Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

Friederike Steenken; Sonny Bovee; Christine Köppl

doi:10.3791/63874

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Method Article

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

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DOI:

10.3791/63874

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April 21st, 2022

Friederike Steenken¹ , Sonny Bovee¹ , Christine Köppl¹

¹Cluster of Excellence "Hearing4all" and Research Centre Neurosensory Science, Department of Neuroscience, School of Medicine and Health Science, Carl von Ossietzky University Oldenburg

Summary

A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

Abstract

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The most common method for detecting the loss of ribbon synapses is immunolabeling because it allows for quantitative sampling from several tonotopic locations in an individual cochlea. However, the structures of interest are buried deep inside the bony cochlea. Gerbils are used as an animal model for age-related hearing loss. Here, routine protocols for fixation, immunolabeling gerbil cochlear whole mounts, confocal imaging, and quantifying ribbon synapse numbers and volumes are described. Furthermore, the particular challenges associated with obtaining good material from valuable aging individuals are highlighted.

Gerbils are euthanized and either perfused cardiovascularly, or their tympanic bullae are carefully dissected out of the skull. The cochleae are opened at the apex and base and directly transferred to the fixative. Irrespective of the initial method, the cochleae are postfixed and subsequently decalcified. The tissue is then labeled with primary antibodies against pre- and postsynaptic structures and hair cells. Next, the cochleae are incubated with secondary fluorescence-tagged antibodies that are specific against their respective primary ones. The cochleae of aged gerbils are then treated with an autofluorescence quencher to reduce the typically substantial background fluorescence of older animals' tissues.

Finally, cochleae are dissected into 6-11 segments. The entire cochlear length is reconstructed such that specific cochlear locations can be reliably determined between individuals. Confocal image stacks, acquired sequentially, help visualize hair cells and synapses at the chosen locations. The confocal stacks are deconvolved, and the synapses are either counted manually using ImageJ, or more extensive quantification of synaptic structures is carried out with image analysis procedures custom-written in Matlab.

Introduction

Age-related hearing loss is one of the world's most prevalent diseases that affects more than one-third of the world's population aged 65 years and older¹. The underlying causes are still under debate and actively being investigated but may include the loss of the specialized synapses connecting inner hair cells (IHCs) with afferent auditory nerve fibers². These ribbon synapses comprise a presynaptic structure that has vesicles filled with the neurotransmitter glutamate tethered to it, as well as postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors³