Modulation of methamphetamine-induced nitric oxide production by neuropeptide Y in the murine striatum

Abstract

Methamphetamine (METH) is a potent stimulant that induces both acute and long-lasting neurochemical changes in the brain including neuronal cell loss. Our laboratory demonstrated that the neuropeptide substance P enhances the striatal METH-induced production of nitric oxide (NO). In order to better understand the role of the striatal neuropeptides on the METH-induced production of NO, we used agonists and antagonists of the NPY (Y1R and Y2R) receptors infused via intrastriatal microinjection followed by a bolus of METH (30 mg/kg, ip) and measured 3-NT immunofluorescence, an indirect index of NO production. One striatum received pharmacological agent while the contralateral striatum received aCSF and served as control. NPY receptor agonists dose dependently attenuated the METH-induced production of striatal 3-NT. Conversely, NPY receptor antagonists had the opposite effect. Moreover, METH induced the accumulation of cyclic GMP and activated caspase-3 in approximately 18% of striatal neurons, a phenomenon that was attenuated by pre-treatment with NPY2 receptor agonist. Lastly, METH increased the levels of striatal preproneuropeptide Y mRNA nearly five-fold 16 h after injection as determined by RT-PCR, suggesting increased utilization of the neuropeptide. In conclusion, NPY inhibits the METH-induced production of NO in striatal tissue. Consequently, production of this second messenger induces the accumulation of cyclic GMP and activated caspase-3 in some striatal neurons, an event that may precede the apoptosis of some striatal neurons.

Figure 1

NPY1 and 2 receptor agonists attenuate the METH-induced NO production in striatal neurons. All animals received aCSF injections in left striata and agonist in right striata 30 minutes prior to systemic injection of METH (30 mg/kg, ip). 3-NT was detected by immunohistofluorescence with the confocal microscope. Note that both agonists demonstrate a significant dose dependent effect on the production of 3-NT in the striatum at 4 hours post-drug treatment (n=6 per group). Analysis was performed from mean ±SEM. Differences between groups and concentrations were analyzed by Two-Way ANOVA. (* p<0.05; **p<0.01; ***p<0.001; **** p<0.0001 relative to aCSF + Saline).

Figure 2

NPY1 and 2 receptor antagonists enhance the METH-induced 3-NT production in striatal neurons. Low, Mid and High doses under the x-axis refer to concentrations of NPY1 receptor antagonist (BIBP3226: 340, 680 and 1360 μmol) or NPY2 receptor antagonist (BIIE0246: 1, 2 and 4 nmol). 3-NT fluorescent intensity increases in a dose-dependent manner with NPY receptor antagonist administration at 4 hours post-METH (30 mg/kg, ip) treatment (n=6 per group). Analysis was performed from mean ±SEM. Differences between groups and concentrations were analyzed by Two-Way ANOVA and Bonferroni’s post-hoc test. **p<0.01, ***p<0.001 relative to aCSF plus saline. !!p<0.01, !!!p<0.001 relative to aCSF plus antagonist.

Figure 3

Immunohistochemical co-localization of cyclic GMP with select markers of striatal projection (A) and interneurons (B-D). A), DARPP-32 in projection neurons; B), parvalbumin; C), nitric oxide synthase; and D) cholineacetyl transferase. Mice (n=6) were injected with METH (30 mg/kg, ip) and sacrificed at 4 and 8 hours post-injection. Stained neurons were counted using computerized unbiased stereology and the results normalized relative to saline-treated controls. Note that the percentage of striatal neurons staining positive for cyclic GMP increase between 4 and 8 hours in all neuronal populations except the SST/NPY/NOS interneurons where staining decreased between 4 and 8 hours. Differences between groups were analyzed by Two-Way ANOVA and Bonferroni’s post-hoc test. ***p<0.001; ****p<0.0001.

Figure 4

Exposure to METH significantly increases the number of striatal cells that co-localize cyclic GMP with activated caspase-3 and effect of NPY2R agonist. Control and experimental animals were given aCSF infusion into one striatum and 1.0 μl of 20μM NPY2R agonist into the contralateral striatum. Mice (n=6) received an injection of METH (30 mg/kg, ip) 30 minutes after the intrastriatal infusions and were sacrificed 8 hours after the injection. Activated caspase-3 and cyclic GMP were visualized in the same section of striatal tissue by immunofluorescence in the confocal microscope. Note that the number of striatal cells co-expressing these two markers increases nearly five-fold in the group exposed to METH and the NPY2R agonist significantly attenuates this effect of METH. Differences between groups were analyzed by t-test and Mann Whitney’s post-hoc test (**p<0.01).

Figure 5

Preproneuropeptide Y mRNA production is elevated at 4 and 16 hours after METH treatment. Mice (n=5) received an injection of METH (30 mg/kg, ip) and were sacrificed 4 or 16 hours after the injection. Striatal preproneuropeptide Y mRNA in control and METH-treated groups was measured by Taqman One-Step RT-PCR and normalized to GAPDH. Differences between groups were analyzed by Two-Way ANOVA and Bonferroni’s post-hoc test. There is a significant effect from control and between time points (**p<0.01; ***p<0.001).