Extracellular N-acetylaspartylglutamate released in the nucleus accumbens modulates the pain sensation: Analysis using a microdialysis/mass spectrometry integrated system

Abstract

Various small molecules act as neurotransmitters and orchestrate neural communication. Growing evidence suggests that not only classical neurotransmitters but also several small molecules, including amino acid derivatives, modulate synaptic transmission. As conditions of acute and chronic pain alter neuronal excitability in the nucleus accumbens, we hypothesized that small molecules released in the nucleus accumbens might play important roles in modulating the pain sensation. However, it is not easy to identify possible pain modulators owing to the absence of a method for comprehensively measuring extracellular small molecules in the brain. In this study, through the use of an emerging metabolomics technique, namely ion chromatography coupled with high-resolution mass spectrometry, we simultaneously analyzed the dynamics of more than 60 small molecules in brain fluids collected by microdialysis, under both the application of pain stimuli and the administration of analgesics. We identified N-acetylaspartylglutamate as a potential pain modulator that is endogenously released in the nucleus accumbens. Infusion of N-acetylaspartylglutamate into the nucleus accumbens significantly attenuated the pain induced by the activation of sensory nerves through optical stimulation. These findings suggest that N-acetylaspartylglutamate released in the nucleus accumbens could modulate pain sensation.

Keywords: N-acetylaspartylglutamate; Pain; analgesia; dopamine; imaging mass spectrometry; in vivo microdialysis; mass spectrometry; morphine; nucleus accumbens; optogenetics.

Figure 1.

Effects of the optical activation of sensory nerves on the pain threshold. (a) Construction of the AAV. (b) Expression of ChR2 (ET/TC)-EYFP and substance P in the lumbar DRG (ipsilateral side). Scale bar = 20 µm. (c) Expressions of ChR2 (ET/TC)-EYFP and myelin in the sciatic nerve. Scale bar = 20 µm. (d) von Frey thresholds in response to optical stimulation of the plantar surface (n = 8, t(14) = 7.183). Data are presented as mean ± SEM. ***p < 0.001; by two-tailed Student’s t-test. AAV: adeno-associated virus; EYFP: enhanced yellow fluorescent protein; GFP: green fluorescent protein; hSyn: human synapsin; DRG: dorsal root ganglion.

Figure 2.

Visualization of dopamine concentration changes by optogenetic stimulation in the mouse brain sections of noxious-stimulated mice. Imaging mass spectrometry visualized distribution of dopamine in coronal brain sections those containing N.Acc., from ChR2-expressing noxious-stimulated (middle) and EGFP-expressing control (upper) mice. Representative dopamine image of noxious-stimulated brain in high magnification and its corresponding reference atlas (Allen Brain Atlas, http://www.brain-map.org/) were also shown. Note that endogenous dopamine signal was normalized by an internal standard (deuterium-labeled dopamine signal homogeneously spayed by a robotic sprayer device, see also Methods section). The normalized signal intensities from caudoputamen (CP) and N.Acc. regions were quantified (lower right). Data are presented as mean ± SD. EGFP: enhanced green fluorescent protein; N.Acc.: nucleus accumbens.

Figure 3.

Profiling of dynamic changes of small molecules in the cerebral fluids of the N.Acc. in response to the optical activation of sensory nerves (pain stimuli) or systemic morphine injection (analgesia) by IC-HRMS analysis. (a, b) Experimental timeline of microdialysis experiment for mice with optogenetically induced pain (a) and morphine-induced analgesia (b). (c) Hierarchical clustering revealed a group of molecules shows up-regulation by pain stimuli in N.Acc., but not PL, and reduction by analgesia (yellow square). The clustering analysis was performed using log2 fold change values (pre/post). Red indicates increase and blue indicates decrease compared to the pre. (d) Time-course concentration changes of the clustered metabolites into the yellow square shown in (c). (e) The Venn diagram shows the number of decreased molecules by pain (blue) and increased molecules by analgesia (pink). Only NAAG and galactose-1-phosphate were identified as those significantly reduced by pain as well as elevated by analgesia. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; by one-way ANOVA with Bonferroni post hoc analysis. N.Acc.: nucleus accumbens; IC-HRMS: ion chromatography coupled with high-resolution mass spectrometry; PL: prelimbic cortex; ANOVA: analysis of variance; AAV: adeno-associated virus; NAAG: N-acetylaspartylglutamate.

Figure 4.

Effect of NAAG in the N.Acc. on optical activation of sensory nerves-mediated sensitivity to mechanical stimuli. (a) Experimental timeline. (b, c) Effect of sustained infusion of NAAG (0.05 or 0.1 mg/mL, n = 6) (b) or PMPA (1 mg/mL, n = 5) (c) into the N.Acc. on pain score in response to optical activation of sensory nerves and von Frey filament. Data are mean ± SEM. ***p < 0.001, ##p < 0.01, ###p < 0.001, ^§§§p < 0.001; by one-way ANOVA with Bonferroni post hoc analysis. NAAG: N-acetylaspartylglutamate; N.Acc.: nucleus accumbens; ANOVA: analysis of variance; CW: continuous wave; PMPA: 2-phosphonomethyl-pentanedioic acid; AAV6: adeno-associated virus serotype 6.