Distinct mechanisms mediating methamphetamine-induced neuronal apoptosis and dopamine terminal damage share the neuropeptide substance p in the striatum of mice

Abstract

Methamphetamine (METH) is an addictive psychostimulant that induces damage to the dopamine terminals and the apoptosis of some neurons of the striatum. Our laboratory demonstrated using either a single bolus dose (30 mg/kg) or a binge (10 mg/kg 4x at 2-h intervals) of METH that pharmacological blockade of the substance P receptor (neurokinin-1) attenuates METH-induced damage to both the presynaptic dopamine terminals and the apoptosis of some neurons of the striatum. To determine the phenotype of striatal neuron ablated by METH, we combined TUNEL (Terminal Deoxyncleotidyl Transferase-Mediated dUTP Nick End Labeling) with immunofluorescence for selective markers of projection and interneurons. METH induces the loss of approximately 20% of the projection neurons. The cholinergic and gamma-aminobutyric acid (GABA)-parvalbumin interneurons sustain losses of 30% and 50%, respectively. The somatostatin/neuropeptide Y (NPY)/nitric oxide synthase (NOS) interneurons are not impacted by METH. To investigate the mechanism by which substance P mediates METH-induced damage in this part of the brain, we ablated the striatal interneurons that express the neurokinin-1 receptor (NK-1R) with the selective neurotoxin substance P-SAP. Ablation of the NK-1R-expressing interneurons prevented METH-induced apoptosis in the striatum but was without effect on depletion of dopamine terminal markers. We propose that substance P mediates the apoptosis of some striatal neurons via the intrastriatal activation of nitric oxide synthesis. In contrast, substance P may mediate damage of the dopamine terminals via an extrastriatal mechanism involving the substantia nigra and cortical glutamate release.

FIGURE 1

The NK-1R antagonist WIN-51,708 (WIN) prevents METH-induced reduction of striatal dopamine transporter sites at day 3 post- METH (autoradiographic study). The mice (n = 6) received one injection of METH (30 mg/kg, i.p.). A separate group received WIN (5 mg/kg, i.p.) 30 min prior to METH. Binding of [¹²⁵I]RTI-121 to striatal dopamine transporter sites was quantitated by image analysis of X ray autoradiograms. VEH, vehicle; SEM, standard error of the mean values. *P < 0.05 (ANOVA).

FIGURE 2

Pharmacological blockade of the NK-1Rs with WIN-51,708 (WIN, 5 mg/kg) prevents METH-induced reduction of TH in the striatum at day 3 post-METH (Western blot analysis). The mice were treated as described in the legend to Figure 1. SEM, standard error of the mean values. *P < 0.05 (ANOVA).

FIGURE 3

Pretreatment with WIN-51,708 (5 mg/kg, i.p.) attenuates METH-induced apoptosis in the striatum. METH was injected i.p. at a dose of 30 mg/kg. Apoptosis was assessed at 24 h post-METH by TUNEL. SEM, standard error of the mean values. *P < 0.05 compared to METH.

FIGURE 4

Intrastriatal injection of SSP-SAP (B) depletes immunostaining for the NK-1Rs (D) rendering the striatal tissue resistant to METH-induced apoptosis (TUNEL, F). The contralateral side that received SAP (A), retains NK-1R immunoreactivity (C) and remains vulnerable to METH (E). The mice (n = 6) received one intrastriatal injection of SSP-SAP or just SAP in the contralateral striatum. The animals received METH (30 mg/kg, i.p.) 3 weeks after the intrastriatal injection of SSP-SAP and were sacrificed 24 h after METH. NK-1Rs were visualized by immunofluorescence with an antibody conjugated to Cy3.

FIGURE 5

Ablation of striatal NK-1R-expressing interneurons does not prevent METH-induced depletion of TH. The mice (n = 6) received an intrastriatal injection of SSP-SAP into one side and SAP into the contralateral side. Then, 3 weeks later, the mice received a single injection of METH (30 mg/kg, i.p.) and were sacrificed 3 days after METH. Striatal sections were processed for NK-1R and TH immunofluorescence in the same section. Note that SSP-SAP abolishes NK-1R staining without affecting staining for TH (b and d). NK-1Rs were visualized with an antibody conjugated to FITC (a, b, e & f) and TH with Cy3 (c, d, g & h).