Figure 1. Anatomy of the olfactory system. (a) Odours, chemosignals and pheromones are captured in two different divisions of the olfactory system, the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). The VNO is encapsulated by cartilage, contained within the intermaxillary bone, and is accessible to the external environment through the vomeronasal duct. The nasal passages of the MOE are comprised of turbinates that provide a large surface area for odorant binding and detection. OB, olfactory bulb, AOB, accessory olfactory bulb. (b) General, volatile odour molecules are detected in the MOE and this information is processed in the OB as the axons of the olfactory sensory neurons project across the cribriform plate into the brain. This information is then transmitted to the olfactory cortex, a higher brain region that includes the anterior olfactory nucleus (AON), the piriform cortex (PC), the olfactory tubercle (OT), the entorhinal cortex (EC) and the lateral cortical amygdala (LA). Species-specific pheromones and more specialized chemosignals are mostly detected in the VNO and this information is processed in the AOB and then propagates to the vomeronasal amygdala (VA) and then on to the hypothalamus (H) that controls endocrine and hormonal responses. Modified from Dulac and Torello (2003) Nature Publishing and Dr. Michael Meredith, Florida State University (FSU), Program in Neuroscience, VNO website, http://www.neuro.fsu.edu/research/vomeronasal/

Figure 2. Microscopic visualization of the vomeronasal organ (VNO) system. (a) Low- (left) and high- (right) magnification photomicrograph of a 10 m frozen coronal section of the VNO. Four prominent structures are visible in the high-magnification view as labelled: cartilage (C), vomeronasal axon bundles (A), vomeronasal epithelium (VNE), and the microvillar layer (M). Bars,=25 m. (b) Vomeronasal sensory neurons (VSNs) can be enzymatically dissociated from the VNE and these isolated neurons can be electrically stimulated with pheromones as in Figure 4. Visible in the photomicrograph are the filaments of the microvilli (M) that contain the pheromone transduction machinery (Figure 5), a long modified dendrite (De) to carry electrical signals to the cell body or soma (S), and a portion of the axon (Ax), which will propagate the electrical signals to the brain, specifically to the AOB. Bar,=10 m. (c) A coronal frozen section of the VNO labelled with a vital dye (rhodamine-conjugated dextran) that is introduced into one of the vomeronasal ducts to visualize the VSNs. Note that the contralateral VNE is not labelled. (d) The dye was permitted to migrate for approximately 2 weeks in the animal to visualize the first site of synaptic connection into the AOB at the level of the glomeruli (G). Photographs with permission from Murphy FA, Tucker K and Fadool DA (2001) Sexual dimorphism and developmental expression of signal-transduction machinery in the vomeronasal organ. Journal of Comparative Neurology. 432: 61–74. Wiley-Liss Inc. and Fadool DA, Wachowiak M and Brann JH (2001) Patch-clamp analysis of voltage-activated and chemically activated currents in the vomeronasal organ of Sternotherns odoratus (Stinkpot/musk turtle). Journal of Experimental Biology 204: 4199–4212. Company of Biologists Limited.

Figure 3. Segregation of VNO neural pathways in rodents. (a) Two classes of G-protein coupled receptors, V1R and V2R, have been identified as putative pheromone receptors in the mammalian VNO. In rodents, the G_i2 G protein is co-localized with pheromone receptors of the V1R family and the G_o G-protein is co-localized with receptors of the V2R family. The molecular structure of the pheromone molecule (purple diamond) is thought to be recognized by hypervariable transmembrane domains or a large extracellular N-terminus in the V1R and V2R class of receptors, respectively. (b) The soma of the VSNs containing the V2R/G_i2 combination (blue) are found in the basal region of the VNO and send their axonal projections to the posterior (P) portion of the AOB. In contrast, exhibiting anatomical and perhaps functional segregation, the V1R/G_o combination (yellow) are found in the apical region of the VNO and send their axonal projections to the anterior (A) portion of the AOB.

Figure 4. Patch-clamp electrophysiology in the VNO. Electrical currents elicited from isolated VSN (as in Figure 2a) can be monitored through a technique called patch-clamp recording. A recording electrode is fabricated from a glass capillary tube and an internal silver-chloride wire is connected to an apparatus to capture and record currents as ions flow across the lumen of an ion channel protein in the membrane of the VSN. A stimulating glass pipette is positioned in close proximity to the VSN to deliver one of six different pheromones to the neuron at the tip of the microvilli/dendritic knob. A typical current response evoked by pheromone but not application of control saline is shown here. Note that the electrophysiological equipment can measure a biological current response in the VSN on the scale of picoamperes (10^–12 A). For comparison, a light bulb operates in the milliampere range (10^–3 A).

Figure 5. Putative signal transduction machinery of the VNO. Pheromone molecules (purple diamond) are received at the external face of the VSN membrane by binding to a pheromone receptor that is a member of the seven transmembrane spanning G-protein coupled receptor (GPCR) family. The pheromone receptor undergoes a conformational change upon binding the pheromone and activates a G protein (green; either G_o or G_i2). The G protein in turn activates the effector enzyme phospholipase C (PLC) that acts to convert the substrate PIP₂ to two different second messengers, diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). There is evidence for the presence of three different ion channels that conduct calcium, namely the transient receptor potential channel (TRPC2), the type III IP₃ receptor (IP₃R3), and a nonselective cation channel that is activated by calcium (CaCN). The regulation and potential interaction of these three channels is currently under investigation to determine the source of the evoked electrical change in the VSN that encodes the information contained in the pheromone molecule. ER, endoplasmic reticulum; Homer, adaptor protein; CaM, calcium calmodulin; N,N-terminus; C,C-terminus. All of this transduction machinery is thought to reside at or in close proximity to the plasma membrane (mustard colour) of the microvilli.