Dopamine receptor activation reveals a novel, kynurenate-sensitive component of striatal N-methyl-D-aspartate neurotoxicity

Abstract

The N-methyl-d-aspartate (NMDA) subtype of glutamate receptors plays an important role in brain physiology, but excessive receptor stimulation results in seizures and excitotoxic nerve cell death. NMDA receptor-mediated neuronal excitation and injury can be prevented by high, non-physiological concentrations of the neuroinhibitory tryptophan metabolite kynurenic acid (KYNA). Here we report that endogenous KYNA, which is formed in and released from astrocytes, controls NMDA receptors in vivo. This was revealed with the aid of the dopaminergic drugs d-amphetamine and apomorphine, which cause rapid, transient decreases in striatal KYNA levels in rats. Intrastriatal injections of the excitotoxins NMDA or quinolinate (but not the non-NMDA receptor agonist kainate) at the time of maximal KYNA reduction resulted in two- to threefold increases in excitotoxic lesion size. Pre-treatment with a kynurenine 3-hydroxylase inhibitor or with dopamine receptor antagonists, i.e., two classes of pharmacological agents that prevented the reduction in brain KYNA caused by dopaminergic stimulation, abolished the potentiation of neurotoxicity. Thus, the present study identifies a previously unappreciated role of KYNA as a functional link between dopamine receptor stimulation and NMDA neurotoxicity in the striatum.

Figure 1

Pretreatment (1 h) with d-Amph dose-dependently decreases striatal KYNA levels and potentiates the striatal neurotoxicity of NMDA (1.4 nmol/1 μl). Controls received i.p. phosphate-buffered saline (PBS) instead of d-Amph. (A) Tissue KYNA content; (B) Behavioral assessment: Apo (1 mg/kg)-induced ipsilateral rotations; (C) Striatal lesion volume in animals tested in B. Data are the mean + SEM (n = 6 per group). *p < 0.05 vs. PBS.

Figure 2

(A) Representative cytochrome oxidase-stained tissue sections from three rostro-caudal levels of the NMDA-lesioned striatum. Sections were taken from animals pretreated with PBS or d-Amph (5 mg/kg). Animals were killed 4 days after the NMDA infusion; (B) Microscopic appearance of the striatum 4 days after an intrastriatal NMDA injection without and with d-Amph pretreatment. Representative cresyl violet (Nissl)-stained (top row) and tyrosine hydroxylase (TH)-stained (bottom row) sections of control (C), NMDA-lesioned (N), and d-Amph + NMDA-lesioned (AN) striata. Photomicrographs of Nissl-stained and TH-immunoreactive tissue were taken from nearby sections. Scenes in N and AN were photographed at the center of the lesioned area. Scale bar: 0.5 μm.

Figure 3

Prevention of d-Amph-induced effects on striatal KYNA levels and NMDA neurotoxicity by pre-treatment with the kynurenine 3-hydroxylase inhibitor Ro 61-8048. Ro 61-8048 (2 mg/kg) was administered 5 h before PBS or d-Amph (5 mg/kg). All animals received an intrastriatal injection of NMDA (1.4 nmol/1 μl) 1 h after PBS or d-Amph. (A) Ro 61-8048 increases striatal KYNA levels and reverses the effect of d-Amph on KYNA levels; (B) Ro 61-8048 prevents the pro-excitotoxic effect of d-Amph assessed behaviorally (Apo-induced rotations); (C) In the same animals tested in B, Ro 61-8048 prevents the pro-excitotoxic effect of d-Amph. Data are the mean + SEM (n = 6 per group). *p < 0.05 versus PBS, #p < 0.05 versus d-Amph.

Figure 4

Role of free radicals and NMDA receptor activation in the pro-excitotoxic effect of d-Amph in the striatum. In all animals, excitotoxins (NMDA: 1.4 nmol; QUIN: 90 nmol; kainate: 5 nmol) were injected (1 μl) intrastriatally 1 h after PBS or d-Amph (5 mg/kg). (A) The free radical scavenger IPA (80 mg/kg, i.p., 15 min before d-Amph) fails to prevent the potentiation of NMDA neurotoxicity by d-Amph (p > 0.05 vs. d-Amph); (B) The NMDA receptor antagonist GCP 40116 (20 mg/kg, i.p., 15 min before NMDA) blocks both the neurotoxicity of NMDA alone and the potentiation of NMDA neurotoxicity by d-Amph; (C) d-Amph potentiates QUIN- but not kainate-induced neurotoxicity. Ro 61-8048 (2 mg/kg, 5 h before d-Amph) prevents the potentiation of QUIN neurotoxicity by d-Amph. Data are the mean + SEM (n = 6 per group). *p < 0.05 vs. PBS, #p < 0.05 vs. d-Amph.

Figure 5

Dopamine receptors mediate the reduction of KYNA and the potentiation of striatal NMDA (1.4 nmol/1 μl) neurotoxicity. (A) Pretreatment with the D1 antagonist SCH 23390 (1 mg/kg) or the D2 antagonist raclopride (2 mg/kg), both administered 15 min before d-Amph (5 mg/kg), prevents the d-Amph-induced reduction of KYNA levels and the potentiation of NMDA neurotoxicity; (B) Dopamine receptor stimulation by Apo (1 mg/kg) causes a reduction in striatal KYNA after 1 h, and potentiates the striatal neurotoxicity of NMDA (injected 1 h after Apo). Ro 61-8048 (2 mg/kg, 5 h before Apo) prevents the potentiation of NMDA neurotoxicity by Apo. Data are the mean + SEM (n = 6 per group). *p < 0.05 versus PBS, #p < 0.05 vs. d-Amph or Apo.