1. Structural changes of the photoreceptor ribbon synapse observed via confocal and transmission electron microscopy. In retina tissues from awake squirrels, about 20 to 25 ribbons cluster at active zones located at the bottom of each cone pedicle . In hibernating tissues, however, a large proportion of ribbon proteins disengage from the active zones and aggregate into a sphere that resides several microns above the active zone. Notably, there remains some faint labeling of ribeye at the active zones, indicating residual ribbon structures. To gather further details at the nanoscale resolution and to quantify the structural changes of photoreceptor ribbons under hibernating conditions, we utilized transmission electron microscope (EM). In concert with the confocal microscope images, the EM images show that ribbons at active zones are significantly reduced in size. Ribbon clusters away from the active zone are apparent, presumably corresponding to the large spheres observed in confocal images. To quantify the change in the amount of ribbon at active zones, we reconstructed the cone pedicles from hibernating (n=3) and awake (n=4) tissues with serial thin sections. The number of ribbons in each cone pedicle is reduced under hibernating conditions (240.8 vs. 162.0). For individual ribbons, the height of the ribbon is significantly reduced (144.424.5 vs. 66.711.0nm). The structure of the invagination and postsynaptic processes appear to be normal leading to the assessment that the plasticity is mostly presynaptic. Other presynaptic proteins examined include kinesin II, which has a similar re-distribution pattern as ribeye and Munc 13, which remains at the active zones. Another important presynaptic protein, bassoon, will be studied in the future. In hibernating tissues, postsynaptic receptors, both ionotropic and metabotropic (that are expressed on Off and On CBC dendrites respectively), have been examined via confocal microscopy and appear to be comparable to the awake tissues as expected from the EM images. Hence, the presynaptic ribbon is the major site of the photoreceptor synaptic plasticity during hibernation. 2. To assess the function of the photoreceptor ribbon synapse in the hibernating ground squirrel retina. To probe the function of the photoreceptor synapse under hibernating conditions, we measured the response from postsynaptic b2 Off CBCs to presynaptic cone depolarization. The fast AMPA receptor mediated EPSC provided a faithful readout of the kinetic properties of vesicle release from the photoreceptor synapse. In hibernating tissue, when depolarizing voltage steps were applied to cones, large EPSCs could still be elicited from b2 cells, even though immune-staining performed after the recording indicates that the labeling of ribeye at active zones is greatly reduced . Quantal miniature EPSCs in hibernating tissues were comparable in size and kinetics with those in awake tissues (the mean amplitude of mEPSCs is smaller in hibernating tissues, mostly due to fewer multivesicular events). To reveal more subtle functional changes of the photoreceptor ribbon synapse during hibernation, we measured two important features of a ribbon synapse, the size of the readily releasable pool (RRP) of vesicles and the rate of vesicle replenishment for the RRP. To estimate the RRP, we deconvolved the quantal miniature EPSC (mEPSC) from the EPSC waveform to acquire the release rate profile. The kinetics of the release rate profile was then used to isolate the transient component of the EPSC, the integral of which can be normalized to mEPSC integral to calculate RRP. An additional critical step was to reconstruct the synapse after recording and acquire the number of synaptic contacts in order to estimate RRP per synapse. Our results indicated that the RRP per ribbon in hibernating condition were significantly smaller than that of the awake condition (7.40.6, n=4 vs. 12.7 1.5, n=5 respectively). However, an alternative scenario could be that the RRP per ribbon is not changed in hibernating condition, rather some of the synaptic contacts lack ribbon. Currently, we cannot differentiate these two possibilities. Given that RRP is highly correlated with the length of the ribbon and our EM reconstruction revealed a decreased number of ribbons in each cone, the latter is possible. Nonetheless, an essential feature of the ribbon synapse in cone photoreceptors is that, regardless of variations among individual ribbons, the sum of the length of ribbons in each cone is highly consistent. Therefore, it is reasonable to conclude that the overall RRP of a cone synapse is reduced in hibernating conditions. To measure vesicle replenishment rate in hibernating tissues, we applied the pair-pulse paradigm, in which a series of double voltage pulses were given to a cone with the first pulse designed to deplete the RRP and the second pulse, with an increasing inter-pulse interval, to assess the fraction of RRP refilled. The time constant of the recovery curve gives an estimate of the rate of vesicle turnover for the RRP. We found roughly three fold decrease in the rate of vesicle replenishment of the RRP in hibernating tissues. This result implicates that reduced ribbon size, particularly in height;profoundly affects the vesicle refilling process of the RRP. It is conceivable that the upper portion of the ribbon is involved in vesicle recruitment, a necessary step before vesicles enter the RRP. 3. Summery. In this project, we found that synaptic ribbon, a key synaptic structure in photoreceptors shows remarkable plasticity during hibernation. By correlating functional changes with the structural alterations of the ribbon, we identified functional compartmentalization of the ribbon. The residual ribbon component in hibernating tissue may represent the basic functional compartment that provides essential synaptic functions such as docking and releasing of the readily releasable pool of vesicles. The ribbon component that is eradicated during hibernation functions to facilitate vesicle replenishment. This additional compartment speeds up the synapse, ensuring high frequency signaling at the photoreceptor synapse.
