chemoaffinity hypothesis, while the evidence on mapping robustness has largely been neglected. By having a fresh look on the old data, we expound that they indicate, in addition to fiber-target chemoaffinity, the existence of a second autonomous guidance influence, which we call fiber-fiber chemoaffinity. Classical in vitro observations suggest both influences be composed of opposing monofunctional guidance activities. Based on the molecular evidence, we propose that those might be ephrin/Eph forward and reverse signaling, not only in fiber-target but also in fiber-fiber interactions. In fact, computational models based on this assumption can reconcile the seemingly conflicting findings on rigid and flexible topographic mapping. Supporting the suggested parsimonious and powerful mechanism, they contribute to an understanding of the evolutionary success of robust topographic mass wiring of axons.

Practical limitations of superresolution imaging due to conventional sample preparation revealed by a direct comparison of CLSM, SIM and dSTORM.

We evaluate the suitability of conventional sample preparation and labelling methods for two superresolution techniques, structured illumination microscopy and direct stochastic optical reconstruction microscopy, by a comparison to established confocal laser scanning microscopy. We show that SIM is compatible with standard fixation procedures and immunofluorescence labelling protocols and improves resolution by a factor of two compared to confocal laser scanning microscopy. With direct stochastic optical reconstruction microscopy, fluorophores can theoretically be localized with much higher precision. However, in practice, with indirect immunofluorescence labelling density can be insufficient due to the bulky probes to reveal biological structures with high resolution. Fine structures like single actin fibres are in fact resolved with direct stochastic optical reconstruction microscopy when using small affinity probes, but require proper adjustment of the fixation protocol. Finally, by a direct comparison of immunofluorescent and genetic labelling with fluorescent proteins, we show that target morphology in direct stochastic optical reconstruction microscopy data sets can differ significantly depending on the labelling method and the molecular environment of the target.

Tools for efficient analysis of neurons in a 3D reference atlas of whole mouse spinal cord.

To fill the prevailing gap in methodology for whole spinal cord (SC) analysis, we have (1) designed scaffolds (SpineRacks) that facilitate efficient and ordered cryo-sectioning of the entire SC in a single block, (2) constructed a 3D reference atlas of adult mouse SC, and (3) developed software (SpinalJ) to register images of sections and for standardized analysis of cells and projections in atlas space. We have verified mapping accuracies for known neurons and demonstrated the usefulness of this platform to reveal unknown neuronal distributions. Together, these tools provide high-throughput analyses of whole mouse SC and enable direct comparison of 3D spatial information between animals and studies.

SpineRacks and SpinalJ for efficient analysis of neurons in a 3D reference atlas of the mouse spinal cord.

Spatial analysis of spinal neurons is currently limited by a lack of tools for efficient preparation and imaging of the whole spinal cord (SC) and the absence of a 3D reference atlas. Here, we describe protocols for efficient sectioning of whole SC using SpineRacks and subsequent image registration, atlas mapping, and 3D analysis of cells and projections, using SpinalJ. Together, these tools enable high-throughput analyses of adult mouse SC and direct comparison of spatial information of neurons between animals and studies. For complete details on the use and execution of this protocol, please refer to Fiederling et al. (2021).

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance.

Genetic hardwiring during brain development provides computational architectures for innate neuronal processing. Thus, the paradigmatic chick retinotectal projection, due to its neighborhood preserving, topographic organization, establishes millions of parallel channels for incremental visual field analysis. Retinal axons receive targeting information from quantitative guidance cue gradients. Surprisingly, novel adaptation assays demonstrate that retinal growth cones robustly adapt towards ephrin-A/EphA forward and reverse signals, which provide the major mapping cues. Computational modeling suggests that topographic accuracy and adaptability, though seemingly incompatible, could be reconciled by a novel mechanism of coupled adaptation of signaling channels. Experimentally, we find such 'co-adaptation' in retinal growth cones specifically for ephrin-A/EphA signaling. Co-adaptation involves trafficking of unliganded sensors between the surface membrane and recycling endosomes, and is presumably triggered by changes in the lipid composition of membrane microdomains. We propose that co-adaptative desensitization eventually relies on guidance sensor translocation into cis-signaling endosomes to outbalance repulsive trans-signaling.