Embryonic Methamphetamine Exposure Inhibits Methamphetamine Cue Conditioning and Reduces Dopamine Concentrations in Adult N2 Caenorhabditis elegans

Abstract

Methamphetamine (MAP) addiction is substantially prevalent in today's society, resulting in thousands of deaths and costing billions of dollars annually. Despite the potential deleterious consequences, few studies have examined the long-term effects of embryonic MAP exposure. Using the invertebrate nematode Caenorhabditis elegans allows for a controlled analysis of behavioral and neurochemical changes due to early developmental drug exposure. The objective of the current study was to determine the long-term behavioral and neurochemical effects of embryonic exposure to MAP in C. elegans. In addition, we sought to improve our conditioning and testing procedures by utilizing liquid filtration, as opposed to agar, and smaller, 6-well testing plates to increase throughput. Wild-type N2 C. elegans were embryonically exposed to 50 μM MAP. Using classical conditioning, adult-stage C. elegans were conditioned to MAP (17 and 500 μM) in the presence of either sodium ions (Na+) or chloride ions (Cl-) as conditioned stimuli (CS+/CS-). Following conditioning, a preference test was performed by placing worms in 6-well test plates spotted with the CS+ and CS- at opposite ends of each well. A preference index was determined by counting the number of worms in the CS+ target zone divided by the total number of worms in the CS+ and CS- target zones. A food conditioning experiment was also performed in order to determine whether embryonic MAP exposure affected food conditioning behavior. For the neurochemical experiments, adult worms that were embryonically exposed to MAP were analyzed for dopamine (DA) content using high-performance liquid chromatography. The liquid filtration conditioning procedure employed here in combination with the use of 6-well test plates significantly decreased the time required to perform these experiments and ultimately increased throughput. The MAP conditioning data found that pairing an ion with MAP at 17 or 500 μM significantly increased the preference for that ion (CS+) in worms that were not pre-exposed to MAP. However, worms embryonically exposed to MAP did not exhibit significant drug cue conditioning. The inability of MAP-exposed worms to condition to MAP was not associated with deficits in food conditioning, as MAP-exposed worms exhibited a significant cue preference associated with food. Furthermore, our results found that embryonic MAP exposure reduced DA levels in adult C. elegans, which could be a key mechanism contributing to the long-term effects of embryonic MAP exposure. It is possible that embryonic MAP exposure may be impairing the ability of C. elegans to learn associations between MAP and the CS+ or inhibiting the reinforcing properties of MAP. However, our food conditioning data suggest that MAP-exposed animals can form associations between cues and food. The depletion of DA levels during embryonic exposure to MAP could be responsible for driving either of these processes during adulthood.

Figure 1. Effect of embryonic methamphetamine (MAP) exposure (50 μM) on MAP cue conditioning in adult N2 C. elegans

When the CS+ was Na⁺ (top panel), a two-way ANOVA found a significant effect of conditioning dose [F(2, 94) = 5.5; p < 0.006] and a significant interaction between pre-exposure and conditioning dose [F(2, 94) = 6.1; p < 004] on PI. For the vehicle pre-exposed groups, a one-way ANOVA revealed significant effect of MAP conditioning dose on PI [F(2, 46) = 8.8; p < 0.002]. Post-hoc tests found significant differences between the vehicle conditioned and either the 17 μM (p < 0.007) or 500 μM (p < 0.001) MAP conditioned groups (top panel). However, for the MAP pre-exposed groups, a one-way ANOVA did not find a significant effect of MAP conditioning dose on PI [F(2, 46) = 2.8; p < 0.07] (top panel). When the CS+ was Cl^-(bottom panel), a two-way ANOVA found a significant effect of pre-exposure [F(1, 91) = 4.4; p < 0.05], conditioning dose [F(2, 91) = 8.1; p < 0.002] and a significant interaction between pre-exposure and conditioning dose [F(2, 91) = 6.6; p < 0.003] on PI. For the vehicle pre-exposed groups, a one-way ANOVA revealed significant effect of MAP conditioning dose on PI [F(2, 46) = 13.5; p < 0.001]. Post-hoc tests found significant differences between the vehicle conditioned and either the 17 μM (p < 0.001) or 500 μM (p < 0.002) MAP conditioned groups (bottom panel). However, for the MAP pre-exposed groups, a one-way ANOVA did not find a significant effect of MAP conditioning dose on PI for Cl [F(2, 43) = 2.2; p < 0.124] (bottom panel). * indicates significant (p < 0.05) increases in PI compared to respective vehicle conditions.

Figure 2. Effect of embryonic MAP exposure (50 μM) on food conditioning in adult N2 C. elegans

When the CS+ was Na⁺ (top panel), a two-way ANOVA found a significant effect of pre-exposure [F(1, 48) = 13.0; p < 0.002], and conditioning [F(1, 48) = 8.7; p < 006], without a significant interaction between pre-exposure and conditioning [F(1,48) = 0.2; ns] on PI. These findings indicate that MAP pre-exposure increased Na⁺ preference and that both the vehicle and MAP pre-exposed groups conditioned to Na⁺, when previously paired with food. When the CS+ was Cl- (bottom panel), a two-way ANOVA found a significant effect of pre-exposure [F(1, 47) = 5.6; p < 0.05] on PI. However, no significant effect of conditioning [F(1, 47) = 0.6; ns] and no significant interaction between pre-exposure and conditioning [F(1, 47) = 0.1; ns] were found on PI. These findings indicate that MAP pre-exposure decreased Cl- preference, and that both the vehicle and MAP pre-exposed groups did not condition to Cl-, when previously paired with food. * indicates significant (p < 0.05) increase in PI compared to respective control condition.

Figure 3. Effect of embryonic MAP exposure (50 and 177 μM) on mean (± SEM) percent (%) of respective vehicle treated control dopamine (DA; nM) concentrations in adult N2 C. elegans

DA concentrations (nM) were normalized to % of respective vehicle control condition (50 or 177 μM). A one-way ANOVA examining the effect of MAP dose (0, 50 and 177 μM) found a main effect of MAP dose on % of vehicle treated control DA concentrations [F(2, 48) = 4.6; p < 0.02]. Specifically, embryonic exposure to 50 and 177 μM MAP reduced DA concentrations compared to vehicle treated controls. * indicates a significant decrease in DA concentrations compared to vehicle controls.