A novel high-speed sectioning fluorescence microscope reveals patterned neural responses to pheromones.

摘要

In mice, most of the sensory neurons for pheromone detection form a layered epithelium in a special compartment of the nasopharynx known as the vomeronasal organ (VNO). Each of these sensory neurons expresses a single pheromone receptor type out of approximately 200 possible choices from two distinct gene families (V1R and V2R). From the layered cell bodies of the VNO to their terminal projection sites in the two lobes of the accessory olfactory bulb (AOB), V1R and V2R-expressing neurons are segregated. Optical recording methods can take advantage of this spatial organization by associating stimulated activity changes with spatial location to decode receptor/stimulus interactions.;Mouse pheromone receptor neurons were selectively loaded with a calcium-sensitive fluorescent dye and optical microscopy was employed to record from large numbers of these cells during stimulation with the natural pheromones in urine. Calcium imaging was conducted on their cellular somas in the excised VNO neuroepithelium as well as their presynaptic terminals in the AOB in vivo. For recordings in excised VNO, a novel high-speed sectioning fluorescence microscope was built which simultaneously recorded from thousands of individual neurons in scanned three-dimensional volumes. Highly efficient photon collection to a CCD detector allowed long-term high-speed recordings with only limited photobleaching effects, making this instrument potentially valuable in a broad range of biological applications. In vivo recordings from the presynaptic terminals employed a wide-field, low-magnification fluorescence microscope with high numerical aperture and fast-switching LED-based illuminators, able to acquire images from the full extent of the labeled AOB.;Optical recordings captured spatial patterns of activity in sensory neurons stimulated by mouse urine pheromones. These pheromones activated cells from the apical regions of the VNO neuroepithelium and excited terminals in the rostral lobe of the AOB. Both activity patterns indicate that urine pheromones are predominately V1R receptor family ligands and point toward the interesting possibility that the two receptor families receive information from different sources. Future experiments utilizing single pheromone stimuli may uncover more refined patterns of activity, reveal differences in receptor affinities, and provide insights about the classes of chemicals detected by V1R and V2R family receptors.