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Inhibition of spinal monosynaptic reflex in newborn rats by aurintricarboxylic acid
Neuroscience Research 27 (1997) 371 – 376
Inhibition of spinal monosynaptic reflex in newborn rats by aurintricarboxylic acid Xiaofen Sun a, Yoshio Harada b, Yuhei Miyata a,* a
Department of Pharmacology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113, Japan b Department of Physiology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113, Japan Received 9 December 1996; accepted 4 February 1997
Abstract The effect of aurintricarboxylic acid (ATA), an inhibitor of nuclease, on glutamatergic synaptic transmission was examined electrophysiologically in the isolated spinal cords of newborn rats. Monosynaptic reflex (MSR) was depressed about 20%, 50 min after exposure to 100 mM of ATA. Pretreatment with APV, a N-methyl-D-aspartate (NMDA) type receptor antagonist, depressed MSR by about 10%, but additional application of ATA did not affect the MSR further. In contrast, the remaining MSR following treatment with DNQX, a non-NMDA type receptor antagonist, in the Mg2 + -free medium was almost completely inhibited by addition of ATA. ATA depressed NMDA- but not D,L-a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA)- or kainate-induced depolarization in the medium containing normal ionic composition. Thus it is concluded that the reduction of MSR by ATA is due to blockade of NMDA type but non-NMDA type glutamate receptors. The present study also confirmed the previous conclusion that Ia monosynaptic transmission in the spinal cord of the newborn rat is mediated by NMDA as well as non-NMDA type glutamate receptors. © 1997 Elsevier Science Ireland Ltd. Keywords: Aurintricarboxylic acid (ATA); Glutamate receptors; N-methyl-D-aspartate (NMDA); methyl-4-isoxalone propionic acid (AMPA); Kainate (KA); Monosynaptic reflex (MSR); Spinal cord
1. Introduction Triphenylmethane dye aurintricarboxylic acid (ATA) is the general inhibitor of nuclease in vitro (Hallick et al., 1977). ATA is used to protect various kinds of cells in vitro from apoptotic cell death in which DNA fragmentation occurs (Batistatou and Greene, 1991; Kure et al., 1991; McConkey et al., 1989; Roberts-Lewis et al., 1993; Samples and Dubinsky, 1993). Besides this action, recent binding studies have revealed that ATA selectively binds to N-methyl-D-aspartate (NMDA) receptors, a subtype of glutamate receptors (Zeevalk et al., 1993; 1995). Furthermore, a whole-cell patch-clamp
* Corresponding author. Tel.: + 81 3 38222131 (5277); fax: +81 3 58141684; e-mail: [email protected]
D,L-a-amino-3-hydroxy-5-
study has shown that ATA depresses NMDA-induced currents in cortical neurons (Csernansky et al., 1994). In the central nervous system (CNS), glutamate is a major excitatory transmitter (Cotman and Monaghan, 1987; Dagani and D’Angelo, 1992; Fagg and Foster, 1983; Fonnum, 1984; Greenamyre and Porter, 1994). Overstimulation of glutamate receptors, particularly the NMDA type, causes an influx of Ca2 + ions, which in turn activates Ca2 + -dependent endonuclease and results in neuronal death (Choi, 1990; 1992; Greenamyre and Porter, 1994). Thus, it is suggested that ATA prevents the neuronal death induced by NMDA receptor activation through two different mechanisms: inhibition of endonuclease and blockade of NMDA receptors (Zeevalk et al., 1993). Therefore, if ATA is therapeutically used to prevent the neuronal death, it is possible that ATA may inhibit glutamatergic synaptic transmission mediated by NMDA type receptors in the
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CNS. However, such an action of ATA is not known yet. Group Ia sensory fibers mediating signals from the muscle spindle make monosynaptic connections with spinal motoneurons. The transmitter of the Ia synapses is supposed to be glutamate (Jessell et al., 1986; Jiang et al., 1990). In newborn rats, previous pharmacological studies have shown that glutamate receptors on motoneurons for the monosynaptic transmission are NMDA as well as non-NMDA types (Harada, 1992; Jiang et al., 1990). In the present study, two questions are posed: (1) does ATA inhibit the glutamatergic monosynaptic transmission of the newborn rat spinal cord? and (2) if so, is the action mediated by blocking of NMDA type glutamate receptors? A preliminary account of this study has been communicated elsewhere (Sun et al., 1995).
2. Materials and methods
of the preamplifier was stored in a transient memory device and then recorded with a pen recorder using an expanded time-scale (Fig. 1). Agonists used were NMDA (10 mM), D,L-a-amino-3hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) (1–3 mM) and kainate (KA) (3–10 mM). In our preliminary experiments, doses of the agonists applied were selected for motoneurons to depolarize submaximally. (9 ) 2-amino-5-phosphonopentanoic acid (APV, 100 mM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 6 mM) were used as antagonists, of which doses were selected to reduce the glutamate-induced response significantly. The dose of ATA used was 100 mM, which is preferentially used to prevent excitotoxic cell death (Kure et al., 1991; Zeevalk et al., 1993; 1995) and apoptosis caused by nerve growth factor (NGF) deprivation (Batistatou and Greene, 1991). ATA and/or the antagonists dissolved in either the normal or the modified ACSF were continuously applied to the preparation by perfusion, whereas the agonists were applied for 1 min. Experiments were ended within 1 h after ATA application (see Section 3).
2.1. Preparation 2.3. Statistical analysis Experiments were done on the isolated spinal cord of newborn rats (Otsuka and Konishi, 1974). Newborn rats of Wistar strain (3 – 5 days old) were anaesthetized with ether. The spinal cord below the thoracic region with roots was isolated and placed in a small chamber of which volume was about 0.4 ml. The cord was perfused with artificial cerebrospinal fluid (ACSF) saturated with 100% O2 at a flow rate of 4 ml/min. The composition of the ACSF (pH 7.4) was as follows (mM): NaCl 157.7, KCl 3.4, CaCl2 2.5, MgCl2 1.0, Glucose 10.0 and HEPES 5.0. This medium is referred to as the ‘normal ACSF ’ in the text. All the experiments were performed at room temperature (24–25°). In some experiments, the ACSF without Mg2 + ions was used to release Mg2 + -block of NMDA receptors (Ault et al., 1980; Mayer et al., 1984; Nowak et al., 1984; Reynolds, 1990); the ACSF without Ca2 + ions was used to block synaptic transmission.
Amplitudes of MSR and agonist-induced depolarizations under test were expressed as percent of the control in the normal ACSF just before applying a test solution. The mean values of the results obtained under
2.2. Electrophysiology Potentials generated in the motoneurons either by stimulation of a dorsal root or by application of drugs were recorded extracellularly from the corresponding ventral root with a conventional suction electrode which was connected to a preamplifier (Otsuka and Konishi, 1974). The output was fed to a D.C. penrecorder. Monosynaptic reflex (MSR) was elicited at every 60 s by stimulation of either a lumbar fourth or fifth dorsal root with a rectangular pulse of 0.1 ms in duration. The intensity to the root was sufficient to evoke maximal MSR. For recording MSR, the output
Fig. 1. Sample records of MSR (A) and NMDA-induced depolarizations (B). (a) Control in the normal ACSF; (b) Under 100 mM of ATA. Ab and Bb were taken 60 and 20 min after ATA application, respectively. NMDA (10 mM) was applied for 1 min as indicated by the horizontal bars in B. Sharp vertical traces in each record are artifacts of DR stimulation. Calibration bars indicate 1 mV and 10 ms in A or 1 min in B.
X. Sun et al. / Neuroscience Research 27 (1997) 371–376
Fig. 2. Effects of ATA on MSR. (A) A typical example of ATA action on MSR with time. (B) Time course of MSR reduction within 50 min after ATA (100 mM) application (n= 6). Circles with vertical bars indicate mean9 S.D. () MSR in the normal ACSF. () MSR under the treatment with ATA. Ordinate expresses MSR amplitude in per cent. (*) Significantly different from the control at P B0.05.
ATA treatment and those of the control just before ATA application were statistically examined by the paired t-test with the significance level of P B0.05 or PB 0.01.
3. Results
3.1. Effects of ATA on MSR As shown in sample records of Fig. 1A, 100 mM of ATA depressed MSR (Ab). A typical example of ATA action on MSR with time is illustrated in Fig. 2A. It was reduced to about 40%, 150 min after the application. The time course of the reduction by ATA was not monotonic, that is, initially MSR was reduced gradu-
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ally but after about 60 min steeply. The MSR once reduced was hardly recovered for more than a few hours after cessation of ATA treatment (Fig. 2A). The different reduction rates may suggest that the underlying mechanism to reduce MSR within 1 h is different from that of the later phase. This notion is supported by the following observation (data not shown). The depolarization induced by g-aminobutyric acid (GABA) or acetylcholine (ACh) was not affected within 1 h of ATA treatment. However, thereafter, responses to GABA or ACh were gradually decreased and did not restore the control level after washing. Therefore, in this study, analysis of ATA action on the MSR was restricted only to the initial phase within 1 h after ATA application (see Section 4). Fig. 2B shows the time course of the MSR reduction within 50 min after ATA application. MSR at 20 min after ATA application was 96.393.2% (mean9S.D.) of the control (PB 0.05, n= 6). It was further reduced with a similar rate for another 30 min. The amplitudes at 30 and 50 min after ATA application were 93.095.5 and 81.299.8%, respectively. The reduction of MSR may be due to blockade of glutamate receptors by ATA (see Section 1). To test this possibility, the spinal cords were treated with either NMDA or non-NMDA type receptor antagonists. APV, a NMDA type receptor antagonist, at a dose of 100 mM reduced MSR by about 10% as early as 10 min after the application and the MSR remained at this level thereafter (Fig. 3A) (90.69 2.9% at 0 min in Fig. 3A, PB 0.05, n=6). ATA did not affect it further (Fig. 3A). In the next experiments, the spinal cords were treated with 6 mM of DNQX, a non-NMDA type receptor antagonist, under the Mg2 + -free condition. As can be seen in Fig. 3B, the Mg2 + -free medium did not affect MSR. Under this condition, DNQX immediately reduced MSR to about half of the control. This can be interpreted that the reduction is due to blocking of non-NMDA type receptors, while the NMDA type component supposedly remains to be activated. Under this condition, ATA strongly reduced MSR with time (Fig. 3B), which was significant at 20 min. MSR 50 min after ATA treatment was only 21.69 2.9% of the value just before ATA application (PB 0.01, n=3). Thus, these results indicate that ATA reduces MSR by blocking the NMDA type receptors without affecting the non-NMDA type receptors.
3.2. Effects of ATA on depolarizations induced by glutamate agonists To verify further the notion that the NMDA type receptor is the target of ATA, we examined antagonisms between ATA and exogenously applied agonists for each subtype of glutamate receptors. All the glutamate agonists depolarized motoneurons. The response
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to NMDA in the normal ACSF was markedly inhibited by 100 mM of ATA (Fig. 1B). The mean peak amplitude was 16.19 20.0% of the control (P B 0.01, n= 8) (Fig. 4). On the other hand, the depolarization induced by a non-NMDA type agonist, AMPA or KA, was little depressed (Fig. 4). Thus, these results further support the notion that ATA specifically inhibits NMDA type receptors. The site of ATA action is supposed to be on the motoneurons. To ascertain this, the effect of ATA on the NMDA-induced depolarization was examined un-
Fig. 4. Effects of ATA on the depolarizations induced by glutamate agonists in the normal ACSF. The open columns illustrate the depolarizations induced by each agonist (100%) and stippled columns those under ATA. Observations were made 20, 30 and 40 min after ATA application for NMDA, AMPA and KA, respectively. Each column with a vertical bar indicates mean 9 S.D. The numbers of preparations for NMDA, AMPA and KA experiments were 8, 6 and 6, respectively. (**) Significantly different from the control at PB 0.01.
der the Ca2 + -free condition in which synaptic transmission to motoneurons is blocked. NMDA depolarized motoneurons even in the Ca2 + -free ACSF, although it was about 30% of the control in the normal ACSF (29.79 4.8%, n=3) (Fig. 5). Thus, the result indicates that NMDA depolarizes motoneurons directly and indirectly. The response to NMDA under the Ca2 + -free condition was also inhibited by ATA. It was 15.19 1.4% of the value in the Ca2 + -free medium (PB0.01, n= 3) (Fig. 5). The degree of the reduction was similar to that in the normal ACSF (see above, Fig. 4). Unexpectedly, it was found that ATA depressed the depolarization induced by AMPA in the Ca2 + -free medium. The reduction was 32.7 9 12.8% (PB 0.01) and 27.8 9 7.7% (PB0.05) at 1 and 3 mM of AMPA controls in the Ca2 + -free ACSF, respectively (n= 5) (Fig. 5). KAinduced depolarization was not affected by ATA (Fig. 5).
4. Discussion
Fig. 3. Effects of glutamate receptor antagonists and ATA on MSR. (A) Effects of APV and ATA on MSR. () MSR amplitude (100%) in the normal ACSF. (,) MSR amplitudes from the spinal cords treated with APV (100 mM ). () MSR under the treatment with APV and ATA. (B) Effects of DNQX and ATA on MSR. () MSR amplitude (100%) in the normal ACSF. (,) MSR in the Mg2 + free ACSF. (2) MSR under the treatment with DNQX (6 mM) in the Mg2 + -free ACSF. () MSR under the treatment with DNQX and ATA in the Mg2 + -free ACSF. (**) Significantly different from the control just before ATA application (PB 0.01). Each symbol with a vertical bar indicates mean 9 S.D. (n= 3). Abscissae indicate time after ATA applied (0 min). Ordinates indicate MSR amplitude (%).
The present study revealed that ATA inhibits monosynaptic transmission in the in vitro spinal cord. The MSR reduction is supposed to be composed of two phases with respect to the time course: initial slow and later steep reduction. The depolarization induced by GABA or ACH was not affected by ATA during the initial phase, whereas it was later. It has been reported that ATA at a dose of 100 mM does not inhibit phosphofructokinase (PFK) activity of retinal cells treated for 1 h (Zeevalk et al., 1995), whereas it inhibits the enzyme in cell-free system (McCune et al., 1989; Zeevalk et al., 1995). These may suggest that ATA can
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not penetrate cells within 1 h. Thus, the latter phase seems to be complicated. Therefore, we did not examine the mechanism for the latter phase of MSR reduction, only the initial phase. It is generally accepted that MSR in the spinal cord is mediated by glutamate as a transmitter of group Ia sensory fibers (see Section 1). The present study showed that MSR was reduced by antagonists for both types of glutamate receptors, confirming the notion that both are involved in monosynaptic transmission in the newborn rats (Harada, 1992; Jiang et al., 1990), though non-NMDA type is dominant. To reduce synaptic transmission, a drug must act on sites either presynaptic to reduce transmitter release or postsynaptic to inhibit generation of excitatory postsynaptic potentials. In the spinal cord, GABA-mediated presynaptic inhibitory mechanism is operating (Miyata and Otsuka, 1975; Watson, 1992). ATA did not affect membrane potentials of the motoneurons (data not shown) like GABA depolarizes them in embryonic and neonatal rats (Wu et al., 1992; Gao and Ziskind-Conhaim, 1995). Thus, ATA can not activate the presynaptic inhibition. ATA did not reduce MSR further in the
Fig. 5. Effects of ATA on the depolarizations induced by glutamate agonists in the Ca2 + -free ACSF. Each open column illustrates the amplitude of agonist-induced depolarization in the normal ACSF. Stippled and hatched columns indicate depolarizations with and without ATA in the Ca2 + -free ACSF. They express per cent of the corresponding control values in the normal ACSF. In cases of AMPA and KA, tha non-NMDA type receptor antagonist, in the Mg2 + -free medium was almost completely inhibited by addition of ATA. ATA depressed NMDA- but not AMPA- or kainate-induced depolarization in the medium containing normal ionic composition. Thus, it is concluded that the reduction of MSR by ATA is due to blockade of NMDA type but not non-NMDA type glutamate receptors. And, the present study confirmed the previous conclusion that Ia monosynaptic transmission in the spinal cord of the newborn rat is mediated byisolated spinal cords of newborn rats. Monosynaptic reflex (MSR) was depressed about 20%, 50 min after exposure to 100 mM of ATA. Pretreatment with APV, a NMDA type receptor antagonist, depressed MSR by about 10%, but additional application of ATA did not affect the MSR further. In contrast, the remaining MSR following treatment with DNQX, DA type as well as non-NMDA type glutamate receptors.
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spinal cord pretreated with APV (Fig. 3A), indicating that the transmitter release is not affected by ATA. Finally, ATA blocked NMDA-induced depolarization in the Ca2 + -free medium, indicating that ATA exerts direct action on the motoneurons. Therefore, it is concluded that the MSR reduction by ATA is, if not all, due to blockade of transmitter action on the motoneurons. The following results indicate that the target of ATA is NMDA type glutamate receptors. The MSR depressed by APV, a NMDA type receptor antagonist, was not affected further by ATA. In contrast, ATA greatly diminished the MSR in the spinal cord treated with DNQX and the Mg2 + -free ACSF, by which NMDA-type receptors remained to be activated. Furthermore, ATA greatly reduced the depolarization induced by NMDA, but did not that by AMPA or KA in the normal ACSF. Thus, it is concluded that ATA blocks NMDA type receptors specifically in the normal ionic condition. It was found that ATA reduced the depolarization induced by AMPA in the preparation perfused with the Ca2 + -free medium but not with the normal ACSF. The underlying mechanism for the reduction is not clear from the present study. A binding study has shown that IC50 for displacement of AMPA by ATA requires a 10-fold higher concentration than that for a NMDA ligand, whereas a much higher dose of ATA is necessary for displacement of KA (Zeevalk et al., 1995). Thus, it is likely that AMPA but not KA has affinity to the NMDA type receptor, though weak (Zeevalk et al., 1995). This affinity might be strengthened under the Ca2 + -free condition by some unknown mechanisms. Glutamate is known to be excitotoxic on many types of neurons to cause neuronal death (Ankarcrona et al., 1995; Choi, 1990; Csernansky et al., 1994; Kure et al., 1991; Portera-Cailliau et al., 1995; Roberts-Lewis et al., 1993; Samples and Dubinsky, 1993; Zeevalk et al., 1993; 1995). ATA inhibits Ca2 + -dependent endonuclease by which DNA fragmentation occurs and leads cells to apoptosis (McConkey et al., 1989). Therefore, the present study supports the notion that apoptotic cell death induced by excitotoxic glutamate action can be prevented by ATA through two different mechanisms, blockade of NMDA type glutamate receptors and inhibition of Ca2 + -dependent endonuclease (Zeevalk et al., 1993). Furthermore, it casts a caution that therapeutic use of ATA may cause adverse reaction due to blockade of glutamatergic synaptic transmission. References Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. and Nicotera, P. (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron, 15: 961–973.
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Ault, B., Evans, R.H., francis, A.A., Oakes, D.J. and Watkins, J.C. (1980) Selective depression of excitatory amino acid depolarizations by magnesium ions in isolated spinal cord preparations. J. Physiol., 307: 413–428. Batistatou, A. and Greene, L.A. (1991) Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J. Cell Biol., 115: 461–471. Choi, D.W. (1990) The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci., 13: 171 – 182. Choi, D.W. (1992) Excitotoxic cell death. J. Neurobiol., 23: 1261 – 1276. Cotman, C.W. and Monaghan, D.T. (1987) Chemistry and anatomy of excitatory amino acid systems. In: H.Y. Meltzer (Ed.), Psychopharmacology, The Third Generation of Progress, Raven Press, New York, pp. 197–210. Csernansky, C.A., Canzoniero, L.M.T., Sensi, S.L. and Choi, D.W. (1994) Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury. J. Neurosci. Res., 38: 101 – 108. Dagani, F. and D’Angelo, E. (1992) Glutamate metabolism, release, and quantal transmission at central excitatory synapses: Implications for neural plasticity. Funct. Neurol., 7: 315–336. Fagg, G.E. and Foster, A.C. (1983) Amino acid neurotransmitters and their pathway in the mammalian central nervous system. J. Neurosci., 9: 701–719. Fonnum, F. (1984) Glutamate: A neurotransmitter in mammalian brain. J. Neurochem., 42: 1–11. Gao, BX. and Ziskind-Conhaim, L. (1995) Development of glycineand GABA-gated currents in rat spinal motoneurons. J. Neurophysiol., 74: 113–121. Greenamyre, J.T. and Porter, R.H.P. (1994) Anatomy and physiology of glutamate in the CNS. Neurology, 44 (suppl. 8): S7 – S13. Hallick, R.B., Chelm, B.K., Gray, P.W. and Orozco, E.M. (1977) Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res., 4: 3055–3064. Harada, Y. (1992) Two types of glutamate receptor mediate Ia synaptic transmission in spinal cord of newborn rat. Jpn. J. Physiol., 42 (suppl): S140. Jessell, T.M., Yoshioka, K. and Jahr, C.E. (1986) Amino acid receptor-mediated transmission at primary afferent synapses in rat spinal cord. J. Exp. Biol., 124: 239–258. Jiang, Z.G., Shen, E. and Dun, N.J. (1990) Excitatory and inhibitory transmission from dorsal root afferents to neonate rat motoneurons in vitro. Brain Res., 535: 110–118. Kure, S., Tominaga, T., Yoshimoto, T., Tada, K. and Narisawa, K. (1991) Glutamate triggers internucleosomal DNA cleavage in neuronal cells. Biochem. Biophys. Res. Commun., 179: 39 – 45.
Mayer, M.L., Westbrook, G.L. and Guthrie, P.B. (1984) Voltagedependent block by Mg2 + of NMDA responses in spinal cord neurones. Nature, 309: 261 – 263. McConkey, D.J., Hartzell, P., Nicotera, P. and Orrenius, S. (1989) Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J., 3: 1843 – 1849. McCune, S.A., Foe, L.G., Kemp, R.G. and Jurin, R.R. (1989) Aurintricarboxylic acidis a potent inhibitor of phosphofructokinase. Biochem. J. 259: 925 – 927. Miyata, Y. and Otsuka, M. (1975) Quantitative histochemistry of g-aminobutyric acid in cat spinal cord with special reference to presynaptic inhibition. J. Neurochem., 25: 239 – 244. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A. (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature, 307: 462 – 465. Otsuka, M. and Konishi, S. (1974) Electrophysiology of mammalian spinal cord in vitro. Nature, 252: 733 – 734. Portera-Cailliau, C., Hedreen, J.C., Price, D. and Koliatsos, V.E. (1995) Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci., 15: 3775–3787. Reynolds, I. J. (1990) Modulation of NMDA receptor responsiveness by neurotransmitters, drugs and chemical modification. Life Sci., 47: 1785 – 1792. Roberts-Lewis, J.M., Marcy, V.R., Zhao, Y. and Vaught, J.L., Siman, R. and Lewis, M.E. (1993) Aurintricarboxylic acid protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo. J. Neurochem., 61: 378 – 381. Samples, S.D. and Dubinsky, J.M. (1993) Aurintricarboxylic acid protects hippocampal neurons from glutamate excitotoxicity in vitro. J. Neurochem., 61: 382 – 385. Sun, X., Harada, Y. and Miyata, Y. (1995) Differential actions of aurintricarboxylic acid on glutamate receptors of motoneurons of newborn rats. Fourth IBRO World Congr. Neurosci. Abstr., A3.54. Watson, A.H.D. (1992) Presynaptic modulation of sensory afferents in the invertebrate and vertebrate nervous system. Comp. Biochem. Physiol., 103A: 227 – 239. Wu, WL., Ziskind-Conhaim, L. and Sweet, M.A. (1992) Early development of glycine- and GABA-mediated synapses in rat spinal cord. J. Neurosci., 12: 3935 – 3945. Zeevalk, G.D., Schoepp, D. and Nicklas, W.J. (1993) Aurintricarboxylic acid prevents NMDA-mediated excitotoxicity: Evidence for its action as an NMDA receptor antagonist. J. Neurochem., 61: 386 – 389. Zeevalk, G.D., Schoepp, D. and Nicklas, W.J. (1995) Excitotoxicity at both NMDA and non-NMDA glutamate receptors is antagonized by aurintricarboxylic acid: Evidence for differing mechanisms of action. J. Neurochem., 64: 1749 – 1758.
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Inhibition of spinal monosynaptic reflex in newborn rats by aurintricarboxylic acid Xiaofen Sun a, Yoshio Harada b, Yuhei Miyata a,* a
Department of Pharmacology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113, Japan b Department of Physiology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113, Japan Received 9 December 1996; accepted 4 February 1997
Abstract The effect of aurintricarboxylic acid (ATA), an inhibitor of nuclease, on glutamatergic synaptic transmission was examined electrophysiologically in the isolated spinal cords of newborn rats. Monosynaptic reflex (MSR) was depressed about 20%, 50 min after exposure to 100 mM of ATA. Pretreatment with APV, a N-methyl-D-aspartate (NMDA) type receptor antagonist, depressed MSR by about 10%, but additional application of ATA did not affect the MSR further. In contrast, the remaining MSR following treatment with DNQX, a non-NMDA type receptor antagonist, in the Mg2 + -free medium was almost completely inhibited by addition of ATA. ATA depressed NMDA- but not D,L-a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA)- or kainate-induced depolarization in the medium containing normal ionic composition. Thus it is concluded that the reduction of MSR by ATA is due to blockade of NMDA type but non-NMDA type glutamate receptors. The present study also confirmed the previous conclusion that Ia monosynaptic transmission in the spinal cord of the newborn rat is mediated by NMDA as well as non-NMDA type glutamate receptors. © 1997 Elsevier Science Ireland Ltd. Keywords: Aurintricarboxylic acid (ATA); Glutamate receptors; N-methyl-D-aspartate (NMDA); methyl-4-isoxalone propionic acid (AMPA); Kainate (KA); Monosynaptic reflex (MSR); Spinal cord
1. Introduction Triphenylmethane dye aurintricarboxylic acid (ATA) is the general inhibitor of nuclease in vitro (Hallick et al., 1977). ATA is used to protect various kinds of cells in vitro from apoptotic cell death in which DNA fragmentation occurs (Batistatou and Greene, 1991; Kure et al., 1991; McConkey et al., 1989; Roberts-Lewis et al., 1993; Samples and Dubinsky, 1993). Besides this action, recent binding studies have revealed that ATA selectively binds to N-methyl-D-aspartate (NMDA) receptors, a subtype of glutamate receptors (Zeevalk et al., 1993; 1995). Furthermore, a whole-cell patch-clamp
* Corresponding author. Tel.: + 81 3 38222131 (5277); fax: +81 3 58141684; e-mail: [email protected]
D,L-a-amino-3-hydroxy-5-
study has shown that ATA depresses NMDA-induced currents in cortical neurons (Csernansky et al., 1994). In the central nervous system (CNS), glutamate is a major excitatory transmitter (Cotman and Monaghan, 1987; Dagani and D’Angelo, 1992; Fagg and Foster, 1983; Fonnum, 1984; Greenamyre and Porter, 1994). Overstimulation of glutamate receptors, particularly the NMDA type, causes an influx of Ca2 + ions, which in turn activates Ca2 + -dependent endonuclease and results in neuronal death (Choi, 1990; 1992; Greenamyre and Porter, 1994). Thus, it is suggested that ATA prevents the neuronal death induced by NMDA receptor activation through two different mechanisms: inhibition of endonuclease and blockade of NMDA receptors (Zeevalk et al., 1993). Therefore, if ATA is therapeutically used to prevent the neuronal death, it is possible that ATA may inhibit glutamatergic synaptic transmission mediated by NMDA type receptors in the
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CNS. However, such an action of ATA is not known yet. Group Ia sensory fibers mediating signals from the muscle spindle make monosynaptic connections with spinal motoneurons. The transmitter of the Ia synapses is supposed to be glutamate (Jessell et al., 1986; Jiang et al., 1990). In newborn rats, previous pharmacological studies have shown that glutamate receptors on motoneurons for the monosynaptic transmission are NMDA as well as non-NMDA types (Harada, 1992; Jiang et al., 1990). In the present study, two questions are posed: (1) does ATA inhibit the glutamatergic monosynaptic transmission of the newborn rat spinal cord? and (2) if so, is the action mediated by blocking of NMDA type glutamate receptors? A preliminary account of this study has been communicated elsewhere (Sun et al., 1995).
2. Materials and methods
of the preamplifier was stored in a transient memory device and then recorded with a pen recorder using an expanded time-scale (Fig. 1). Agonists used were NMDA (10 mM), D,L-a-amino-3hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) (1–3 mM) and kainate (KA) (3–10 mM). In our preliminary experiments, doses of the agonists applied were selected for motoneurons to depolarize submaximally. (9 ) 2-amino-5-phosphonopentanoic acid (APV, 100 mM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 6 mM) were used as antagonists, of which doses were selected to reduce the glutamate-induced response significantly. The dose of ATA used was 100 mM, which is preferentially used to prevent excitotoxic cell death (Kure et al., 1991; Zeevalk et al., 1993; 1995) and apoptosis caused by nerve growth factor (NGF) deprivation (Batistatou and Greene, 1991). ATA and/or the antagonists dissolved in either the normal or the modified ACSF were continuously applied to the preparation by perfusion, whereas the agonists were applied for 1 min. Experiments were ended within 1 h after ATA application (see Section 3).
2.1. Preparation 2.3. Statistical analysis Experiments were done on the isolated spinal cord of newborn rats (Otsuka and Konishi, 1974). Newborn rats of Wistar strain (3 – 5 days old) were anaesthetized with ether. The spinal cord below the thoracic region with roots was isolated and placed in a small chamber of which volume was about 0.4 ml. The cord was perfused with artificial cerebrospinal fluid (ACSF) saturated with 100% O2 at a flow rate of 4 ml/min. The composition of the ACSF (pH 7.4) was as follows (mM): NaCl 157.7, KCl 3.4, CaCl2 2.5, MgCl2 1.0, Glucose 10.0 and HEPES 5.0. This medium is referred to as the ‘normal ACSF ’ in the text. All the experiments were performed at room temperature (24–25°). In some experiments, the ACSF without Mg2 + ions was used to release Mg2 + -block of NMDA receptors (Ault et al., 1980; Mayer et al., 1984; Nowak et al., 1984; Reynolds, 1990); the ACSF without Ca2 + ions was used to block synaptic transmission.
Amplitudes of MSR and agonist-induced depolarizations under test were expressed as percent of the control in the normal ACSF just before applying a test solution. The mean values of the results obtained under
2.2. Electrophysiology Potentials generated in the motoneurons either by stimulation of a dorsal root or by application of drugs were recorded extracellularly from the corresponding ventral root with a conventional suction electrode which was connected to a preamplifier (Otsuka and Konishi, 1974). The output was fed to a D.C. penrecorder. Monosynaptic reflex (MSR) was elicited at every 60 s by stimulation of either a lumbar fourth or fifth dorsal root with a rectangular pulse of 0.1 ms in duration. The intensity to the root was sufficient to evoke maximal MSR. For recording MSR, the output
Fig. 1. Sample records of MSR (A) and NMDA-induced depolarizations (B). (a) Control in the normal ACSF; (b) Under 100 mM of ATA. Ab and Bb were taken 60 and 20 min after ATA application, respectively. NMDA (10 mM) was applied for 1 min as indicated by the horizontal bars in B. Sharp vertical traces in each record are artifacts of DR stimulation. Calibration bars indicate 1 mV and 10 ms in A or 1 min in B.
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Fig. 2. Effects of ATA on MSR. (A) A typical example of ATA action on MSR with time. (B) Time course of MSR reduction within 50 min after ATA (100 mM) application (n= 6). Circles with vertical bars indicate mean9 S.D. () MSR in the normal ACSF. () MSR under the treatment with ATA. Ordinate expresses MSR amplitude in per cent. (*) Significantly different from the control at P B0.05.
ATA treatment and those of the control just before ATA application were statistically examined by the paired t-test with the significance level of P B0.05 or PB 0.01.
3. Results
3.1. Effects of ATA on MSR As shown in sample records of Fig. 1A, 100 mM of ATA depressed MSR (Ab). A typical example of ATA action on MSR with time is illustrated in Fig. 2A. It was reduced to about 40%, 150 min after the application. The time course of the reduction by ATA was not monotonic, that is, initially MSR was reduced gradu-
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ally but after about 60 min steeply. The MSR once reduced was hardly recovered for more than a few hours after cessation of ATA treatment (Fig. 2A). The different reduction rates may suggest that the underlying mechanism to reduce MSR within 1 h is different from that of the later phase. This notion is supported by the following observation (data not shown). The depolarization induced by g-aminobutyric acid (GABA) or acetylcholine (ACh) was not affected within 1 h of ATA treatment. However, thereafter, responses to GABA or ACh were gradually decreased and did not restore the control level after washing. Therefore, in this study, analysis of ATA action on the MSR was restricted only to the initial phase within 1 h after ATA application (see Section 4). Fig. 2B shows the time course of the MSR reduction within 50 min after ATA application. MSR at 20 min after ATA application was 96.393.2% (mean9S.D.) of the control (PB 0.05, n= 6). It was further reduced with a similar rate for another 30 min. The amplitudes at 30 and 50 min after ATA application were 93.095.5 and 81.299.8%, respectively. The reduction of MSR may be due to blockade of glutamate receptors by ATA (see Section 1). To test this possibility, the spinal cords were treated with either NMDA or non-NMDA type receptor antagonists. APV, a NMDA type receptor antagonist, at a dose of 100 mM reduced MSR by about 10% as early as 10 min after the application and the MSR remained at this level thereafter (Fig. 3A) (90.69 2.9% at 0 min in Fig. 3A, PB 0.05, n=6). ATA did not affect it further (Fig. 3A). In the next experiments, the spinal cords were treated with 6 mM of DNQX, a non-NMDA type receptor antagonist, under the Mg2 + -free condition. As can be seen in Fig. 3B, the Mg2 + -free medium did not affect MSR. Under this condition, DNQX immediately reduced MSR to about half of the control. This can be interpreted that the reduction is due to blocking of non-NMDA type receptors, while the NMDA type component supposedly remains to be activated. Under this condition, ATA strongly reduced MSR with time (Fig. 3B), which was significant at 20 min. MSR 50 min after ATA treatment was only 21.69 2.9% of the value just before ATA application (PB 0.01, n=3). Thus, these results indicate that ATA reduces MSR by blocking the NMDA type receptors without affecting the non-NMDA type receptors.
3.2. Effects of ATA on depolarizations induced by glutamate agonists To verify further the notion that the NMDA type receptor is the target of ATA, we examined antagonisms between ATA and exogenously applied agonists for each subtype of glutamate receptors. All the glutamate agonists depolarized motoneurons. The response
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to NMDA in the normal ACSF was markedly inhibited by 100 mM of ATA (Fig. 1B). The mean peak amplitude was 16.19 20.0% of the control (P B 0.01, n= 8) (Fig. 4). On the other hand, the depolarization induced by a non-NMDA type agonist, AMPA or KA, was little depressed (Fig. 4). Thus, these results further support the notion that ATA specifically inhibits NMDA type receptors. The site of ATA action is supposed to be on the motoneurons. To ascertain this, the effect of ATA on the NMDA-induced depolarization was examined un-
Fig. 4. Effects of ATA on the depolarizations induced by glutamate agonists in the normal ACSF. The open columns illustrate the depolarizations induced by each agonist (100%) and stippled columns those under ATA. Observations were made 20, 30 and 40 min after ATA application for NMDA, AMPA and KA, respectively. Each column with a vertical bar indicates mean 9 S.D. The numbers of preparations for NMDA, AMPA and KA experiments were 8, 6 and 6, respectively. (**) Significantly different from the control at PB 0.01.
der the Ca2 + -free condition in which synaptic transmission to motoneurons is blocked. NMDA depolarized motoneurons even in the Ca2 + -free ACSF, although it was about 30% of the control in the normal ACSF (29.79 4.8%, n=3) (Fig. 5). Thus, the result indicates that NMDA depolarizes motoneurons directly and indirectly. The response to NMDA under the Ca2 + -free condition was also inhibited by ATA. It was 15.19 1.4% of the value in the Ca2 + -free medium (PB0.01, n= 3) (Fig. 5). The degree of the reduction was similar to that in the normal ACSF (see above, Fig. 4). Unexpectedly, it was found that ATA depressed the depolarization induced by AMPA in the Ca2 + -free medium. The reduction was 32.7 9 12.8% (PB 0.01) and 27.8 9 7.7% (PB0.05) at 1 and 3 mM of AMPA controls in the Ca2 + -free ACSF, respectively (n= 5) (Fig. 5). KAinduced depolarization was not affected by ATA (Fig. 5).
4. Discussion
Fig. 3. Effects of glutamate receptor antagonists and ATA on MSR. (A) Effects of APV and ATA on MSR. () MSR amplitude (100%) in the normal ACSF. (,) MSR amplitudes from the spinal cords treated with APV (100 mM ). () MSR under the treatment with APV and ATA. (B) Effects of DNQX and ATA on MSR. () MSR amplitude (100%) in the normal ACSF. (,) MSR in the Mg2 + free ACSF. (2) MSR under the treatment with DNQX (6 mM) in the Mg2 + -free ACSF. () MSR under the treatment with DNQX and ATA in the Mg2 + -free ACSF. (**) Significantly different from the control just before ATA application (PB 0.01). Each symbol with a vertical bar indicates mean 9 S.D. (n= 3). Abscissae indicate time after ATA applied (0 min). Ordinates indicate MSR amplitude (%).
The present study revealed that ATA inhibits monosynaptic transmission in the in vitro spinal cord. The MSR reduction is supposed to be composed of two phases with respect to the time course: initial slow and later steep reduction. The depolarization induced by GABA or ACH was not affected by ATA during the initial phase, whereas it was later. It has been reported that ATA at a dose of 100 mM does not inhibit phosphofructokinase (PFK) activity of retinal cells treated for 1 h (Zeevalk et al., 1995), whereas it inhibits the enzyme in cell-free system (McCune et al., 1989; Zeevalk et al., 1995). These may suggest that ATA can
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not penetrate cells within 1 h. Thus, the latter phase seems to be complicated. Therefore, we did not examine the mechanism for the latter phase of MSR reduction, only the initial phase. It is generally accepted that MSR in the spinal cord is mediated by glutamate as a transmitter of group Ia sensory fibers (see Section 1). The present study showed that MSR was reduced by antagonists for both types of glutamate receptors, confirming the notion that both are involved in monosynaptic transmission in the newborn rats (Harada, 1992; Jiang et al., 1990), though non-NMDA type is dominant. To reduce synaptic transmission, a drug must act on sites either presynaptic to reduce transmitter release or postsynaptic to inhibit generation of excitatory postsynaptic potentials. In the spinal cord, GABA-mediated presynaptic inhibitory mechanism is operating (Miyata and Otsuka, 1975; Watson, 1992). ATA did not affect membrane potentials of the motoneurons (data not shown) like GABA depolarizes them in embryonic and neonatal rats (Wu et al., 1992; Gao and Ziskind-Conhaim, 1995). Thus, ATA can not activate the presynaptic inhibition. ATA did not reduce MSR further in the
Fig. 5. Effects of ATA on the depolarizations induced by glutamate agonists in the Ca2 + -free ACSF. Each open column illustrates the amplitude of agonist-induced depolarization in the normal ACSF. Stippled and hatched columns indicate depolarizations with and without ATA in the Ca2 + -free ACSF. They express per cent of the corresponding control values in the normal ACSF. In cases of AMPA and KA, tha non-NMDA type receptor antagonist, in the Mg2 + -free medium was almost completely inhibited by addition of ATA. ATA depressed NMDA- but not AMPA- or kainate-induced depolarization in the medium containing normal ionic composition. Thus, it is concluded that the reduction of MSR by ATA is due to blockade of NMDA type but not non-NMDA type glutamate receptors. And, the present study confirmed the previous conclusion that Ia monosynaptic transmission in the spinal cord of the newborn rat is mediated byisolated spinal cords of newborn rats. Monosynaptic reflex (MSR) was depressed about 20%, 50 min after exposure to 100 mM of ATA. Pretreatment with APV, a NMDA type receptor antagonist, depressed MSR by about 10%, but additional application of ATA did not affect the MSR further. In contrast, the remaining MSR following treatment with DNQX, DA type as well as non-NMDA type glutamate receptors.
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spinal cord pretreated with APV (Fig. 3A), indicating that the transmitter release is not affected by ATA. Finally, ATA blocked NMDA-induced depolarization in the Ca2 + -free medium, indicating that ATA exerts direct action on the motoneurons. Therefore, it is concluded that the MSR reduction by ATA is, if not all, due to blockade of transmitter action on the motoneurons. The following results indicate that the target of ATA is NMDA type glutamate receptors. The MSR depressed by APV, a NMDA type receptor antagonist, was not affected further by ATA. In contrast, ATA greatly diminished the MSR in the spinal cord treated with DNQX and the Mg2 + -free ACSF, by which NMDA-type receptors remained to be activated. Furthermore, ATA greatly reduced the depolarization induced by NMDA, but did not that by AMPA or KA in the normal ACSF. Thus, it is concluded that ATA blocks NMDA type receptors specifically in the normal ionic condition. It was found that ATA reduced the depolarization induced by AMPA in the preparation perfused with the Ca2 + -free medium but not with the normal ACSF. The underlying mechanism for the reduction is not clear from the present study. A binding study has shown that IC50 for displacement of AMPA by ATA requires a 10-fold higher concentration than that for a NMDA ligand, whereas a much higher dose of ATA is necessary for displacement of KA (Zeevalk et al., 1995). Thus, it is likely that AMPA but not KA has affinity to the NMDA type receptor, though weak (Zeevalk et al., 1995). This affinity might be strengthened under the Ca2 + -free condition by some unknown mechanisms. Glutamate is known to be excitotoxic on many types of neurons to cause neuronal death (Ankarcrona et al., 1995; Choi, 1990; Csernansky et al., 1994; Kure et al., 1991; Portera-Cailliau et al., 1995; Roberts-Lewis et al., 1993; Samples and Dubinsky, 1993; Zeevalk et al., 1993; 1995). ATA inhibits Ca2 + -dependent endonuclease by which DNA fragmentation occurs and leads cells to apoptosis (McConkey et al., 1989). Therefore, the present study supports the notion that apoptotic cell death induced by excitotoxic glutamate action can be prevented by ATA through two different mechanisms, blockade of NMDA type glutamate receptors and inhibition of Ca2 + -dependent endonuclease (Zeevalk et al., 1993). Furthermore, it casts a caution that therapeutic use of ATA may cause adverse reaction due to blockade of glutamatergic synaptic transmission. References Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. and Nicotera, P. (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron, 15: 961–973.
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Ault, B., Evans, R.H., francis, A.A., Oakes, D.J. and Watkins, J.C. (1980) Selective depression of excitatory amino acid depolarizations by magnesium ions in isolated spinal cord preparations. J. Physiol., 307: 413–428. Batistatou, A. and Greene, L.A. (1991) Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J. Cell Biol., 115: 461–471. Choi, D.W. (1990) The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci., 13: 171 – 182. Choi, D.W. (1992) Excitotoxic cell death. J. Neurobiol., 23: 1261 – 1276. Cotman, C.W. and Monaghan, D.T. (1987) Chemistry and anatomy of excitatory amino acid systems. In: H.Y. Meltzer (Ed.), Psychopharmacology, The Third Generation of Progress, Raven Press, New York, pp. 197–210. Csernansky, C.A., Canzoniero, L.M.T., Sensi, S.L. and Choi, D.W. (1994) Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury. J. Neurosci. Res., 38: 101 – 108. Dagani, F. and D’Angelo, E. (1992) Glutamate metabolism, release, and quantal transmission at central excitatory synapses: Implications for neural plasticity. Funct. Neurol., 7: 315–336. Fagg, G.E. and Foster, A.C. (1983) Amino acid neurotransmitters and their pathway in the mammalian central nervous system. J. Neurosci., 9: 701–719. Fonnum, F. (1984) Glutamate: A neurotransmitter in mammalian brain. J. Neurochem., 42: 1–11. Gao, BX. and Ziskind-Conhaim, L. (1995) Development of glycineand GABA-gated currents in rat spinal motoneurons. J. Neurophysiol., 74: 113–121. Greenamyre, J.T. and Porter, R.H.P. (1994) Anatomy and physiology of glutamate in the CNS. Neurology, 44 (suppl. 8): S7 – S13. Hallick, R.B., Chelm, B.K., Gray, P.W. and Orozco, E.M. (1977) Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res., 4: 3055–3064. Harada, Y. (1992) Two types of glutamate receptor mediate Ia synaptic transmission in spinal cord of newborn rat. Jpn. J. Physiol., 42 (suppl): S140. Jessell, T.M., Yoshioka, K. and Jahr, C.E. (1986) Amino acid receptor-mediated transmission at primary afferent synapses in rat spinal cord. J. Exp. Biol., 124: 239–258. Jiang, Z.G., Shen, E. and Dun, N.J. (1990) Excitatory and inhibitory transmission from dorsal root afferents to neonate rat motoneurons in vitro. Brain Res., 535: 110–118. Kure, S., Tominaga, T., Yoshimoto, T., Tada, K. and Narisawa, K. (1991) Glutamate triggers internucleosomal DNA cleavage in neuronal cells. Biochem. Biophys. Res. Commun., 179: 39 – 45.
Mayer, M.L., Westbrook, G.L. and Guthrie, P.B. (1984) Voltagedependent block by Mg2 + of NMDA responses in spinal cord neurones. Nature, 309: 261 – 263. McConkey, D.J., Hartzell, P., Nicotera, P. and Orrenius, S. (1989) Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J., 3: 1843 – 1849. McCune, S.A., Foe, L.G., Kemp, R.G. and Jurin, R.R. (1989) Aurintricarboxylic acidis a potent inhibitor of phosphofructokinase. Biochem. J. 259: 925 – 927. Miyata, Y. and Otsuka, M. (1975) Quantitative histochemistry of g-aminobutyric acid in cat spinal cord with special reference to presynaptic inhibition. J. Neurochem., 25: 239 – 244. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A. (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature, 307: 462 – 465. Otsuka, M. and Konishi, S. (1974) Electrophysiology of mammalian spinal cord in vitro. Nature, 252: 733 – 734. Portera-Cailliau, C., Hedreen, J.C., Price, D. and Koliatsos, V.E. (1995) Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci., 15: 3775–3787. Reynolds, I. J. (1990) Modulation of NMDA receptor responsiveness by neurotransmitters, drugs and chemical modification. Life Sci., 47: 1785 – 1792. Roberts-Lewis, J.M., Marcy, V.R., Zhao, Y. and Vaught, J.L., Siman, R. and Lewis, M.E. (1993) Aurintricarboxylic acid protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo. J. Neurochem., 61: 378 – 381. Samples, S.D. and Dubinsky, J.M. (1993) Aurintricarboxylic acid protects hippocampal neurons from glutamate excitotoxicity in vitro. J. Neurochem., 61: 382 – 385. Sun, X., Harada, Y. and Miyata, Y. (1995) Differential actions of aurintricarboxylic acid on glutamate receptors of motoneurons of newborn rats. Fourth IBRO World Congr. Neurosci. Abstr., A3.54. Watson, A.H.D. (1992) Presynaptic modulation of sensory afferents in the invertebrate and vertebrate nervous system. Comp. Biochem. Physiol., 103A: 227 – 239. Wu, WL., Ziskind-Conhaim, L. and Sweet, M.A. (1992) Early development of glycine- and GABA-mediated synapses in rat spinal cord. J. Neurosci., 12: 3935 – 3945. Zeevalk, G.D., Schoepp, D. and Nicklas, W.J. (1993) Aurintricarboxylic acid prevents NMDA-mediated excitotoxicity: Evidence for its action as an NMDA receptor antagonist. J. Neurochem., 61: 386 – 389. Zeevalk, G.D., Schoepp, D. and Nicklas, W.J. (1995) Excitotoxicity at both NMDA and non-NMDA glutamate receptors is antagonized by aurintricarboxylic acid: Evidence for differing mechanisms of action. J. Neurochem., 64: 1749 – 1758.
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