13-lined ground squirrels hibernate during winter season in order to survive the shortage of food supply. During hibernation, metabolic changes in the body affect the central nervous system profoundly. Our preliminary data indicated that in the hibernating ground squirrel retina, photoreceptor synaptic ribbons undergo drastic morphological changes. In each cone terminal, the ribbon structures fragment into small spheres, and form a large aggregate that resides several microns above the base of the photoreceptor terminals (shown by light microscopy and EM results). This form of synaptic ribbon plasticity provides an excellent model to study the function of the ribbon in synaptic transmission. By studying the functional changes of the ribbon synapse and correlating them with the structural changes of the ribbon synapse, we will be able to infer functions of the synaptic ribbon. In addition, since this is a form of adult plasticity, there are no confounding factors of developmental abnormalities that are commonly associated with models using genetically manipulated animals. To study the synaptic activities of the altered ribbon synapse, we applied direct whole-cell patch-clamp paired recordings on cone photoreceptors and their connected bipolar cells in the hibernating ground squirrel retina. We found, surprisingly, that large excitatory postsynaptic potentials (EPSCs) can be elicited from the postsynaptic bipolar cells by applying depolarizing voltage steps to the cone photoreceptors;even though immunocytochemical staining performed after the recording indicated that the labeling of synaptic ribbon was largely diminished. Compared to the control animals, the distribution of the spontaneous miniature EPSCs (mEPSCs) in hibernating tissues were skewed towards smaller events and reduced in frequency, but the kinetics was largely unchanged. The instantaneous vesicle release rate was calculated by deconvolving the mEPSC waveform from the EPSC waveform. The readily releasable pool (RRP) of vesicles at the hibernating cone ribbon synapse was estimated by integrating the transient component of the EPSC which were then divided by the number of synapses counted from the reconstructed synapses after recordings. The RRP calculated to be 7 vesicles per synapse. To assess the rate of vesicle replenishment, we estimated the recovery rate of the paired-pulse depression (PPD) to be 500 ms, which is slightly slower than the control condition. The ability of the vesicle turnover appeared to be temperature dependent. In accordance with the results from the synaptic responses, we found that the hibernating animals have relative normal flash ERG responses when measured at the room temperature, indicating that the cone - bipolar synaptic responses are transmitted successfully through the retina (there are significant changes in cone flick ERG that will be studied in more detail). By correlating ribbon structure with the synaptic activity, we conclude that at the photoreceptor ribbon synapse, synaptic transmission can occur with ribbons significantly reduced in size. The residual ribbons in the hibernating ground squirrel retina represent the basic operational unit of the ribbon synapse. A potential function of synaptic ribbon may be to facilitate highly synchronized release in forms such as muti-vesicular release or compound fusion. This will be the focus of our future work.
