Methamphetamine induces striatal neurokinin-1 receptor endocytosis primarily in somatostatin/NPY/NOS interneurons and the role of dopamine receptors in mice

Abstract

Methamphetamine (METH) is a psychostimulant that induces long-term deficits of dopamine terminal markers and apoptotic cell death in the striatum. Our laboratory demonstrated that pharmacological blockade of the neurokinin-1 receptor attenuated the METH-induced damage to the striatal dopamine terminals and the apoptotic cell death of some striatal neurons. Here, we used histological methods to assess the effect of METH on neurokinin-1 receptor trafficking in the striatum as an indirect index of signaling by the neuropeptide substance P (natural ligand for this receptor). Male mice received a single injection of METH (30 mg/kg, i.p.) and were sacrificed 30 min later. Immunohistofluorescence confocal microscopy confirmed that the neurokinin-1 receptor is located on cholinergic and somatostatin interneurons of the striatum. METH induced the trafficking of the neurokinin-1 receptor from the membrane into cytoplasmic endosomes primarily in the somatostatin/NPY/NOS interneurons, and this phenomenon was attenuated by antagonists of the dopamine D1 (SCH-23390), D2 (raclopride), or neurokinin-1 (WIN-51,708) receptors. These data demonstrate that METH induces the trafficking of the striatal neurokinin-1 receptors principally in the somatostatin/NPY/NOS interneurons and that this phenomenon is dependent on the activity of dopamine D1 and D2 receptors.

Figure 1

Striatal neurokinin-1 receptors (NK1R, A&D) are expressed by cholinergic (ChAT, B) and somatostatin (SOM, E) interneurons. Striatal sections were processed for immunofluorescence with antibodies against the neurokinin-1 receptor (FITC, green) and choline acetyltransferase (ChAT) or somatostatin (Cy3, red). Overlay of the images shows the expression of the striatal neurokinin-1 receptor within these two subpopulations of interneuron (C&F). Photomicrographs were obtained with the confocal microscope. Space bar: 100 µm. (n = 6)

Figure 2

METH induces trafficking of the neurokinin-1 receptor in cholinergic and somatostatin interneurons of the striatum. Mice received a single injection of METH (30 mg/kg, i.p.) and were sacrificed 30 minutes after the injection. Coronal sections of striatal tissue were processed for immunofluorescence with antibodies against the neurokinin-1 receptor and choline acetyltransferase (ChAT, A&B) or somatostatin (SOM, C&D). The staining shown is only for the neurokinin-1 receptor (FITC, green). Photomicrographs were obtained with the confocal microscope. Arrows indicate the position of representative endosomes within a somatostatin interneuron. Scale bars: 10 µm. (n = 8)

Figure 3

METH induced the endocytosis of the neurokinin-1 receptor primarily in the somatostain/NPY/NOS interneurons. Mice received a single injection of METH (30 mg/kg, i.p.) and were sacrificed 30 minutes after the injection. Coronal sections of striatal tissue were processed simultaneously for immunofluorescence of the neurokinin-1 receptor (NK1R) and somatostatin (SOM) or choline acetyltransferase (ChAT). Note that the somatostatin/NPY/NOS interneurons in the METH group contain more endosomes per soma than the cholinergic interneurons. The number of endosomes per soma was counted manually. !p<0.0001; *p<0.01; **p<0.05. (n = 8)

Figure 4

Pre-treatment with the neurokinin-1 receptor antagonist WIN-51,708 attenuated the METH-induced trafficking of the neurokinin-1 receptors. Mice received vehicle or a single injection of WIN-51,708 (5 mg/kg, i.p.) prior to METH (30 mg/kg, i.p.) and were sacrificed 30 minutes after the METH injection. Coronal sections through the striatum were processed for immunofluorescence for the neurokinin-1 receptor (FITC, green) and somatostatin (Cy3, red). A–D show a representative neuron from each condition. V/S, vehicle/saline (A); W/S, WIN-51,708/saline (B); V/M, vehicle/METH (C); W/M, WIN-51,708/METH (D). Note that blockade of the neurokinin-1 receptor with WIN-51,708 significantly attenuated the METH-induced trafficking of the neurokinin-1 receptors. Photomicrographs were obtained with the confocal microscope. Images are shown in dark field for greater contrast. Scale bar: 10 µm. (n = 6)

Figure 5

Pre-treatment with dopamine D1 or D2 receptor antagonists attenuated the METH-induced internalization of the neurokinin-1 receptors in the striatum. Animals received vehicle (A) or METH (D, 30 mg/kg, i.p.) and were sacrificed 30 minutes after the injection of METH. Note that METH induces neurokinin-1 receptor trafficking (D). In a separate set of animals, SCH-23390 (0.1 mg/kg, i.p.) was administered alone (B) or 30 minutes prior to METH (E). Similarly, raclopride (1 mg/kg, i.p.) was injected alone (C) or 30 minutes before METH (F). Note that blockade of dopamine D1 or D2 receptors (E & F, respectively) significantly attenuated the METH-induced internalization of the neurokinin-1 receptors. Confocal images were photographed in dark field to enhance contrast. Arrowheads indicate the position of endosomes in the cells. Note in D the position of the endosomes surrounding the perinuclear region (dark oval region surrounded by neurokinin-1 receptor staining). (n = 8)

Figure 6

Histogram showing the effect of dopamine D1 or D2 receptor antagonists on METH-induced trafficking of the neurokinin-1 receptors of the striatum. Treatment groups and conditions were as in Figure 5 above. Note that both SCH-23390 (D1 antagonist) and raclopride (D2 antagonist) were effective in preventing trafficking of the neurokinin-1 receptors in the presence of METH. V, vehicle; R, raclopride; S, saline; SCH, SCH-23390; M, METH; NK1R, neurokinin-1 receptor. !p<0.0001; *p<0.01.