Abstract
Purpose:
The synaptic ribbon is a key synaptic structure for many sensory neurons. In the hibernating ground squirrel retina, photoreceptor synaptic ribbons undergo drastic morphological changes. Thus, in hibernating tissues a large proportion of ribbon proteins disengage from the CAZ (cytomatrix at the active zone) and aggregate into a sphere that resides several microns above the CAZ. We set out to study the molecular and regulatory mechanisms of such adaptive changes.
Methods:
Retinal tissue slices were fixed and processed for immunocytochemistry. Slices were mounted and imaged in slice or flatmount orientations. Images were acquired with Zeiss LSM-510 META confocal microscope and edited with Photoshop CS2 (Adobe Systems). For EM, strips of superior retina were fixed in paraformaldehyde and glutaraldehyde. The tissues were post-fixed in osmium tetroxide, followed by uranyl acetate. EM was done on a JEOL 1010 operating at 80kV. Proteins were separated on SDS-PAGE and western blots were performed with specific antibodies. Free cytoplasmic and total NADH/NAD ratios were estimated by enzymatic assay and metabolomic analysis, respectively.
Results:
In hibernating tissues, immunostaining showed that bassoon largely remained at the CAZ whereas ribeye were detached, suggesting a disassociation between ribeye and bassoon. Co-immunoprecipitation (Co-IP) indicated the interaction between ribeye and bassoon in hibernating tissues is reduced to less than 50% of that in the awake tissues. Knockout of the NAD(H) binding site on RBD (ribeye b-domain) reduced its interaction with bassoon. In addition, increasing the cellular NADH/NAD+ ratio via sodium azide treatment (NaN3) reduced the interaction between ribeye and bassoon. Both free cytoplasmic NADH/NAD+ ratio by enzymatic assay and total NADH/NAD+ ratio by metabolomic analysis were increased in hibernating ground squirrel retina.
Conclusions:
These results suggest the interaction between ribeye and bassoon is reduced in ground squirrel retina during hibernation, which might be the underlying mechanism of the detachment of ribbon from the CAZs during hibernation and is regulated by NAD(H) binding and their ratio. Ribeye, a main structural component of the ribbon, may also act as a redox sensor, monitoring the changes in the metabolic status of the photoreceptor during hibernation and regulating the ribbon structure accordingly.
Keywords: 648 photoreceptors •
728 synapse •
650 plasticity