Abstract

1. FIG 4A FIG 4B FIG 2B FIG 2A FIG 3A FIG 3B 100 100 water/ water 100 80 80 water/ water + 80 1000 METH/ water 1000 0 METH/ water 80 METH + LY (0.1 mM)/ water + 0.01 75 water/ LY (10 mM) + 75 60 60 METH + LY (1 mM)/ water 60 750 METH/ LY (0.1 mM) * + 750 0.1 60 METH + LY (10 mM)/ water METH/ LY (1 mM) * 50 1 Total pLMV (gridlines crossed) * Total pLMV (gridlines crossed) pLMV (gridlines crossed) 50 40 40 Total pLMV (gridlines crossed) pLMV (gridlines crossed) METH/ LY (10 mM) pLMV (gridlines crossed) * * 40 40 500 10 500 * 100 25 25 20 20 20 20 250 250 0 0 0 0 0 0 1 2 3 4 5 6 0 0.01 0.1 1 10 100 0 0 0 1 2 3 4 5 6 0 0 0.01 0.1 1 10 100 0 1 2 3 4 5 water/ water Time (min) Time (min) water/ water METH/ water METH/ water METH/ LY ( 1 mM) water/ LY (10 mM) METH/ LY (10 mM) METH/ LY ( 0.1 mM) METH + LY (1 mM)/ water Time (min) METH + LY (0.1 mM)/ water METH + LY (10 mM)/ water FIG 2C water/ water METH/ water water/ METH METH/ METH * * * * * Glu (pmol/ mg protein) Abstract A role for glutamate and nitric oxide in methamphetamine physical dependence was investigated at the behavioral and neurochemical levels using a simple, established planarian model of drug withdrawal. A behavioral sign of methamphetamine withdrawal (planarian spontaneous locomotor velocity, pLMV) was determined in the presence of four drugs: a NMDA antagonist (LY 235959); an AMPA antagonist (DNQX); a glutamate release inhibitor (riluzole); and a nitric oxide synthase inhibitor (L-NAME). Glutamate levels in whole planarians were measured following spontaneous withdrawal from methamphetamine. Experiments demonstrated that methamphetamine (0.1-100 μM)-exposed planarians placed into drug-free water displayed a dose-related decrease in planarian spontaneous locomotor velocity (pLMV). No change in pLMV occurred when methamphetamine (10 μM)-exposed planarians were placed into water containing methamphetamine (10 μM). For combined administration, methamphetamine (10 μM)-exposed planarians placed into water containing LY 235959 (1-10 μM), L-NAME (10 μM) or riluzole (0.01 μM) did not display withdrawal. Withdrawal was not observed in planarians that were co-exposed to solutions containing methamphetamine (10 μM) and LY 235959 (0.1-10 μM), L-NAME (10 μM) or riluzole (0.01 μM). The AMPA receptor antagonist DNQX did not alter methamphetamine-evoked physical dependence. Glutamate levels increased in direct proportion to the magnitude of the dose-related abstinence-induced withdrawal.The present experiments demonstrate that methamphetamine physical dependence develops in planarians and that NMDA-like receptor activation and nitric oxide production is required for both the development and expression of the dependence. FIG 1A FIG 1B 100 water/ water 100 METH/ water 80 75 METH/ METH water/ METH 60 Total pLMV (gridlines crossed) pLMV (gridlines crossed) * 50 40 25 20 0 0 0 1 2 3 4 5 FIG 1C water/ water METH/ water water/ METH METH/ METH Time (min) * + * Glu (pmol/ mg protein) + FIG 5 FIG 6B FIG 6A water / water water / water water / L-NAME water / water 60 60 60 water / Riluzole 100 µM METH / water water / Riluzole 100 µM METH + L-NAME / water 10µM METH / water 100 µM METH / water 100 µM METH / Riluzole 10µM METH / Riluzole 40 pLMV (gridlines crossed) 40 40 pLMV (gridelines crossed) pLMV (gridlines crossed) 20 20 20 0 0 0 1 2 3 4 5 0 0 1 2 3 4 5 0 1 2 3 4 5 Time (min) Time (min) Fig. 5. Inhibition of abstinence-induced methamphetamine withdrawal by LY 235959 pLMV, expressed as the cumulative number of gridline crossings of planarians co-exposed to 100 μM methamphetamine and 10 μM L-NAME Time (min) Fig. 6. (A) Inhibition of abstinence-induced methamphetamine withdrawal by riluzole, expressed as the cumulative number of gridline crossings of planarians pretreated in 10 μM methamphetamine and tested in 0.01 μM riluzole. (B) Inhibition of abstinence-induced methamphetamine withdrawal by riluzole, expressed as the cumulative number of gridline crossings of planarians pretreated in 100 μM methamphetamine then tested in 0.01 μM riluzole. 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 Results 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. 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 (* P<0.05) (** P<0.001) 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 (21˚C). 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 14,000×g 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 (O’Shea 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. O’Shea, 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.