Hernandez-Varas, P. | bioRxiv Science

bioRxiv2023

Spinal inhibitory neurons degenerate before motor neurons and excitatory neurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of somatic motor neurons. Major focus has been directed to motor neuron intrinsic properties as a cause for degeneration while less attention has been given to the contribution of spinal interneurons. In the present work, we applied multiplexing detection of transcripts and machine learning-based image analysis to investigate the fate of multiple spinal interneuron populations during ALS progression in the SOD1G93A mouse model. The analysis showed that spinal inhibitory interneurons are affected early in disease, before motor neurons death, and are characterized by a slow progressive degeneration, while excitatory interneurons are affected later with a steep progression. Moreover, we report differential vulnerability within inhibitory and excitatory subpopulations, with interneurons directly projecting onto motor neurons being preferentially affected. Our study reveals a strong interneuron involvement in ALS development with interneuron specific degeneration. This points to differential involvement of diverse spinal neuronal circuits that eventually may be determining for motor neuron degeneration. TeaserA new approach to study the motor neuron disorder Amyotrophic Lateral Sclerosis shows the early and differential dysregulation of spinal interneurons.

neuroscience↗

bioRxiv2019

High photon count rate super-resolution fluorescence fluctuation spectroscopy

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of up to 40 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 M. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

biophysics↗