1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

309 Poster Chemosensory Molecular Genetics andVNO/PheromoneREGULATION OF THE VNO BY A MOLECULAR CLOCKKatz R. 1 , Firestein S. 2 1 Center for Neurobiology and Behavior,Columbia University, New York, NY; 2 Dept. of Biological Sciences,Columbia University, New York, NYBehavior and physiology coordinated to the 24-hour day aremaintained through an internal timing mechanism. Time is maintainedby a molecular clock driven by recurrent rhythms in RNA and proteinlevels through transcriptional feedback loops of “clock genes”.Rhythmic expression of clock genes in peripheral tissues drives localrhythms. The rhythmic expression of clock genes and their role in theVomeronasal Organ (VNO) are examined in this study. Initially weidentified the expression profile of Per1 and Per2 in the VNO and foundthat these clock proteins are expressed by Vomeronasal SensoryNeurons. We examined their temporal expression profile, as well as theclock gene Bmal1, using Real Time PCR. The rhythmic expression ofBmal1 mRNA occurred with a nadir in the early evening or subjectivenight and a peak in the early morning or subjective day. In an opposingrhythm the expression of Per1 and Per2 mRNA occurred with a nadir inthe early morning or subjective day and a peak in the early evening orsubjective night. The rhythmic expression of clock genes identifies theVNO as the locus of a peripheral clock. We further evaluated the effectof a peripheral clock on pheromone sensitivity. A urine preferenceassay revealed a diurnal rhythm in pheromone sensitivity. These resultsindicate the importance of VNO oscillators in mediating daily rhythmsin pheromone detection and in behavior. Support Contributed By:NIDCD311 Poster Chemosensory Molecular Genetics andVNO/PheromoneARACHIDONIC ACID PLAYS A ROLE IN THE ODORRESPONSES OF MOUSE VOMERONASAL NEURONSZhang P. 1 , Delay R. 1 1 Biology Department, University of Vermont,Burlington, VTMouse vomeronasal neurons detect pheromones, as well as somegeneral odorants. The odor responses appear to be transduced through Gprotein coupled receptors linked to PLC cascade. The activation of PLCcauses a Ca 2+ influx that depolarizes the cells. It was recently proposedthat DAG activates a Ca 2+ -permeable channel, TRPC2. However, DAGcan be hydrolyzed to arachidonic acid (AA) by DAG lipase. We foundthat AA also plays a role in odor responses. We used a mixture of maleand female urine to stimulate odor responses in isolated vomeronasalneurons. With perforated patch clamp recordings (gramicidin),application of diluted urine induced an inward Ca 2+ current (-80 mV).Only part of the current was decreased by a DAG lipase inhibitor,indicating that DAG is not the only second messenger that activatesodor responses and AA might play a role. To test this, TRPC2 inhibitorswere used. The inhibitors did not completely eliminate the odor-inducedinward current, supporting a role for AA in odor responses. Directstimulation with AA also induced a Ca 2+ transient, which was sensitiveto Cd 2+ , but not to TRPC2 inhibitors, suggesting AA activates a channeldifferent from TRPC2. Moreover, the effect of AA could be mimickedby a non-metabolizable AA analogue, oleic acid (OA). Inhibition of anAA metabolic pathway by 12-LOX (increased [AA]i) activated aninward current. These data suggest the Ca 2+ channel activation was notcaused by AA metabolites. Together, our data indicate that both DAGand AA mediate the excitatory odor responses of vomeronasal neurons,by activating different Ca 2+ channels. Supported by NIH-DC006939,NSF-EPS0236976.310 Poster Chemosensory Molecular Genetics andVNO/PheromoneATTENUATION OF THE PRODUCTION OF INOSITOL 1,4,5TRISPHOSPHATE IN THE VOMERONASAL ORGAN BYANTIBODIES AGAINST THE &ALPHAQ/11 SUBFAMILY OFG- PROTEINS.Thompson R.N. 1 , Napier A. 1 , Wekesa K. 1 1 Biological Sciences,Alabama State University, Montgomery, ALThe social and reproductive behaviors of most mammals aremodulated by pheromones, which are perceived by the vomeronasalorgan. Vomeronasal transduction in vertebrates is activated through Gprotein coupled receptors, which in turn leads to the generation of IP3and DAG by the activity of phospholipase C. DAG has been shown togate the transient receptor potential channel 2 (TRPC2) which allowsthe subsequent increase of calcium. The role of IP3 in this transductioncascade and the G protein that is activated in the process has yet to bedetermined. To investigate the role of the alpha subunits of Go, Gi2 andGq/11 in the transduction process, microvillar membranes from femaleVNO were pre-incubated with selective C-terminal peptide antibodiesand then stimulated with adult male urine. Incubation of VNOmembranes with antibodies against Gq/11 blocked the production ofIP3 in a dose dependent manner. Furthermore pre-incubation withantibodies against Go also significantly impaired the production of IP3whereas the use of Gi2 antibodies did not impair the production of IP3.Thus our observations indicate that the alpha subunits of Gq/11 and Goplay a role in pheromonal signaling in the VNO. Supported by NIGMSgrant GM08219 and NCMHD grant 5P20MD000547.312 Poster Chemosensory Molecular Genetics andVNO/PheromoneFEMALE SNAKE SEX PHEROMONE INDUCES MEMBRANERESPONSES IN VOMERONASAL SENSORY NEURONS OFMALE SNAKESHuang G. 1 , Zhang J. 2 , Wang D. 3 , Mason R. 4 , Halpern M. 2 1 Anatomy andCell Biology, State University of New York (SUNY), Brooklyn, NY;2 Anatomy and Cell Biology, SUNY Downstate Medical Center,Brooklyn, NY; 3 Biochemistry, SUNY Downstate Medical Center,Brooklyn, NY; 4 Zoology, Oregon State University, Oregon, ORThe vomeronasal organ (VNO) is important for activating accessoryolfactory pathways that are involved in sexually dimorphic matingbehavior. The vomeronasal system of male garter snakes is criticallyimportant for detection of, and response to, female sex pheromones. Inthe present study, under voltage-clamp conditions, male snake VNOneurons were stimulated with female sexual attractiveness pheromone.Thirty-nine of 139 neurons exhibited inward current responses (reversalpotential: 10.6 ± 2.8 mV). The amplitude of the inward current wasdose-dependent, and the relationship could be fitted by the Hillequation. Under current-clamp conditions, application of pheromoneproduced membrane depolarizing responses and increases in firingfrequency. These results suggest that the female pheromone directlyaffects male snake VNO neurons and results in opening of ion channels,thereby converting the pheromone signal to an electrical signal. Theresponse to female pheromone is sexually dimorphic, i.e., thepheromone does not evoke responses in VNO neurons of female snakes.An associated finding of the present study is that the female sexpheromone which is insoluble in aqueous solutions became soluble inthe presence of Harderian gland homogenate.78