Abstract
Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders.
Highlights
Recent advances in stem cell biology have overturned the long-held belief that neurons do not regenerate
Samples obtained at different developmental stages of B6J, rd1, and rd1 after miPSC-retina transplantation were co-stained for pre- and post-synaptic markers, Ctbp2 (RIBEYE) and Metabotropic glutamate receptor type 6 (mGluR6) (Figure 1A)
We have successfully evaluated developmental synaptogenesis of the wildtype B6J mouse retina, the degenerative process of the rd1 mouse retina, and regenerative synaptogenesis of the miPSC-retina after transplantation
Summary
Recent advances in stem cell biology have overturned the long-held belief that neurons do not regenerate. It has been established beyond doubt that neural networks can be reconstructed after injury or degeneration either by endogenous regeneration (Jorstad et al, 2017; Yao et al, 2018) or by cell or tissue transplantation (Singh et al, 2013; Barnea-Cramer et al, 2016; Mandai et al, 2017). The gold standard to assess the state of a synapse is by electron microscopy (EM), where subcellular pre- and post-synaptic components can be directly observed (Geinisman et al, 1996). Visualization of pre- and post-synaptic markers by immunohistochemistry (IHC) allows for a robust and high throughput analysis, while simultaneously obtaining some qualitative information
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