Single and binge methamphetamine administrations have different effects on the levels of dopamine D2 autoreceptor and dopamine transporter in rat striatum

Abstract

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum. Dopamine D2 receptor antagonists and dopamine transporter (DAT) inhibitors protect against neurotoxicity of the drug by decreasing intracellular dopamine content and, consequently, dopamine autoxidation and production of reactive oxygen species. In vitro, amphetamines regulate D2 receptor and DAT functions via regulation of their intracellular trafficking. No data exists on axonal transport of both proteins and there is limited data on their interactions in vivo. The aim of the present investigation was to examine synaptosomal levels of presynaptic D2 autoreceptor and DAT after two different regimens of METH and to determine whether METH affects the D2 autoreceptor-DAT interaction in the rat striatum. We found that, as compared to saline controls, administration of single high-dose METH decreased D2 autoreceptor immunoreactivity and increased DAT immunoreactivity in rat striatal synaptosomes whereas binge high-dose METH increased immunoreactivity of D2 autoreceptor and had no effect on DAT immunoreactivity. Single METH had no effect on D2 autoreceptor-DAT interaction whereas binge METH increased the interaction between the two proteins in the striatum. Our results suggest that METH can affect axonal transport of both the D2 autoreceptor and DAT in an interaction-dependent and -independent manner.

Figure 1.

Dopamine D2 receptor and dopamine transporter (DAT) species in rat striatal synaptosomes. (A) The immunoreactivity of the antibody recognizing both D2S and D2L isoforms of D2 receptor; (B) the immunoreactivity of the antibody recognizing the D2L receptor; and (C) the immunoreactivity of the antibody recognizing the DAT. Abbreviations: D2L, D2long receptor; D2S, D2short receptor; H, homogenate; M, membrane synaptosomal fraction; T, total synaptosomal fraction; V, vesicular synaptosomal fraction.

Figure 2.

The effect of binge and single dose METH on core body temperature. (A) In the binge dose regimen, core body temperatures (°C) were measured before the first METH or saline injection and 1 h after each METH or saline injection. Values are expressed as mean ± SEM. The arrows indicate METH injections. Binge METH caused hyperthermia lasting for at least 6 h; (B) in the single dose regimen, core body temperatures were measured immediately before sacrifices. Single injection of METH caused transient hyperthermia at 10, 30, and 60 min that peaked at 30 min. *** Significant difference (p < 0.001) between saline and METH. Abbreviations: METH, methamphetamine; SAL, saline.

Figure 3.

The effect of single intravenous (i.v.) dose of METH on the immunoreactivity of dopamine pre-synaptic D2 autoreceptor (D2S) in rat striatal synaptosomes (total fraction). The time course of D2S receptor immunoreactivity after 6 mg/kg METH shows a decrease (−28%, * p < 0.05) in D2S signal at 10 min after METH injection. Abbreviations: METH, methamphetamine; PonS, Ponceau S; SAL, saline.

Figure 4.

The effect of single intravenous (i.v.) dose of METH on the immunoreactivity of dopamine transporter (DAT) in rat striatal synaptosomes (total fraction). The time course of DAT immunoreactivity after 6 mg/kg METH shows an increase (+37%, ** p < 0.01) in DAT immunoreactivity at 5 h after METH injection. Abbreviations: METH, methamphetamine; PonS, Ponceau S; SAL, saline.

Figure 5.

The effect of binge intraperitoneal (i.p.) METH on the immunoreactivity of pre-synaptic dopamine D2 autoreceptor (D2S) in striatal synaptosomal fractions. Binge METH (4 × 10 mg/kg, every 2 h, i.p.) increased D2 receptor immunoreactivity at 1 h in total synaptosomal fraction (+32%, * p < 0.05) and decreased D2 receptor immunoreactivity at 24 h in all three fractions, total, membrane-endosomal, and cytosol-vesicular fraction (−34% (** p < 0.01), −28%, and −11%, respectively). A trend for an increase in D2S immunoreactivity was observed in cytosol-vesicular fraction (+48%, p = 0.069). Abbreviations: M, membrane fraction; METH, methamphetamine; PonS, Ponceau S; SAL, saline; T, total fraction; V, vesicular fraction.

Figure 6.

The effect of binge intraperitoneal (i.p.) METH on the immunoreactivity of dopamine transporter (DAT) in striatal synaptosomal fractions. Binge METH (4 × 10 mg/kg, every 2 h, i.p.) decreased DAT immunoreactivity at 24 h in total synaptosomal (−21%, *** p < 0.001) and membrane-endosomal (−25%, * p < 0.05) fraction. Abbreviations: M, membrane fraction; METH, methamphetamine; PonS, Ponceau S; SAL, saline; T, total fraction; V, vesicular fraction.

Figure 7.

The assessment of dopamine pre-synaptic D2 autoreceptor-dopamine transporter (D2S-DAT) interactions after single and binge METH. Synaptosomes from saline- and METH-treated rats were subjected to co-immunoprecipitation using antibodies against DAT or D2 receptor (D2R). (A) Single METH (6 mg/kg, i.v.) did not cause any apparent changes to the levels of D2S-DAT complex (lane 3 vs. 4); (B) binge METH (4 × 10 mg/kg, every 2 h, i.p.) increased the levels of D2S-DAT complex (top: lane 2 vs. 4; bottom: lane 3 vs. 4). Controls: IP Control—negative control, Synaptosome—positive control. Abbreviations: IP, immunoprecipitation; i.p., intraperitoneal; i.v., intravenous; METH, methamphetamine; SAL, saline; WB, western blotting.