1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

241 Poster Central Olfaction and Chemical EcologyMEASURING OLFACTORY SENSORY NEURON SYNAPTICVESICLE RELEASE IN ZEBRAFISH USING THEGENETICALLY-ENCODED EXOCYTOSIS MARKERSYNAPTOPHLUORINSakata Y. 1 , Greig A. 1 , Michel W.C. 1 1 Physiology, University of Utah,Salt Lake City, UTUse of the genetically-encoded exocytosis indicator synaptopHluorin(spH) permits direct examination of presynaptic vesicle releasedynamics in targeted neurons. In acidic synaptic vesicles spHfluorescence is quenched; upon neutralization following exocytosisfluorescence increases approximately 20-fold. We have developed azebrafish line stably expressing spH under control of the zebrafisholfactory marker protein (OMP) promoter to examine olfactory input tothe adult and developing olfactory bulb (OB). spH expression in thedeveloping OB is detectable 28-48 hpf in F1 or F2 embryos. Labeling ishighest in the presynaptic terminals, evident in the distal axonalprocesses but nearly undetectable in the OSN soma. An increase influorescence following neutralization of the synaptic vesicles withNH 4 Cl confirmed function. An odor mixture, forskolin (an adenylatecyclase activator) and electrical stimulation of the olfactory nerve elicitOSN synaptic vesicle exocytosis at developmental stages as early as 48hpf. Suppression of the second response was observed during pairedpulse stimulation (ISI 400 msec) of olfactory nerve bundles entering theglomerular layer of F1 adult OBs indicating that the intrinsic inhibitorymechanisms previously noted in the mouse pOMP-spH transgenic lineare likely functional in the zebrafish OB. Ionotropic glutamate receptorantagonists partially reduced the suppression. The pOMP-spHtransgenic zebrafish line provides an important tool for investigations ofbulbar circuitry development. This work was supported by NationalInstitutes of Health grants DC01418 and NS-07938. We wish to thankDr. Matt Wachowiak for assistance.242 Poster Central Olfaction and Chemical EcologyTYROSINE HYDROXLASE AND CFOS EXPRESSION INMOUSE OLFACTORY BULB SLICE CULTURES REQUIRESAN L-TYPE CALCIUM CHANNELAkiba Y. 1 , Cave J.W. 1 , Baker H. 1 1 Burke Medical Research Institute,Weill Med. Coll., Cornell, White Plains, NYExpression of the olfactory bulb (OB) dopamine (DA) phenotype, asreflected by the level of the first enzyme in DA biosynthesis, tyrosinehydroxylase (TH), requires either receptor afferent stimulation orequivalent depolarizing conditions. Previous studies suggested a role forL-type calcium channels in development of the DA phenotype as wellas a causal relationship between cFOS and TH expression. To show thatcFOS is involved in the signal transduction mechanisms underlying theactivity-dependent expression of TH in OB, forebrain slices wereprepared from postnatal day 2-3 transgenic mice expressing enhancedgreen fluorescent protein (GFP) driven by 9 kb of TH promoter(TH/GFP). Slices were treated with: (1) a depolarizing concentration ofpotassium chloride (KCl, 50mM) to simulate receptor afferent activity;(2) KCl plus an L-type calcium channel blocker, Nifedipine (10µM); or(3) as a control, sodium chloride (NaCl, 50mM). cFOS and TH/GFPexpression, detected immunohistochemically, were quantitated usingMetaMorph Imaging software. cFOS expression was widespread in OB,peaking at 3 hours (h) after stimulation, whereas, TH/GFP levels werehighest at 48 h. The increase in the number of TH/GFP expressing cellswas greater in the superficial granule than in periglomerular regions.Nifedipine prevented the increase in both cFOS and TH/GFPexpression. These findings suggest that the same signal transductionpathway regulates cFOS and TH expression and, despite the temporaldisparity, supports the hypothesis that cFOS plays a role in OB THexpression. Supported by AG09686.243 Poster Central Olfaction and Chemical EcologyOLFACTORY BULB SPECIFIC REGULATION OF TYROSINEHYDROXYLASE GENE EXPRESSION BY ER81 IN MICECave J.W. 1 , Akiba Y. 1 , Berlin R. 1 , Baker H. 1 1 Burke Medical ResearchInstitute, Weill Med. Coll., Cornell, White Plains, NYTyrosine hydroxylase (TH) is both the rate limiting enzyme in thebiosynthesis of the neurotransmitter dopamine (DA) and a wellestablished marker for DA neurons. The DA phenotype shows regionspecific brain development and regulation. In the mouse olfactory bulb(OB), peak generation of DA neurons occurs primarily in earlypostnatal development, and new DA neurons are produced throughoutthe adult lifespan from stem cells maintained in the subventricular zone.Recent evidence suggests that the transcription factors, Pax6 and ER81,may be necessary for the OB-specific expression of TH in DA neurons.To determine whether either Pax6 or ER81 act directly to regulate THexpression, we have examined the upstream mouse, rat and human THpromoters for potential Pax6 and ER81 binding sites. The analysisidentified consensus ER81, but not Pax6, binding sites in the THpromoter. Chromatin immunoprecipitation pull down assays withmouse OBs suggested that these sites in the TH promoter are bound byER81 in vivo. Immunohistochemical staining revealed that ER81 isbroadly expressed in most periglomerular cells and overlaps with thesubpopulation that also contains TH. Perturbations that profoundlyreduce TH expression in the OB, such as odor deprivation, decrease, butdo not eliminate, ER81 expression. Together these results suggest thatER81, but not Pax6, is a direct regulator of TH. ER81 expression is notsufficient to activate TH expression, however, and OB-specific THexpression may require a combinatorial set of transcription factors thatinclude ER81. Supported by AG09686.244 Poster Central Olfaction and Chemical EcologySEROTONIN INCREASES GABA RELEASE FROMPERIGLOMERULAR CELLS IN MOUSE OLFACTORY BULBAungst J.L. 1 , Shipley M.T. 2 1 Anatomy & Neurobiology, University ofMaryland at Baltimore, Baltimore, MD; 2 University of Maryland atBaltimore, Baltimore, MDPeriglomerular (PG) cells, the most populous neuron type in theglomerular layer, have physiological and morphological properties thatdistinguish them from external tufted (ET) and short axon (SA) cells.PG cells are small interneurons whose dendrites are generally restrictedto a single glomerulus. Subpopulations of PG cells express GABAand/or dopamine. Proposed functions of PG cells are (i) presynapticinhibition of ON terminals and (ii) postsynaptic inhibition ofmitral/tufted cells, including ET cells. PG cells receive monosynapticglutamatergic input from and monosynaptically feed back onto ET cells.This glomerular circuit suggests that modulation of PG cell activityaffects ET cell activity. Glomeruli are heavily targeted by 5-HT fibersarising from the raphe nuclei. We have shown that 5-HT, via 5-HT 2Creceptors, causes a depolarizing current in ET cells whenpharmacologically isolated from excitatory and inhibitory inputs. Herewe show that when PG cells are isolated from ET and otherglutamatergic inputs, 5-HT, via 5-HT 2A receptors, induces GABArelease from PG cells observed as IPSCs in postsynaptic ET cells. Thisincreased inhibitory input is action potential independent as it isunaffected by TTX. 5-HT modulation of PG cells may function toinhibit glomerular excitation through suppression of bursting activity inET cells. Alternatively, 5-HT's combined actions on PG and ET cellsmay enhance the signal to noise ratio of glomerular throughput.Supported by NIH NIDCD DC 36940 & DC02173.61