Givaudan-Roure Lecture - Association for Chemoreception Sciences

147 Poster [ ] Olfactory Development
CELL TYPES EXPRESSING OMP IN THE OLFACTORY
EPITHELIUM OF LARVAL ZEBRAFISH
Sakata Y. 1, Michel W.C. 1 1Physiology, University of Utah, Salt Lake
City, UT
The olfactory epithelium of zebrafish contained ciliated, microvillar
and crypt-type sensory neurons. While there appears to be some overlap
in specificity across the cell types the ciliated OSNs appear to respond
preferentially to bile salts and the microvillar cells to amino acids.
Recently, Celik et al (Euro. J. Neurosci. 15:798, 2002) reported the
expression of GFP under the zebrafish OMP promoter was found
primarily in ciliate OSNs but also found in cells with shorter and stouter
dendrites, presumably microvillar cells. In the current investigation we
produced a genetic construct with the zebrafish OMP promoter driving
expression of eYFP to quantitatively examine the distribution of eYFP
in the OSN populations of larval zebrafish. The zOMP promoter was
cloned from genomic DNA using primers described by Celik et al
(2002), ligated into an pEYFP vector (Clontech). The vector was
subsequently linearized and injected into 1 cell stage zebrafish
embryos. OMP promoter driven expression of eYFP was noted by 48
hours. The transiently transfected embryos were reared for 48-72 hours
post-fertilization, fixed and processed for whole embryo
immunocytochemistry using the anti-GFP antibody. After
immunostaining the embryos were embedded in Eponate plastic and
sectioned for electron microscopy. Preliminary counts indicate that the
majority of the eYFP expressing cells are ciliated OSNs but confirm the
expression was also observed in some microvillar OSNs. Until a stable
transgenic line is produced we cannot provide reliable estimates of the
proportions of ciliated or microvillar OSNs expressing. Supported by
NIH DC01418 and NS-07938.
148 Poster [ ] Olfactory Development
TYROSINE HYDROXYLASE-LIKE IMMUNOREACTIVE
CELLS IN THE OLFACTORY TRACTS OF GOLDFISH
Hansen A. 1, Finger T.E. 2 1University of Colorado Health Sciences
Center, Denver, CO; 2Cell and Developmental Biology, University of
Colorado Health Sciences Center, Denver, CO
Goldfish possess a substantial population of tyrosine hydroxylase
(TH)-like-immunoreactive cells within the olfactory tracts (OT) as well
as TH-like-ir cells in the olfactory bulbs (OB). The TH-like-ir neurons
in the OB have round cell bodies and large round nuclei with a single
dendritic process extending towards the margin of the OB. These
neurons were identified as a subset of granule cells (Alonso et al.,
1989). The nature and origin of the TH-like-ir cells in the OT are
unknown. The aim of the present study was to clarify whether the THlike-ir
cells are newly generated cells which migrate into the OB, or
whether they are more mature cells that are stationary in the OT.
Goldfish was chosen as a model since they have long OTs, and ample
information is available as to anatomy, physiology, and behavior.
BrdU-injections were used to visualize newly generated cells; and
antibodies used to characterize the nature of the cells in question. The
TH-like-ir cells in the OT are similar in shape to those in the OB,
however, the tract cells are bipolar, extending one long process towards
the OB and one towards the telencephalon. The majority of these cells
occur in the medial OT, fewer in the lateral OT. One day after injection,
BrdU-positive nuclei occur along the medial and lateral OTs. Six days
after BrdU-injection, labeled nuclei are present at the whole length of
the OTs and also in the OB. Further experiments are underway to test
whether the TH-positive cells of the OT are equivalent to the rostral
migratory stream of rodents.
This study was supported by NIDCD grant RO1 DC033792 to J.
Caprio and P30 DC04657 to D. Restrepo.
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149 Slide [ ] Olfactory Development
POSTNATAL CHANGES IN THE RAT MODIFIED
GLOMERULAR COMPLEX: A QUANTITATIVE
CYTOCHROME OXIDASE STUDY
Meisami E. 1 1Molecular and Integrative Physiology, University of
Illinois at Urbana-Champaign, Urbana, IL
The modified glomerular complex (MGC) has been described as a
set of glomeruli on the dorsomedial side of the rat olfactory bulb (OB),
associated with suckling behavior in the neonate. We further explored
the morphometric and anatomical features of MGC during postnatal
development, using coronal series of cytochrome oxidase (CO) stained
sections of the OB at postnatal ages of 1-, 3 -, 10- and 25 -days and
analyzed with regard to lateral and medial distribution of the glomeruli,
their size and number, and the anterior-posterior (AP) length of MGC.
The MGC stained intensely for CO even in the newborn animal,
compared to main OB glomeruli. In addition to the originally described
medial complex, a lateral complex was also noted, particularly in rat
pups 3 days and older. This complex had fewer glomeruli and was
shorter than the medial complex, but its glomeruli were similar in size
and stain intensity. Anteriorly the lateral and medial portions appear to
fuse along the midline, forming an asymmetrical horseshoe structure.
Total glomeruli number in the entire complex (medial and lateral)
increased postnatally, from about 15 at birth to about 20 at 3 days, 40 at
10 days, and about 70 at day 25. During the same period, mean
glomerular diameter increased from about 60 mm at birth to about 85
mm in the weanling. These changes were accompanied by marked
increases in total glomerular volume. Results indicate that MGC is well
developed at birth, but continues to develop postnatally in terms of
number and size of its glomeruli, indicating possible olfactory functions
of this complex in the weanling and older animals.
Support: University of Illinois Research Funds
150 Poster [ ] Odor Activation and Modulation
OMP IS A MODULATOR OF CA2+ CLEARANCE PROCESSES
IN MOUSE OLFACTORY RECEPTOR NEURONS (ORNS)
Kwon H.J. 1, Leinders-Zufall T. 1, Zufall F. 1, Margolis F.L. 1 1Dept.
Anatomy & Neurobiology, Program in Neuroscience, University of
Maryland, Baltimore, MD
Ca 2+ entry to ORNs is a well-studied event in chemosensory
transduction. However, restoration of intracellular Ca 2+ levels to the
pre-stimulus level is less well understood. Recent behavioral and
electrophysiological analyses of OMP–null mice led us to hypothesize
that OMP participates in the recovery phase of olfactory signaltransduction.
To investigate this we compared Ca 2+ transients in ORNs
of wild-type and OMP-null mice by activating various pathways to
increase the cytoplasmic [Ca 2+ ]. KCl, 3-isobutyl-1-methylxanthine
(IBMX), and caffeine were used to induce Ca 2+ influx from voltagegated
Ca 2+ channels, cyclic-nucleotide gated channels, and intracellular
Ca 2+ stores, respectively. Confocal laser images were recorded from
dendritic knobs of mouse ORNs loaded with the Ca 2+ -indicator dye
fluo-4 AM. In OMP-nulls in response to IBMX, the rate of Ca 2+ entry
to reach peak was 5-fold slower, and to achieve half-recovery to basal
level was 4-fold slower compared to controls. Following KCl
depolarization, the OMP-null mice showed a 2-fold delay in the halfrecovery
time of the Ca 2+ peak. Similar results were obtained following
Ca 2+ store depletion by stimulating ryanodine receptors with caffeine.
These results suggest that the cytoplasmic Ca 2+ clearance of ORNs is
significantly impaired in the OMP-null mice. These data imply that
OMP is a modulator of Ca 2+ extrusion or sequestration processes in
ORNs. Characterization of the mechanism by which OMP modulates
Ca 2+ flux in ORNs is under investigation.
Supported by NIH DC03112 (FLM), NIH DC DC00347 (FLM, FZ),
NIH DC003773 (TL-Z).