Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity

Abstract

Production of nitric oxide (NO) has been implicated in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. The source of this NO has not been clearly delineated, but recent evidence suggests that it arises from activation of neuronal nitric oxide synthase (nNOS), which is selectively expressed in a subpopulation of striatal interneurons. Our objective was to determine whether inhibiting activation of nNOS-containing interneurons in the striatum blocks METH-induced neurotoxicity. These interneurons selectively express the neurokinin-1 (NK-1) receptor, which is activated by substance P. One particular toxin, a conjugate of substance P to the ribosome-inactivating protein saporin (SSP-SAP), selectively destroys neurons expressing the NK-1 receptor. Thus, we examined the extent to which depletion of the nNOS-containing interneurons alters production of NO and attenuates METH-induced neurotoxicity. The SSP-SAP lesions resulted in significant loss of nNOS-containing interneurons throughout striatum. Surprisingly, this marked deletion did not confer resistance to METH-induced DA neurotoxicity, even in areas devoid of nNOS-positive cells. Furthermore, these lesions did not attenuate NO production, even in areas lacking nNOS. These data suggest that nNOS-containing interneurons either are not necessary for METH-induced DA neurotoxicity or produce NO that can diffuse extensively through striatal tissue and thereby still mediate neurotoxicity.

Fig. 1

Body temperatures during METH administration. a Rectal temperatures over time for each of the four treatment groups sacrificed 7 days after the last injection: Blank-SAP/Saline (n = 2), Blank-SAP/METH (n = 2), SSP–SAP/Saline (n = 2), SSP–SAP/METH (n = 4). Rats were given intrastriatal infusions of Blank-SAP (control) or SSP–SAP (toxin) into one striatum followed by systemic administration of saline (4 × 1 ml/kg) or (±)-METH (4 × 7.5 mg/kg, s.c. at 2-hr intervals) 3 weeks later. * METH-treated rats significantly greater than saline-treated rats at each indicated time point, p < 0.05. b Rats were treated as described above for (a). Shown are rectal temperatures over time for each of the four treatment groups sacrificed 1 h after the last injection: Blank-SAP/Saline (n = 6), Blank-SAP/METH (n = 8), SSP–SAP/Saline (n = 6), SSP–SAP/METH (n = 9). * METH-treated rats significantly greater than saline-treated rats at each indicated time point, p < 0.05. Arrows indicate injection times

Fig. 2

Representative photomicrographs from an animal that received SSP–SAP into the infused striatum followed by a systemic injection of saline three weeks later. a NADPH diaphorase histochemistry reveals a clear loss of nNOS-containing interneurons in the SSP–SAP-infused hemisphere. b NeuN IHC performed on an adjacent section confirms the specificity of the SSP–SAP-induced lesion. No gross loss of neurons was evident in the infused striatum other than mechanical damage from the cannulae. c Data are NADPH diaphorase histochemical staining in the striata of rats sacrificed 7 days after treatment with METH or saline (4 weeks after infusion of Blank-SAP or SSP–SAP into one striatum), and are represented as number of NADPH diaphorase-positive cell bodies per mm² (mean ± SEM). * Significant main effect of pretreatment, p < 0.05

Fig. 3

Effects of SSP–SAP infusions on METH-induced loss of DAT immunohistochemical staining in the striatum. Rats were treated as described in the legend to Fig. 1 and in the Methods. Data are DAT immunohistochemical staining in the striata of animals sacrificed 7 days after treatment with METH or saline (4 weeks after infusion of Blank-SAP or SSP–SAP into one striatum), and are represented as average gray values (mean ± SEM; arbitrary units). * Significant main effect of treatment, p < 0.05

Fig. 4

Effects of SSP–SAP infusions on NADPH diaphorase histochemical staining in the striatum. Rats were treated as described in the legend to Fig. 1 and in the Methods. Data are NADPH diaphorase histochemical staining in the striata of animals sacrificed 1 h after treatment with METH or saline, and are represented as percent area of the imaged field with NADPH diaphorase histochemical signal (mean ± SEM). * Significant differences, p < 0.05

Fig. 5

Effects of SSP–SAP infusions on nitrotyrosine (NT) immunohistochemical staining in the striatum. Rats were treated as described in the legend to Fig. 1 and in the Methods. Data are NT immunohistochemical staining in the striata of animals sacrificed 1 h after treatment with METH or saline, and are represented as average gray values (mean ± SEM; arbitrary units). * Significant main effect of treatment, p < 0.05