Long-term consequences of methamphetamine exposure in young adults are exacerbated in glial cell line-derived neurotrophic factor heterozygous mice

Abstract

Methamphetamine abuse in young adults has long-term deleterious effects on brain function that are associated with damage to monoaminergic neurons. Administration of glial cell line-derived neurotrophic factor (GDNF) protects dopamine neurons from the toxic effects of methamphetamine in animal models. Therefore, we hypothesized that a partial GDNF gene deletion would increase the susceptibility of mice to methamphetamine neurotoxicity during young adulthood and possibly increase age-related deterioration of behavior and dopamine function. Two weeks after a methamphetamine binge (4 x 10 mg/kg, i.p., at 2 h intervals), GDNF(+/-) mice had a significantly greater reduction of tyrosine hydroxylase immunoreactivity in the medial striatum, a proportionally greater depletion of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum, and a greater increase in activated microglia in the substantia nigra than wild-type mice. At 12 months of age, methamphetamine-treated GDNF(+/-) mice exhibited less motor activity and lower levels of tyrosine hydroxylase-immunoreactivity, dopamine, DOPAC, and serotonin than wild-type mice. Greater striatal dopamine transporter activity in GDNF(+/-) mice may underlie their differential response to methamphetamine. These data suggest the possibility that methamphetamine use in young adults, when combined with lower levels of GDNF throughout life, may precipitate the appearance of parkinsonian-like behaviors during aging.

Figure 1.

METH-induced hyperthermia and locomotor activity did not differ between GDNF^+/− and WT mice initially, but differences in locomotor activity were revealed as the mice aged. A, A significant increase in body temperature occurred in METH versus saline-treated mice that did not differ between genotypes (*p < 0.05 vs WT Saline and GDNF^+/− Saline). n = 5 per group. B, After the first injection, regardless of genotype, mice treated with METH were significantly more active than saline-treated mice (***p < 0.001). n = 5 per group. C, After the fourth injection, mice treated with METH, regardless of genotype, were hypoactive when compared with saline-treated mice (***p < 0.001). n = 5 per group. D, Two weeks after the injections (3 months of age), there were no differences in spontaneous motor activity among the groups challenged with saline. In contrast, METH binge-pretreated mice challenged with nomifensine 2 weeks later exhibited significantly less activity than those pretreated with saline (***p < 0.001). E, At 9 months of age, mice treated with METH, regardless of genotype, exhibited less locomotor activity than saline-treated mice (***p < 0.001). n = 8 per group. F, At 12 months of age, saline-pretreated GDNF^+/− mice displayed less motor activity than WT mice (**p < 0.01). METH-treated GDNF^+/− mice exhibited less motor activity than GDNF^+/− mice treated with saline or METH-treated WT mice (*p = 0.05; **p = 0.01; ***p = 0.001). n = 8 per group. Error bars indicate SEM.

Figure 2.

After METH treatment, 3- and 12-month-old GDNF^+/− mice demonstrated a greater loss of striatal TH-IR than WT mice. A–D, Photomicrographs of TH-IR in coronal hemisections of the striatum (medial to the left; lateral to the right) from 3-month-old saline-treated WT mice (A), saline-treated GDNF^+/− mice (B), METH-treated WT mice (C), and METH-treated GDNF^+/− mice (D). E, Quantitation of the average integrated density from the four treatment groups at 3 months of age confirmed that there were no differences in the lateral region (right) of the striatum between the GDNF^+/− and WT mice treated with saline, and METH induced a similar TH-IR depletion in the lateral striatum of GDNF^+/− and WT mice when compared with saline-treated mice (***p < 0.001). In the medial striatum (left), GDNF^+/− mice expressed less TH-IR than WT mice treated with saline (*p < 0.05), and METH induced a greater depletion in the medial striatum of GDNF^+/− than WT mice (*p < 0.05). F, Quantitation of the average integrated density of TH-immunoreactive sections from the four treatment groups at 12 months of age demonstrated that TH-IR levels were significantly less in both the lateral and medial striatum of GDNF^+/− mice versus WT mice treated with saline (*p < 0.05), that there was a residual deficit in TH-IR in METH- versus saline-treated WT mice in the lateral region of the striatum (right; *p < 0.05), and that there was significantly less TH-IR in the lateral striatum of GDNF^+/− versus WT mice treated with METH (*p = 0.05). Circles denote measurement areas of the lateral (L) and medial (M) regions of the striatum. n = 8 per group. Error bars indicate SEM.

Figure 3.

A–H, GDNF^+/− mice treated with METH exhibited an inflammatory response in the SN 2 weeks after injection. CD45-IR in the SN (A–D) and striatum (E–H) in saline (SAL)-treated WT mice (A, E), saline-treated GDNF^+/− mice (B, F), METH-treated WT mice (C, G), and METH-treated GDNF^+/− mice (D, H). Scale bar, 0.5 mm. I, J, Quantitation of the average integrated density of CD45-ir in the SN (I) and striatum (J). Saline-treated GDNF^+/− mice had more CD45-IR in the SN than saline-treated WT mice (*p < 0.05). METH treatment caused a significantly greater increase in CD45-IR in the SN of GDNF^+/− mice than in saline-treated GDNF^+/− mice or METH-treated WT mice (*p < 0.05). n = 5 per group. Error bars indicate SEM.

Figure 4.

METH treatment resulted in a significant reduction in striatal tissue levels of DA and 5-HT that did not differ between genotypes at 3 months of age (*p < 0.05). A, B, D, Saline (SAL)-treated GDNF^+/− mice had higher tissue levels of DA (A) and DOPAC (B) than WT mice (p < 0.05), but there was no difference in 5-HT (D) tissue levels. METH treatment caused a significant reduction in DA, DOPAC, and 5-HT tissue levels in WT and GDNF^+/− mice (p < 0.05). C, The DA/DOPAC ratio was not different between the genotypes treated with saline, and the ratio was reduced similarly in both genotypes by METH treatment (p < 0.05). D, 5-HT tissue levels were similar in saline-treated GDNF^+/− and WT mice and were decreased to the same extent by METH (*p = 0.05; ***p = 0.001). n = 5 per group. Error bars indicate SEM.

Figure 5.

METH-induced reductions in striatal DA, DOPAC, and 5-HT tissue levels are still present at 12 months of age. A–D, Saline (SAL)-treated GDNF^+/− mice have less striatal tissue levels of DA (A) and 5-HT (D) than WT mice, but the striatal levels of DOPAC (B) and the DA/DOPAC ratio (C) do not differ by genotype. METH-treated WT mice have less tissue DA, DOPAC, DA/DOPAC, and 5-HT than saline-treated WT mice (p < 0.05). Furthermore, DA, DOPAC, and 5-HT levels were significantly lower in METH-treated GDNF^+/− mice versus WT mice (*p = 0.05; ***p = 0.001). n = 8 per group. Error bars indicate SEM.

Figure 6.

A, Representative GC–MS–NiCl-selected ion-monitoring chromatogram of METH (top; retention time, 4.32 min) and internal standard deuterated METH (bottom; 4.28 min) extracted from a striatal microsample. B, C, Striatal METH concentration (micrograms per milligram of tissue) (B) and plasma METH concentration (microgram per milliliter) (C) did not differ between WT and GDNF^+/− mice at 30, 60, 120, or 180 min after the fourth METH injection. n = 5 per group per time point. Error bars indicate SEM.

Figure 7.

GDNF^+/− mice have greater DAT activity, but not DAT protein levels in the striatum than WT mice at 3 and 12 months of age. A, DAT activity in striatal synaptosomes from 3- and 12-month-old WT and GDNF^+/− mice. DA uptake was assayed in purified synaptosomes (n = 6) and the results are expressed as percentage of uptake in synaptosomes derived from WT mice (mean ± SEM). *p < 0.05 versus WT; ⁺p < 0.05 versus 3 month GDNF^+/−. B, DAT protein expression in striatal synaptosomes from 3- and 12-month-old WT and GDNF^+/− mice. Purified striatal synaptosomes were subjected to SDS-PAGE followed by immunoblotting with DAT antibody. Subsequently, the blots were stripped and reprobed with calnexin antibody. A representative DAT and calnexin immunoblot is shown. C, Quantitative analysis of DAT band densities. The density of DAT protein bands was quantified using NIH image (mean ± SEM) after normalization with calnexin. n = 6 per group. Error bars indicate SEM.