Abstract
Purpose :
The recent development of novel optical methods such as genetically encoded calcium indicators has provided an invaluable opportunity to directly assess nociceptive TRP channels function at the nerve terminals. The aim of the present study was to optically record menthol evoked calcium signals at TRPM8+ corneal nerve endings in combination with the obtention of 3D confocal images of their morphological structure in vivo.
Methods :
Stereotaxic surgery was performed in 5-6 weeks old mice. Two viral vectors were combined to express the fluorophores GCaMP6s and TurboRFP in trigeminal ganglion neurons. Then, selective stimulation of the nerve terminal was performed by applying to a micropipette placed in the proximity of the focused nerve terminal pressure pulses that released a small amount of 1 mM menthol solution or its vehicle. Afterwards the calcium dynamics were recorded at 200 Hz with a high-speed camera (RedShirt camera). Recorded nerve endings and their fibers were imaged in high resolution using confocal microscopy. Then, a 3D reconstruction of the full morphology was obtained using Imaris software.
Results :
Reconstruction of several corneal nerve fibers and their terminals are shown in figure A. From the original confocal image (left) a 3D reconstruction was done (middle and right). (B) Example of the GCaMP6 fluorescence time course in response to menthol application (blue trace) or vehicle (black trace) recorded at the nerve ending highlighted in blue in A. (C) GCaMP6 fluorescence transient increases in response to menthol or its vehicle (n=13; p<0.01).
Conclusions :
This work shows for the first time the measurement of calcium signals at an individual cold thermosensitive nerve terminal in vivo along with the acquisition of images of its morphological structure in 3D, thus opening the door to the study of the relationship between the nerve terminal morphology and its function in health and disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.