DETECTION OF RELEASE OF ASPARTATE AND
GLUTAMATE IN CAT SPINAL DORSAL HORN FOLLOWING
NOXIOUS STIMULATION OF CUTANEOUS AND MUSCULAR
AFFERENT FIBERS USING MICRODIALYSIS^*

SONG　XUE-JUN，SHU　YOU-SHENG,YIN　PING-BO^**?,ZHAO　ZHI-QI^***
(Shanghai Brain Research Institute, Chinese Academy of Sciences, Shanghai 200031;
**Key Laboratory of Neurobiology, Shanghai Institute of Physiology,
Chinese Academy of Sciences, Shanghai 200031)

ABSTRACT　　To investigate the possible mechanisms underlying the difference of NMDA and non-NMDA receptors in spinal nociception originating in skin and muscle, release of aspartate (Asp) and glutamate (Glu) in the spinal dorsal horn was detected by stimulation of cutaneous and muscular nerves in cats using microdialysis technique. Asp and Glu were increased respectively by (323±55)% and (169±16)% following stimulation of cutaneous nerve, but by (150±16)% and (218±42)% respectively following stimulation of muscular nerve. Asp increase was approximately three times higher than that of Glu following cutaneous nerve-stimulation (P<0.01), while Glu increase was approximately twice as high as that of Asp following muscular nerve-stimulation (P<0.05). It is likely that nociceptive cutaneous and muscular inputs preferentially elicite release of Asp and Glu respectively, resulting in a functional differentiation of NMDA and non-NMDA receptor in the mediation of different nociceptive information.
Key words:glutamate; aspartate; microdialysis; spinal nociception

微透析测试刺激皮肤和肌肉神经引起的
天门冬氨酸和谷氨酸在脊髓的释放^*

宋学军　舒友生　尹萍波^**　赵志奇^***

摘　要　　为分析NMDA和非NMDA受体在介导脊髓不同性质疼痛的机能分化，应用微透析技术，测量刺激皮肤和肌肉神经引起的天门冬氨酸(Asp)和谷氨酸(Glu)在脊髓背角的释放。电刺激皮肤神经兴奋C 纤维诱发的Asp 和 Glu 的释放分别是基础值的 (323±55)％ (P<0.01) 和 (169±16)%(P<0.05); 电刺激肌肉神经兴奋C纤维诱发的Asp和Glu的释放分别是基础值的(150±16)％(P<0.01)和(218±42)％ (P<0.05)。 兴奋皮肤传入引起的Asp释放明显高于Glu的释放(约3倍); 而兴奋肌肉传入引起的Glu释放明显高于Asp的释放(约2倍)。从而提示，皮肤伤害性传入主要引起Asp的释放增加，而肌肉的伤害性传入则主要引起Glu 的释放增加，它们分别主要作用于NMDA和非NMDA受体而介导不同的痛传入信息。
关键词: 天门冬氨酸; 谷氨酸; 微透析; 脊髓伤害性感受
学科分类号: Q423

　　A line of evidence has shown involvement of excitatory amino acids in spinal nociception^［1～7］. Our previous study demonstrated that NMDA receptor mainly mediates the spinal transmission of cutaneous nociceptive information whereas non-NMDA receptor mainly mediates the transmission of muscular nociceptive information^［8,9］. The present study was designed to investigate the possible mechanisms underlying the difference of NMDA and non-NMDA receptors in transmission of nociceptive information originating in cat skin and muscle. Aspartate (Asp) and glutamate (Glu) were collected from the extracellular space of the spinal dorsal horn (laminae I-VI) in vivo using microdialysis probes and were analyzed by means of high pressure liquid chromatography in combination with fluorescence detection technique.
　　Experiments were performed on 10 adult cats anesthetized with sodium pentobarbitone (40mg/kg, ip, initially). Radialis venous, carotid arterial and tracheal canals were inserted. The lumber and sacral spinal cord was exposed and transected at L_1-2 segments pretreated with 2% procaine. Animals ere mounted in the stereotaxic frame and artificially ventilated after neuromuscular paralysis with gallamine (4mg/kg, iv) and end tidal CO₂ level was kept at 3.5%～5.0%. A slow infusion pump continuously delivered both sodium pentobarbitone ［2mg/(kg^.h)］ and gallamine ［4mg/(kg^.h)］. The sural nerve (SR) and gastrocnemius-soleus nerve (GS) were isolated for electrical stimulation (100V, 0.2ms, 20Hz, 10 pulses/s for a period of 25min), in which C fibers were activated. Asp and Glu were measured by means of microdialysis in vivo. The single cannular probe (200μm o.d.) was inserted by a microdriver into the dorsal horn of the spinal cord (2200μm) at L_6-7. The parameters of dialysis membrane (Eicom, Japan) were 80000kD cut-off and 1.2mm length of dialysis. The ACSF perfusion was performed with a micropump (Eicom) at a speed of 1μl/min. Before being inserted into the spinal cord, the probe was immersed into a beaker of perfusion medium for wet and transferred to a beaker containing a solution of the standard sample for collection calibration. After the probe was inserted into the spinal cord, the perfusates were collected every 25min and stored at -70℃ for HPLC (Waters208) assay next day.

Fig.1　Concentrations of aspartate (Asp, A) and glutamate (Glu, B) in sequential dialysis samples collected during the 25 min epoches
The probe was inserted and positioned in the dorsal horn at time 0. Immediately after insertion, concentrations of Asp and Glu were detected (n=2). And about 90 min later, the concentrations got to a steady state.

　　The basal dialysate concentrations () of Asp and Glu were 52±6 nmol/L and 1?022±105 nmol/L, respectively. As shown in Fig.1, the high concentrations of Asp and Glu were detected and gradually attenuated to the stable level due to the damage of some neurons as the probe was inserted into the dorsal horn. The electrical stimulation with great intensity to activate C fibers significantly increased the release of Asp and Glu in the dorsal horn. The mean increases of Asp and Glu following stimulation of SR (n=10) were (323±55)% and (169±16)% of baseline (), and those of Asp and Glu following stimulation of GS (n=10) were (150±16)% and (218±42)% of baseline () (Fig.2). It is worth to note that the increase of Asp is three times higher than that of Glu following SR-stimulation (P<0.01), while the increase of Glu is twice as high as that of Asp following GS-stimulation (P<0.05).

Fig.2　Release of aspartate (Asp) and glutamate (Glu) by stimulation of C fibers of SR and GS in dorsal horn (Laminae I-VI)
Comparison of the difference of the levels of extracellular Asp and Glu induced by stimulation of the C fibers of SR and GS (n=10), respectively. The differences are statistical significant (^*P<0.05, ^**P<0.01).

　　The present results demonstrated that the different amounts of excitatory amino acids were released from cutaneous and muscular afferent terminals. The percentage of Asp increase by noxious stimulation of cutaneous nerve was much higher than that of Glu, whereas the percentage of Glu increase by noxious stimulation of muscular nerve was higher than that of Asp. It is likely that nociceptive cutaneous and muscular inputs preferentially elicite release of Asp and Glu, respectively. The pharmacological studies showed that Asp has high affinity to NMDA receptors and Glu has higher affinity to non-NMDA receptors in comparison with NMDA receptors^［10］. It is, therefore, conceivable that cutaneous nociceptive inputs induced more Asp release acting on NMDA receptor, and the muscular nociceptive inputs induced more Glu release acting mainly on non-NMDA receptor. This may be one of the mechanisms underlying the functional differentiation of NMDA and non-NMDA receptors. Previous studies demonstrated that inflammation-induced joint pain was reduced by non-NMDA receptor antagonist CNQX, but not by NMDA receptor antagonist APV in the rat^［11,12］. Also Glu release was significantly enhanced in the inflamed joint rat^［13］. These data further support our results. However, due to a number of Asp- and Glu-containing neurons in the dorsal horn, we are unable to get rid of Asp and Glu release from spinal interneurons in addition to the release from afferent terminals.

^*This project was partially supported by National Natural Science Foundation of China.
^***Correspondence author
^*国家自然科学基金资助
^***联系作者

作者单位：宋学军　舒友生　赵志奇　中国科学院上海脑研究所， 上海 200031
　　　　　尹萍波　中国科学院上海生理研究所神经生物学开放实验室， 上海 200031

REFERENCES

　［1］　Watkins JC, Evans RH. Excitatory amino acid transmitters. Ann Rev Pharmacol Toxicol, 1981, 21: 165～204.
　［2］　Aanonsen LM, Lei SZ, Wilcox GL. Excitatory amino acid receptors and nociceptive neurotransmission in rat spinal cord. Pain, 1990, 41: 309～321.
　［3］　Dougherty PM, Palecek J, Paleckova V, et al. The role of NMDA and non-NMDA receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal and electrical stimuli. J Neurosci, 1992, 12: 3025～3041.
　［4］　King AE, Lopez-Garcia JA. Excitatory amino acid receptor-mediate neurotransmission from cutaneous afferents in rat dorsal horn in vitro. J Physiol， 1993, 472: 443～457.
　［5］　Neugebauer V, Lucke T, Schaible HG. N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists block the hyperexcitability of dorsal horn neurons during development of acute arthritis in rat′s knee joint. J Neurophysiol, 1993, 70: 1365～1377.
　［6］　Radhakrishnan V, Henry JL. Excitatory amino acid receptor mediation of sensory inputs to functionally identified dorsal horn neurons in cat spinal cord. Neuroscience， 1993, 55: 531～544.
　［7］　Song XJ, Zhao ZQ. NMDA and non-NMDA receptors mediating nociceptive and non-nociceptive transmission in spinal cord of cat. Acta Pharmacol Sin(中国药理学报), 1993, 14: 481～485.
　［8］　Song XJ, Zhao ZQ. Differential effects of NMDA and non-NMDA receptor antagonists on spinal cutaneous vs muscular nociception in the cat, NeuroReport, 1993, 4: 17～20.
　［9］　Song XJ, Zhao ZQ. Interaction between substance P and excitatory amino acid receptors in modulation of nociceptive responses of cat spinal dorsal horn neurons． Neurosci Lett, 1993, 168: 49～52.
　［10］　Kushner L, Lerma J, Zukin S, et al. Coexpression of N-methyl-D-aspartate and phencyclindine receptors in Xenopus oocytes injected with rat brain mRNA. Pro Natl Acad Sci USA, 1988, 85: 3250～3254.
　［11］　Sluka KA, Westlund KN. Centrally administered non-NMDA but not NMDA receptor antagonists block peripheral knee joint information, Pain， 1993, 55: 217～225.
　［12］　Sluka KA, Jordan HH, Westlund KN. Reduction in joint swelling and hyperalgesia following post-treatment with non-NMDA glutamate receptor antagonist． Pain， 1994, 59: 95～100.
　［13］　Sluka KA, Westlund KN. Spinal cord amino acid release and content in an arthritis model: the effects of pretreatment with non-NMDA, NMDA and NK-1 receptor antagonists. Brain Res, 1993, 627: 89～103.

Received 1998-07-17　　Accepted 1998-09-17