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Vestibulo-Spinal Pathways in Tetrapods: Chapter - 6.26

The vestibulospinal system provides the spinal motor circuits controlling head/neck and limb movements and body posture with rapid reflex adjustments to maintain equilibrium and stability and with a continuous essential excitatory drive, called tonus, to enhance reactive responses to perturbations that force the animal off normal posture. The sensory signals to these reflex circuits originate from hair cells in the inner ear of otolith structures, namely the utricle and saccule, that transduce inertial acceleration and orientation of the head with respect to gravity and in the three orthogonally arranged semicircular canals that transduce angular head rotation. The principal vestibulospinal pathways are 1) the medial vestibulospinal tract that descends in the ventromedial funiculus and innervates inter- and motoneurons located mainly in lamina VII, VIII, and dorsomedial IX throughout the cervical segments; and 2) the lateral vestibulospinal tracts that course in the lateral to ventrolateral funiculi and are distinguished by two divisions: i) a cervical-projecting tract that overlaps many of the targets of medial vestibulospinal tract neurons including the motoneurons in ventromedial IX and also contributes to reflex control of shoulder and forelimb (arm) muscles; and ii) a lumbosacral-projecting tract that provides a rapid input to maintain stable posture and reflex control of the lower body. A striking observation in understanding the functional organization of this sensory-motor system is both that the driving sensory input can be dynamically modified by the behavioral context in which the sensation is made and that it remains able to quickly respond to an external force during self-generated head movements. The structural basis for vestibulospinal inputs to spinal motor control circuits in quadrupeds and bipeds rely in part on the animal's need for coordination between fore- and hind-limb reflex movements. Understanding the sensory-to-motor transformations in the diverse species rely on the correlations of the conserved and unique species behavior, morphology and physiologic function.

Spinal Cord↗

Novel Approaches to Cellular Transplantation from the US Space Program

Research in the treatment of type I diabetes is entering a new era that takes advantage of our knowledge in an ever increasing variety of scientific disciplines. Some may originate from very diverse sources, one of which is the Space Program at National Aeronautics and Space Administration (NASA). The Space Program contributes to diabetes-related research in several treatment modalities. As an ongoing effort for medical monitoring of personnel involved in space exploration activities NASA and the extramural scientific community investigate strategies for noninvasive estimation of blood glucose levels. Part of the effort in the space protein crystal growth program is high-resolution structural analysis insulin as a means to better understand the interaction with its receptor and with host immune components and as a basis for rational design of a "better" insulin molecule. The Space Program is also developing laser technology for potential early cataract detection as well as a noninvasive analyses for addressing preclinical diabetic retinopathy. Finally, NASA developed an exciting cell culture system that affords some unique advantages in the propagation and maintenance of mammalian cells in vitro. The cell culture system was originally designed to maintain cell suspensions with a minimum of hydrodynamic and mechanical sheer while awaiting launch into microgravity. Currently the commercially available NASA bioreactor (Synthecon, Inc., Houston, TX) is used as a research tool in basic and applied cell biology. In recent years there is continued strong interest in cellular transplantation as treatment for type I diabetes. The advantages are the potential for successful long-term amelioration and a minimum risk for morbidity in the event of rejection of the transplanted cells. The pathway to successful application of this strategy is accompanied by several substantial hurdles: (1) isolation and propagation of a suitable uniform donor cell population; (2) management of host immune rejection; (3) protection from the autoimmune component of the disease; and (4) anatomic placement of the engrafted cells that permits timely response to blood sugar levels as well as effective release and deployment of insulin. Bioreactor technology may provide some critical advances for surmounting some of these scientific hurdles. The NASA bioreactor is a horizontally rotating cylinder that is completely filled with culture medium. Gaseous exchange is maintained by a concentric cylinder of permeable silicon. In slow rotation (15-25 rpm) particles of small mass such as cells and tissue aggregates remain suspended in the rotating body of fluid. This novel approach suspends cells without stirring thus, allowing objects of different size mass to colocate and interact in a very low shear environment. In fact, analysis of the forces acting on individual cells in the rotating bioreactor reveals that the cells are continuously falling through the fluid medium. The conditions in the bioreactor permit assembly of cells into aggregates. three-dimensional tissue growth, synthesis of intercellular matrix,10 differentiation, and some sinusoid formations that may serve as a surrogate vasculature for larger tissue segments.

Pellis, Neal R.↗

Optimizing Single Nuclei Sequencing of Brain Samples From Space Flown Mice Across Age and Strain

The NASA GeneLab Sample Processing Laboratory offers high-throughput sequencing services to NASA-funded space biology researchers. Space biology studies have specific challenges such as low sample numbers, introducing susceptibility to batch effects from sample handling. These issues are compounded by complex protocols such as single-nuclei isolation and sequencing, which has recently become an attractive methodology for assessing the cellular diversity within spaceflight samples. High quality single-nuclei sequencing requires reproducible protocols to dissociate tissue and generate clean suspension of intact single nuclei. Producing single-nuclei suspension from brain tissue is particularly challenging due to cell type heterogeneity and the myelin sheath that carries over into the nuclei suspension as debris. Current procedures tend to be time consuming and sometimes include steps that can alter gene expression and create cell-type bias. Commercially available nuclei isolation kits, such as the 10X Genomics nuclei isolation kit, offers a streamlined way to process samples for nuclei isolation, thereby minimizing batch effects and enabling reproducibility. In this study, we report on the performance of the 10X Genomics nuclei isolation kit and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit to generate sequencing libraries from space-flown mouse brain samples. Single nuclei sequencing was performed on frozen mouse brain tissue from two spaceflight missions, Rodent Research-10 (RR-10) and RR Reference Mission-2 (RRRM-2). RR-10 mice were female B6129SF2/J, euthanized at 18-19 weeks whereas RRRM-2 mice were female C57BL/6NTac, euthanized at 20 or 37 weeks. Sequencing data was processed using standard GeneLab data processing pipelines. We report evaluation of the performance of the 10X Genomics nuclei isolation kit for spaceflight samples from mouse brain, and evaluation of reproducibility across different mouse strains and age groups. We also report preliminary scientific results including cell type inference, cell clustering, and differentially expressed genes and pathways between spaceflight and ground control samples.

RR-10↗

BLOOD-BASED MULTI-SCALE MODEL FOR CANCER RISK FROM GCR IN GENETICALLY DIVERSE POPULATIONS

OBJECTIVES AND METHODS This project addresses the challenge of understanding and predicting individual radiation sensitivity by integrating genetics, demographics and biomarker characteristics across species (mice and humans). We hypothesize that ex vivo DNA repair response to GCR components is a central determinant of cancer risk from space radiation and can serve as a biomarker of radiation risk in combination with genetics. Automated image quantification of 53BP1+ radiation-induced foci (RIF) during the first 4-48 h post-irradiation was performed as a function of dose and LET in non-immortalized primary skin fibroblasts derived from 76 mice across 15 strains (5 inbred reference strains and 10 collaborative-cross strains) exposed to X rays (0.1, 1 and 4 Gy), 350 MeV/n 40Ar and 600 MeV/n 56Fe (1.1 and 3 particles/100μm2), as well as in peripheral blood mononuclear cells (PBMCs) from 768 healthy donors (matched ethnicity, 50/50 male/female, 18-70 years old) exposed to gamma rays (0.1 and 1 Gy), 350 MeV/n 28Si, 350 MeV/n 40Ar and 600 MeV/n 56Fe (1.1 and 3 particles/100μm2). QUANTIFICATION OF 53BP1+ FOCI IN VITRO AND ASSOCIATIONS TO IN VIVO RADIATION SUSCEPTIBILITY IN 15 MOUSE STRAINS We reported in vitro repair kinetic and repairable fractions of RIF for the 15 mouse strains and introduced a mathematical model for RIF as a function of time, dose and LET. We noted that the metabolic activity of cells modulates the RIF response, and we introduced the open access tool terRIFic (Tool for Enhanced Results of RIF In Cells, https://radbiolab.shinyapps.io/terrific/) to correct for such bias using confluence level. Notably, at 4h post-irradiation, RIF/Gy decreased with dose or LET: as the dose or LET increases, so does the proximity of DNA double-strand-breaks (DSB) and our data suggest that proximal DSBs are brought together inside isolated RIF for repair. The RIF/Gy trend was inverted at 24h, suggesting RIF with high DSB content are more difficult to repair. We showed that in vitro metrics correlate with in vivo measurements in the same 15 mouse strains, such as survival levels of immune cells or spontaneous cancer incidence, suggesting a relationship between the efficiency of DSB repair and cancer risk or radiation toxicity. In addition to the efficiency of repair and persistent RIF, the amount of spontaneous foci before irradiation was also found to be strain dependent. Finally, we performed genome-wide association study in the same 15 mouse strains using all RIF phenotypes measured in vitro, identifying genes of interest and validating RIF as an ideal biomarker for individual radiation sensitivity. BASELINE 53BP1+ FOCI PREDICTS INDIVIDUAL HUMAN RESPONSE TO GCR COMPONENTS Based on the analysis of radiation responses of 576 donor PBMCs (using quantification of 53BP1+ foci, oxidative stress and cell death), we observed a wide variability of subject- and LET-dependent radiation responses, with radiation-induced DNA repair foci increasing with LET, though oxidative stress being notably reduced by high-LET irradiation, potentially due to a switch between hydrogen peroxide and oxygen radical-based mechanisms. We identified a relationship between few spontaneous DNA foci at baseline and increased DNA repair after irradiation, accompanied by an alteration in immunoregulatory cytokine secretion, which might be adapted as biomarkers to predict ionizing radiation sensitivity. Among demographic variables, only latent cytomegalovirus infection and age were predictive of high baseline foci formation. Finally, we have performed low-throughput whole genome sequencing of all samples and are currently in the process of identifying the genes and pathways associated with low and high-LET ionizing radiation sensitivity in humans.

53BP1↗

Three Paradigms of Lunar Regolith Evolution

Integration of diverse datasets on the Moon may render some paradigms of lunar science either better-defended or vulnerable. We will consider three paradigms commonly used for understanding the processes of lunar regolith evolution in light of new and accumulated data. Our premise is that all data-sets should converge to a single interpretation if a concept or model is to be accepted as a paradigm. If a convergence is lacking, the paradigm needs fresh scrutiny. SteadyState: Lunar regolith evolution is currently understood in terms of comminution, agglutination, and replenishment as described by McKay and coworkers). Briefly, the model envisages continued micrometeoritic bombardment to comminute exposed soil particles to finer sizes while continued agglutination consumes finer sizes to produce larger constructional particles. Eventually, a balance between these two opposing processes achieves a steady state; soils at steady state maintain their mean grain size (M(sub z)). Episodic higher-energy impacts excavate fresh coarse material from below the soil cover, disturb the steady state, and restart the process to achieve a new steady state. It follows that the thickness of the regolith at any site would control the frequency of replenishment; indeed, the thickness of the regolith at Apollo landing sites was predicted by McKay et al. from the average M(sub z) of local soils. However, replenishment may come also from disintegrating boulders and cobbles at the lunar surface, and rates of comminution and agglutination may depend on the properties of target material. Regression between M(sub z) and I(sub s)/Fe(sup 0) (a measure of maturity or total surface exposure) of Apollo soils at different sites shows the following relations and estimated M(sub z) at a high maturity of I(sub s)/Fe(sup 0)= 100. It is possible that Apollo 12 and 15 sites have the thickest regolith and the Apollo 16 site has the thinnest. It is also possible that Apollo 12 and 15 basalts are comminuted faster than Apollo 16 highland rocks and Apollo 14 and 17 soils are products of mixed parentage. If a soil becomes continually finer as it matures until agglutination catches up, and if comminution is differential-dependent on the physical properties of the constituents, then the composition of the bulk soil has to match the composition of some "fulcrum" grain size fraction, say X Grain size fractions >X and <X will complement each other; their mass balance is the bulk soil. It appears that the 10-20-micron size fraction may be the fulcrum. In general, trace-element chemistry and IR reflectance spectra of this size fraction are closest to that of the bulk soil, regardless of maturity that is surprising. Disaggregated products of regolith breccias may also show similar relationships. If the 10-20 gm is the fulcrum (i.e., X as above) for many soil properties (e.g., major element composition, FMR, solar-wind-implanted elements), then this may be the ultimate mean grain size of lunar soils at steady state. However, different properties of soils may find steady states at different grain size fractions. The steady state of solar-wind-implanted elements, on the other hand, will climb up the grain-size scale as agglutinates transfer surface-correlated components into volume correlated components until a saturation level is reached or the rates of replenishment and implantation become equal. The same will be the case with vapor-deposited reduced metals as they too are incorporated inside constructional particles. Properties that are directly affected by soil-maturation processes will thus have different pathways of achieving steady states. Maturity, i.e., cumulative surface exposure, of lunar soils is best quantified by the amount of nanophase superparamagnetic Fe(sup 0) (np-Fe(sup 0)) normalized to Fe content (=I(sub s)/Fe(sup 0). The majority consensus (paradigm?) for the production of np-Fe(sup 0) is associated with the production of agglutinates. Because large doses of solar-wind H are implanted in all lunar soils upon exposure, any melting (e.g., during agglutinate production) triggers a chemical reduction of Fe-bearing minerals resulting in np-Fe(sup 0) production. The quantity of np-Fe(sup 0) is thus dependent on melting events, (i.e., exposure), and limited by the Fe content of the soil. All freshly produced np-Fe(sup 0) resides in agglutinitic glass, as new TEM images show. Apparently, the correction procedure developed by Lucey et al. to estimate the Fe content of the lunar surface from IR-reflectance spectra depends on accepting the above. However, the process of producing np-Fe(sup 0) may be physical rather than chemical. All np-Fe(sup 0) could be deposits from a vapor produced by micrometeoritic impact on lunar soils. If metal-O bonds in target phases are broken, O being "most volatile" will escape leaving an O-deficient vapor to facilitate the production of np-Fe(sup 0). If so, the quantity of np-Fe(sup 0) is dependent on the vaporizing events, (i.e., exposure), and limited by the efficiency of breaking metal-O bonds and the escape of 0. To the extent that strengths of metal-O bonds are dependent on the local crystal field, production of np-Fe(sup 0) may be limited by the mineral composition of target soils and not by their total Fe content. According to this model, vapor-deposited np-Fe(sup 0) should be found at any retentive sites on lunar soil grains. Indeed, TEM images show np-Fe(sup 0) on plagioclase and ilmenite. Incorporation of such pre-irradiated np-Fe(sup 0)-bearing grains into agglutinates may account for eventual increased emplacement of np-Fe(sup 0) in agglutinates. Such a paradigm shift in understanding the origin of np-Fe(sup 0) will raise questions ranging from the unquestionable use of Is/FeO as the universal maturity parameter of lunar soils to global elemental maps of the Moon from remote-sensing data. Additional information is contained in the original.

Basu, A.↗