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#P221
POSTER SESSION VI:
OLFACTION CNS; TASTE PERIPHERY &
CNS; MULTIMODAL RECEPTION
Mapping the Microglial/Immune Response to Two Methods of
Olfactory Bulb Deafferentation in Zebrafish
Amanda K. McKenna, Christine A. Byrd-Jacobs
Dept. of Biological Sciences/Western Michigan University
Kalamazoo, MI, USA
Since the role of the immune system in neural degeneration is not
understood, this study investigated the potential microglial
response to two different olfactory bulb deafferentation methods in
adult zebrafish. We compared complete, permanent olfactory organ
removal by cautery and temporary, partial olfactory epithelium
ablation with Triton X-100. We hypothesized that a microglial
response to axonal degredation would be more aggressive and long
lasting with complete olfactory organ removal than with detergent
treatment. DiA was applied to both olfactory organs to label the
olfactory axons. Three days later, olfactory bulbs were deafferented
on the right side with either a cautery iron injury or a single
application of 0.07% Triton X-100 to the nose. Fish were allowed
to survive for 1, 3, 5, or 7 days. Cryostat sections of the brains were
viewed using both fluorescence and confocal microscopy. As the
olfactory bulb responded to the damage, phagocytic removal of
degenerating axons by presumptive microglial cells caused them to
pick up the fluorescent dye. As early as 1 day after cautery
treatment, a noticeable increase in irregularly shaped, labeled
profiles was visible and persisted through day 7. The profile
labeling appeared to correlate directly with a decrease in axonal
labeling. After treatment with detergent, an increase in profile
labeling was observed as soon as 1 day but was reduced by 3 days.
Thus, treatment with Triton-X-100 elicited a shortened time course
of immune response and axon clearing. This is consistent with our
previous finding of reinnervation occurring quickly after detergent
treatment. This study is one step in better understanding the
immune response of the brain to peripheral damage and the
potential role of microglia in both deafferentation and reinnervation
processes. Acknowledgements: Supported by NIH-NIDCD
#011137 to CBJ.
#P222
POSTER SESSION VI:
OLFACTION CNS; TASTE PERIPHERY &
CNS; MULTIMODAL RECEPTION
Deafferentation Affects the Expression of Glutamate Receptor
Subunits in the Adult Zebrafish Olfactory Bulb
Qi Xiong, Christine A. Byrd-Jacobs
Dept. of Biological Sciences, Western Michigan University
Kalamazoo, MI, USA
Our goal was to understand the role of glutamate receptors in
regulating deafferentation effects in the olfactory bulb (OB).
This study examined both the presence of several glutamate
receptor subunits in the adult zebrafish OB and the potential effect
of deafferentation on their expression. We hypothesized that loss of
afferent input from olfactory axons will alter expression of
glutamate receptor subunits. Antibodies to ionotropic glutamate
receptor 2/3 (iGluR2/3), metabotropic glutamate receptor 2/3
(mGluR2/3) and Kainate 2 receptor (KA2R) were chosen based on
previous studies or protein sequence comparison between rat and
zebrafish. The right olfactory organ was permanently ablated by
cautery, leaving the left side untreated. Following two survival
times, 1 day and 3 weeks, both the right and left sides of the
olfactory bulb and telencephalon from 6 fish were dissected and
homogenized separately. Protein levels were measured, and equal
amounts of protein were separated by SDS-PAGE and transferred
to PVDF membrane. Blots were incubated with the three
antibodies. Densitometry was used to quantify the band densities
and compare to tubulin levels. We found that all three glutamate
receptors exist in both deafferented and untreated zebrafish OB,
with distinct bands at the predicted molecular weights. The
expression of iGluR2/3 appeared to decrease in deafferented tissues
at 3 weeks. The mGluR2/3 seemed to increase 1 day following
deafferentation, but was not different from control level at 3 weeks.
No obvious change of KA2R was observed in deafferented tissues.
Thus, olfactory input may differentially regulate the expression of
various glutamate receptor subtypes in the OB. Future studies will
examine the cellular distribution of these glutamate receptors in the
adult zebrafish OB. Acknowledgements: Supported by FRACAA
#09-019 and SFSA from WMU (CBJ).
#P223
POSTER SESSION VI:
OLFACTION CNS; TASTE PERIPHERY &
CNS; MULTIMODAL RECEPTION
Olfactory Recovery is Altered in Matrix Metalloproteinase-9
Knockout Mice
Stephen R Bakos, Grayson S Pitcher, Richard M Costanzo
Virginia Commonwealth University/Department of Physiology and
Biophysics Richmond, VA, USA
The olfactory system has the remarkable capacity for functional
recovery following injury, however the molecular mechanisms
contributing to the recovery process remain unknown. We
previously reported that matrix metalloproteinase-9 (MMP-9)
expression increases immediately following olfactory nerve
transection. To determine if MMP-9 plays a critical role in
recovery, we compared the return of olfactory function in wild-type
(WT) and MMP-9 knockout (MMP-9KO) mice after injury. An
odor discrimination task was employed to test olfactory function.
Mice were first trained to detect and distinguish between two odors
prior to bilateral nerve transection. Following surgery, olfactory
function was tested at recovery day 4 and subsequently every third
day. Shortly after surgery (day 4), both the WT and MMP-9KO
mice were unable to detect or distinguish between the two odors,
demonstrating that the surgery produced loss of smell. At day 13,
the WT mice showed initial improvement in olfactory
discrimination function, while the MMP-9KO mice were unable to
distinguish between the odors. By day 25, WT mice performed the
discrimination task at near pre-surgical levels. However, the MMP-
9KO mice showed minimal or no improvement. These data suggest
that MMP-9 plays a critical role in olfactory recovery. The early
expression of MMP-9 following injury may represent a novel
therapeutic target for restoring olfaction function.
Abstracts | 101
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