NMDAlike receptor activation and nitric oxide synthesis mediate the development and expression of me

1
NMDA-like receptor activation and nitric oxide
synthesis mediate the development and expression
of methamphetamine physical dependence in
planarians Christopher Roth, Scott Rawls, PhD,
and Robert Raffa, PhD Department of
Pharmaceutical Sciences, Temple University School
of Pharmacy, 3307 North Broad Street,
Philadelphia PA 19140
Conclusion Methamphetamine abstinence-induced
withdrawal Abstinence-induced withdrawal was
shown to occur in response to 1h exposure to
methamphetamine (Fig. 1A) in a dose dependent
manner (Fig. 2A). Levels of glutamate also
increased in response to dose related increases
(Fig. 2C). Abstinence-induced withdrawal, marked
by decreased locomotor velocity (pLMV), and
glutamate levels were at their highest levels
during exposure to the highest (100 µM)
concentrations of methamphetamine. Inhibition
of withdrawal expression A model for inhibition
of expression of methamphetamine induced
withdrawal is illustrated by a 1h period of
exposure to methamphetamine followed by a 5 min
observation period in the presence of one of four
drugs (LY 235959, DNQX, riluzole, L-NAME).
Results show a significant inhibition of
expression of methamphetamine induced withdrawal
for three of the tested drugs (LY 235959,
riluzole, L-NAME). DNQX showed no significant
effect on expression of methamphetamine
withdrawal. Inhibition of withdrawal
development A model for inhibition of development
of methamphetamine induced withdrawal is
illustrated by a 1h period of co-exposure to
methamphetamine and one of four drugs (LY 235959,
DNQX, riluzole, L-NAME) followed by a 5 min
observation period in water. Results show a
significant inhibition of development of
methamphetamine induced withdrawal for three of
the tested drugs (LY 235959, riluzole, L-NAME).
DNQX showed no significant effect on the
development of methamphetamine induced
withdrawal.
Results
Fig. 1. (A) Methamphetamine abstinence-induced
withdrawal, expressed as the total number of
gridline crossings, of planarians treated in
water (water/), or 10 µM methamphetamine (METH/)
and tested in water (/water), or 10 µM
methamphetamine (/METH). (B) Methamphetamine
abstinence-induced withdrawal, significant ()
reduction in total pLMV (mean S.E.M.) (C)
Concentration of glutamate in planarians,
expressed as the mean S.E.M. pmol/mg planarian.
Significance between treatment and water/water
groups indicated by (), significance between
treatment and METH/water group indicated by ()
Fig. 2. (A) pLMV (cumulative means S.E.M.) of
planarians exposed to methamphetamine at the
concentrations (µM) indicated for 1h then tested
in water. (B) Total cumulative number of gridline
crossings S.E.M. of planarian exposed to
methamphetamine (0.01-100 µM) and tested in
water, significant difference from the naïve
planarians is indicated () at all but the lowest
concentration. (C) Concentration of glutamate in
planarians expressed as the mean S.E.M pmol/mg
planarian. Significant ( Plt0.05) ( Plt0.001)
difference from naïve planarians shown at the
three highest concentrations.
Methods Animals Planarians were purchased from
Carolina Biological Co. (Burlington, NC),
shipment containers were aerated and acclimated
to room temperature (21C). Each planarian was
used only once and within 48 hours of shipment.
Methamphetamine and L-NAME was purchased from
Sigma Chemical Co. (St. Louis, MO), LY 235959
was purchased from Lilly Research Laboratories
(Indianapolis, IN), and DNQX and Riluzole was
purchased from Tocris Bioscience (Ellisville,
MO). Behavioral Measurements Quantification
(pLMV) (Raffa et al., 2001) of locomotor velocity
was conducted by placement of individual
planarians into a petri dish containing tap water
treated with Amquel, positioned on top of grid
paper (0.5 cm spaced lines). pLMV was measured
as the cumulative number of gridlines crossed or
re-crossed during a 5-minute observation period.
Planarians were pretreated prior to observation,
in 1ml centrifuge tubes with room temperature
vehicle or test compound(s) for 1h. Each
planarian was tested in Amquel treated tap
water or test compound. Each planarian was
exposed individually for 1 h to one of the
following treatments water, methamphetamine
(0.01-100 µM), then tested individually for pLMV
for 5 min in one of the following water,
methamphetamine (10 µM), LY 235959 (1-10 µM),
DNQX, riluzole (0.01 µM), and L-NAME (10
µM). Glutamate analysis For each extraction and
measurement, one planarian was weighted
(approximately 2 mg), air dried, and homogenized
in 250 µL of ice-cold 0.15 M buffer. The buffer
consisted of perchloric acid containing 0.25 of
L-cystine and Na2EDTA. The homogenate was
centrifuged at 14,000g for 20 min at 4 C and
the supernatant was passed through a 0.2 µm
filter (Rawls et al. 2006). For derivatization
50 µL of planarian filtrate was reacted with 50
µL of sodium borate, 50 µL of potassium cyanide,
and 20 µL of NDA. The mixture was allowed to
react for 10 min (OShea et al. 1992). Following
derivitzation, glutamate was separated on a 5 µm
C18 reverse-phase column (150 4.6 mm
Phenomenex Inc. Torrance, CA, USA). The mobile
phase consisted of 5 mM sodium citrate buffer (pH
7.5) used in a linear gradient with elution with
30 methanol. The flow rate was 0.75 mL/min.
Fluorescence was detected at 440 nm excitation
and 480 emission. Sample injection volumes were
50 µL. Chromotographic peaks were compared to
working standard solutions and converted to
picomoles of glutamate per milligram of planarian
(pmol/mg planaria). The results were expressed
as the mean S.E.M.
References Raffa, R.B., Valdez, J.M., Holland,
L.J. Shulingkamp, R.J., 2001. Quantitative
assesment of dopamine D2 antagonist activity
using invertebrate (Planaria) locomotion as a
functional endpoint. J. Pharmacol. Toxicol.
Methods 45, 223-226. Rawls, S.M., Gomez, T.,
Stagliano, G.W., Raffa, R.B., Measurement of
glutamate and aspartate in Planaria. J.
Pharmacol. Toxicol. Methods 53, 291-295. OShea,
T., Weber, P., Bammel, B., Lunte, C., Lunte, S.
(1992). Monitoring excitatory amino acid release
in vivo by microdyalysis with capillary
electrophoresis-electrochemistry. Journal of
Chematography, 608, 189-195.
Fig. 3. (A) Dose dependent inhibition of
abstinence-induced methamphetamine withdrawal by
LY 235959 pLMV, expressed as the cumulative
number of gridline crossings of planarians
pretreated in 10 µM methamphetamine and tested in
LY 2335959 (at indicated concentrations). (B)
Significant withdrawal () from naïve and treated
(METH / water) groups and between () METH /
water and Meth / LY (1, 10 µM) groups.
Fig. 4. (A) Dose dependent inhibition of
abstinence-induced methamphetamine withdrawal by
LY 235959 pLMV, expressed as the cumulative
number of gridline crossings of planarians
co-exposed to 10 µM methamphetamine and LY 235959
at indicated concentrations. (B) Significant
withdrawal () from naïve and treated (METH /
water) groups and between () METH / water and
METH LY (1, 10 µM) / water groups.
Acknowledgements This work was funded by a grant
(to RBR) from the NIH (NIDA) Bethesda, MD.