Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

Summary

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Abstract

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species ^1,2. These intraspecies signals, the pheromones, as well as signals from some predators ³, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity ⁴. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR ^5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.

The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively ^6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli ^4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs ^3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations ^12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.

Sensory stimulation elicits strong Ca²⁺ influx in VSNs that is indicative of receptor activation ^4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs ^15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca²⁺ imaging experiments. This method has eliminated the dye loading process used in previous studies ^4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

Protocol

1. Solution preparation

1. Prepare 10X R1, 10X R2 and 10X R3 solutions according to the table.
   R1
   

   Chemicals	MW(g/mol)	mM (1X)	10X stock (g/L)
   NaCl	58.44	125	73.05
   KCl	74.55	2.5	1.86
   MgCl2	1 M stock	1	10 ml
   CaCl2·2H2O	147.02	2	2.94
   NaH....

Discussion

The majority of the vomeronasal receptors (VRs) remain as orphan receptors since their discovery by Dulac and Axel ⁵. The pheromone ligands for these chemosensory receptors and their roles in mediating animal behaviors are not well understood. Until now, only one pair of ligand/receptor, the ESP1 peptide and its cognate receptor, Vmn2r116 (V2Rp5), has been identified and shown to convey specific social information ^19,23. Another receptor, V1rb2, has been shown to respond to 2-heptanone, which presum.......