Biology subjects

Baldwin, P. R.

Publications and source records attributed to Baldwin, P. R..

4 recordsLinked to original sources

bioRxiv2025

Primary cortical neurons form calcium-phosphate sheets with a bone-precursor-like ultrastructure

Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimers disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 DIV. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntingtons disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimers disease.

neuroscience↗

bioRxiv2023

TEMPORAL PATTERN OF SYNAPTIC ACTIVATION DIFFERENTIALLY AFFECTS PLASTICITY IN NORMAL AND INJURED BRAIN

Neurostimulation protocols are increasingly used as therapeutic interventions, including for brain injury. In addition to the direct activation of neurons, these stimulation protocols are also likely to have downstream effects on those neurons synaptic outputs. It is well known that alterations in the strength of synaptic connections (long-term potentiation, LTP; long-term depression, LTD) are sensitive to the frequency of stimulation used for induction, however little is known about the contribution of the temporal pattern of stimulation to the downstream synaptic plasticity that may be induced by neurostimulation in the injured brain. We explored interactions of the temporal pattern and frequency of neurostimulation in the normal cerebral cortex and after mild traumatic brain injury (mTBI), to inform therapies to strengthen or weaken neural circuits in injured brains, as well as to better understand the role of these factors in normal brain plasticity. Whole-cell (WC) patch-clamp recordings of evoked postsynaptic potentials (PSPs) in individual neurons, as well as field potential (FP) recordings, were made from layer 2/3 of visual cortex in response to stimulation of layer 4, in acute slices from control (naive), sham operated, and mTBI rats. We compared synaptic plasticity induced by different stimulation protocols, each consisting of a specific frequency (1 Hz, 10 Hz, or 100 Hz), continuity (continuous or discontinuous), and temporal pattern (perfectly regular, slightly irregular, or highly irregular). At the individual neuron level, dramatic differences in plasticity outcome occurred when the highly irregular stimulation protocol was used at 1 Hz or 10 Hz, producing an overall LTD in controls and shams, but a robust overall LTP after mTBI. Consistent with the individual neuron results, the plasticity outcomes for simultaneous FP recordings were similar, indicative of our results generalizing to a larger scale synaptic network than can be sampled by individual WC recordings alone. In addition to the differences in plasticity outcome between control (naive or sham) and injured brains, the dynamics of the changes in synaptic responses that developed during stimulation were predictive of the final plasticity outcome. Our results demonstrate that the temporal pattern of stimulation plays a role in the polarity and magnitude of synaptic plasticity induced in the cerebral cortex while highlighting differences between normal and injured brain responses. Moreover, these results may be useful for optimization of neurostimulation therapies to treat mTBI and other brain disorders, in addition to providing new insights into downstream plasticity signaling mechanisms in the normal brain.

neuroscience↗

bioRxiv2023

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60{degrees} are virtually indistinguishable. These strategies allowed the reconstruction to 3 [A] resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

biophysics↗

bioRxiv2020

Tools for Visualizing and Analyzing Fourier Space Sampling in Cryo-EM

A complete understanding of how an orientation distribution contributes to a cryo-EM reconstruction remains lacking. It is necessary to begin critically assessing the set of views to gain an understanding of its effect on experimental reconstructions. Toward that end, we recently suggested that the type of orientation distribution may alter resolution measures in a systematic manner. We introduced the sampling compensation factor (SCF), which incorporates how the collection geometry might change the spectral signal-to-noise ratio (SSNR), irrespective of the other experimental aspects. We show here that knowledge of the sampling restricted to spherical surfaces of sufficiently large radii in Fourier space is equivalent to knowledge of the set of projection views. Moreover, the SCF geometrical factor may be calculated from one such surface. To aid cryo-EM researchers, we developed a graphical user interface (GUI) tool that evaluates experimental orientation distributions. The GUI returns plots of projection directions, sampling constrained to the surface of a sphere, the SCF value, the fraction of the empty region of Fourier space, and a histogram of the sampling values over the points on a sphere. Finally, a fixed tilt angle may be incorporated to determine how tilting the grid during collection may improve the distribution of views and Fourier space sampling. We advocate this simple conception of sampling and the use of such tools as a complement to the distribution of views to capture the different aspects of the effect of projection directions on cryo-EM reconstructions.

biophysics↗