The Toxicologist - Society of Toxicology

163 MICROPET IMAGING OF [18F]-DFNSH IN RAT BRAIN:
A RADIOLIGAND FOR KETAMINE-INDUCED
NEURONAL DEATH.
X. Zhang 1 , M. G. Paule 1 , G. D. Newport 1 , X. Zou 1 , N. Sadovova 2 , M. S.
Berridge 3 , S. M. Apana 3 , G. Kabalka 4 , W. Slikker 1 and C. Wang 1 . 1 Division of
Neurotoxicology, NCTR/FDA, Jefferson, AR, 2 Toxicologic Pathology Associates,
Jefferson, AR, 3 3D Imaging, LLC, Little Rock, AR and 4 The University of Tennessee,
Knoxville, TN.
Recent reports indicate that 6-12 hours of ketamine anesthesia triggers neuronal
apoptosis in postnatal day (PND) 7 rats. In vitro, ex vivo and confocal fluorescent
imaging studies suggest that the dansyl compounds can accumulate within the cytoplasm
of the apoptotic cell. High-resolution positron emission tomography
(microPET) imaging has been proposed as a minimally invasive method for detecting
apoptosis in the rat brain. Compared with the [18F]-labeled annexin V, which
binds to externalized phosphatidylserine (PS) on the outer membrane of apoptotic
cells, intracellular uptake of the dansylhydrazone of p-fluorobenzaldehyde
(DFNSH) may lead to improved target to background contrast ratios. In this study,
the effect of ketamine on the uptake and retention of [18F]-DFNSH in the rat
brain was investigated using microPET imaging. On PND-7, rat pups in the experimental
group were exposed to 6 subcutaneous injections of ketamine and control
rat pups received 6 injections of saline. On PND-35, [18F]-DFNSH (37 MBq)
was injected into the tail vein of rats and microPET images were obtained over 2
hours following the injection. Radiolabeled tracer accumulation in the region of interest
(ROI) in the frontal cortex was converted into Standard Uptake Values
(SUVs). After the injection, radiotracer was quickly distributed into the brains of
both ketamine- and saline-treated rats. Compared with the control group, the uptake
of [18F] -DFNSH was significantly increased in the ROI of ketamine-treated
rats. Additionally, the duration for wash-out of the tracer was prolonged in the ketamine-treated
animals. This preliminary study demonstrates that microPET imaging
is capable of distinguishing differences in retention of [18F] -DFNSH in different
brain regions and suggests that this approach may provide a minimally invasive
biomarker of neuronal apoptosis.
Supported by NCTR/FDA, E7264
164 IN VITRO ASSESSMENT OF DEVELOPMENTAL
NEUROTOXICITY: USE OF MICROELECTRODE
ARRAYS TO MEASURE FUNCTIONAL CHANGES IN
NEURONAL NETWORK ONTOGENY.
B. Robinette, W. R. Mundy and T. J. Shafer. ISTD, U.S. EPA, Research Triangle
Park, NC .
Because the Developmental Neurotoxicity Testing Battery requires large numbers
of animals and is expensive, development of in vitro approaches to screen chemicals
for potential developmental neurotoxicity is a high priority. Many proposed approaches
for screening are biochemical, and do not assess function in networks of
interconnected neurons. In this study, microelectrode arrays (MEAs) were used to
determine if chemical-induced changes in function could be detected by assessing
the development of spontaneous network activity. MEAs record individual action
potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity
can be assessed repeatedly over days in vitro (DIV). Primary cultures of cortical
neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2,
6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous
spiking and bursting in cortical networks. In addition 2.5 or 5 μM of the PKC inhibitor
bisindolylmaleamide (BIS) was added to MEAs (n= 7-10) on DIV5 to determine
if changes in spontaneous activity could be detected in response to inhibition
of neurite outgrowth. A clear profile of in vitro activity development occurred
in control MEAs, with the number of active channels increasing from 0/MEA on
DIV2 to 46±5/MEA by DIV13; the rate of increase was most rapid between DIV
6 and 13, and activity declined by DIV 20. A similar pattern was observed for the
number of bursting channels, as well as the total number of bursts. BIS caused a
concentration-dependent decrease in the number of active channels/MEA, the
number of bursting channels/MEA the number of bursts/channel and the number
of individual spikes on bursting channels. Other burst characteristics, such as burst
duration and the number of spikes in a burst, were unchanged by BIS. These results
demonstrate that MEAs can be used to assess the development functional neuronal
networks in vitro, as well as chemical-induced dysfunction. (This abstract does not
reflect Agency policy).
165 EVALUATING ALTERATIONS IN ZEBRAFISH RETINO-
TECTAL PROJECTIONS AS AN INDICATION OF
DEVELOPMENTAL NEUROTOXICITY.
J. Cowden 1, 2 , C. Fan 1, 3 , D. Hunter 1 , B. Padnos 1 , K. Jensen 1 , R.
Ramabhadran 1 and S. Padilla 1 . 1 Integrated Systems Toxicology Division, U.S. EPA,
Durham, NC, 2 National Center for Environmental Assessment, U.S. EPA, Durham,
NC and 3 Syngenta Biotechnology Inc., Durham, NC.
The U.S. EPA is developing alternative screening methods to identify putative developmental
neurotoxicants and prioritize chemicals for additional testing. One
method developmentally exposes zebrafish embryos and assesses nervous system
structure at 2 days post fertilization (dpf). Previous data indicated that exposure to
developmental neurotoxicants reduced retino-tectal projection area. To determine
if altered retino-tectal projection area is a useful endpoint for assessing developmental
neurotoxicity, zebrafish embryos were exposed four chemicals (retinoic acid,
cadmium, valproate, and ethanol) with evidence of developmental neurotoxicity in
mammalian systems, and three chemicals (saccharin, amoxicillin, and omeprazole)
without such evidence. All experiments were conducted in 96-well plates at 26 o C.
Zebrafish embryos were exposed to a range of chemical sublethal concentrations
from 6 hours to 2 dpf. At 2 dpf nervous system structure was examined by whole
mount anti-acetylated α-tubulin staining and retino-tectal projection area was
measured. All measurements were made blinded to dose. Of the chemicals examined,
valproate, ethanol, and retinoic acid showed a dose-dependent decrease in
retino-tectal projection area. Furthermore, quantitative real-time PCR (qRT-PCR)
demonstrated that exposure to valproate or ethanol alters levels of brn3c, a transcription
factor important for optic tectum development. These preliminary results
suggest that changes in retino-tectal projection area may correlate with developmental
neurotoxicity in mammals and such changes might be measured rapidly
using qRT-PCR. The contents of this abstract do not necessarily reflect the views of
the U.S. EPA, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
166 METHYLPHENIDATE: A THREE-YEAR ASSESSMENT
ON COMPLEX BRAIN FUNCTION IN JUVENILE
RHESUS MONKEYS.
T. A. Patterson 1 , M. Li 1 , S. M. Morris 1 , N. C. Twaddle 1 , D. R. Doerge 1 , W.
Slikker 1 , D. R. Mattison 2 and M. G. Paule 1 . 1 NCTR/FDA, Jefferson, AR and
2
NICHD, Rockville, MD.
Methylphenidate (MPH) is a prescribed stimulant for the treatment of attentiondeficit
hyperactivity disorder (ADHD), and the widespread use of MPH continues
to raise concern about its safety. This study examined the ability of juvenile rhesus
monkeys to perform complex behavioral tasks contained in the NCTR Operant
Test Battery (OTB) during treatment with MPH that produced clinically-relevant
blood levels. Monkeys (n=10/group) were treated orally (2x/day – morning and afternoon
(M-F)) with either 0.5 ml/kg vehicle (CON group), 2.5 mg/kg MPH (LD
group), or 12.5 mg/kg MPH (HD group). This dosing regimen provided MPH
plasma levels near human therapeutic values (LD group) and five-to-ten times
human therapeutic values (HD group). OTB testing (50 minutes/day (M-F))
began 30 minutes after the morning dose and required the monkeys to press levers
or press-plates to receive food-pellet reinforcers. The OTB includes Progressive
Ratio (PR), Conditioned Position Responding (CPR) and Incremental Repeated
Acquisition (IRA) tasks, which assess aspects of motivation, color-position discrimination
and learning, respectively. Response Rate (RR), Percent Task Completed
(PTC), and Accuracy (ACC) serve as performance metrics. With the exception of
elevated serum alanine aminotransferase (ALT) in the HD group, there were no remarkable
alterations in other health bio-monitoring parameters. However, there
was a significant dose response of MPH on behavior. Monkeys in the HD group
performed much more poorly than the LD or CON groups. The HD group lagged
well behind in task acquisition and had much lower scores for PTC, RR and ACC
in all tasks. MPH is a known appetite suppressant, therefore, much of these effects
could stem from a decreased motivation to perform, although effects on ACC suggest
other mechanisms. Supported in part by the Best Pharmaceuticals for Children
Act 2002 and 2007.
SOT 2010 ANNUAL MEETING 35