The 13-lined ground squirrel hibernates in cold temperatures, providing an attractive model for studying physiological adaptations that occur to cope with metabolically challenging conditions. One such adaptation can be found in the retina, where cone synaptic ribbons undergo structural plasticity. During hibernation, the majority of ribeye proteins dissociate from the synaptic terminal and bundle together higher in the synaptic pedicle, leaving a much smaller ribbon in its place. Previous work from our lab has shown that changes in synaptic function accompany these structural changes. Specifically, while the size of evoked EPSCs in the postsynaptic OFF bipolar cell are normal, recovery of this full response is approximately 3x slower in hibernating retina than in awake retina. Additionally, the frequency of spontaneous EPSCs in bipolar cells is approximately 10x lower in hibernating retina. To determine whether these functional changes can be explained solely by the change in ribbon structure, we constructed a computer model of vesicle diffusion on and near the ribbon. This model is based upon TEM-derived measurements of ribbon geometry in both awake and hibernating cones. Basic physics principles were used to calculate the interactions of vesicles with one another, the ribbon, and the presynaptic membrane. Published measurements of vesicle diameter, density, and diffusion statistics were used to constrain vesicle motion. Importantly, simulations that reproduced experimental conditions could not reproduce the reductions in either spontaneous release or recovery by shortening the synaptic ribbon. Additional modifications to the model were necessary to reproduce the observed impairments, providing two predictions about ribbon function: First, to produce the hibernating-related slowing of recovery of the readily-releasable pool (RRP) of vesicles, a rate-limiting preparation step was imposed upon ribbon-attached vesicles prior to docking. Second, to produce the reduced frequency of spontaneous fusion, a second source of vesicle fusion was required that was not associated with the RRP. We hypothesized that spontaneous fusion of those ribbon-attached vesicles that have not yet entered the RRP could provide such a source. Endowing the model with this feature reproduced the observed hibernating-induced reduction in the rate of spontaneous fusion. Thus, these predictions derived from model simulations provide two separate hypotheses about cone ribbon function that can be tested by further experiments. In addition, we have acquired RNAseq data sets from retinas of awake and hibernating animals. We used Ensembl Gene 78/ Ictidomys tridecemlineatus spetri2 as our annotation database and performed gene set enrichment analysis (GSEA). For 17761 genes identified, pairwise correlation of 10 RNA seq data sets showed good correlation among samples, and principle component analysis (PCA) indicated wide separation between awake and hibernating conditions. We are currently trying to identity genes/pathways of interest for further analysis and verification. In conjunction with other systems biology approaches, this will provide guidance for our future experiments to investigate adaptive changes of the retina during hibernation.
