Modification of the responses of primate spinothalamic neurons to mechanical stimulation by excitatory amino acids and an N-methyl-D-aspartate antagonist.

Excitatory amino acids (EAAs) are likely to play a key role in sensory transmission in the spinal cord. In the present study, the microiontophoresis technique was used to investigate the effects of L-glutamate (GLUT), N-methyl-D-aspartate (NMDA), and quisqualate (QUIS), as well as an NMDA receptor antagonist, AP-7, on the discharges evoked in nociceptive primate spinothalamic tract (STT) neurons by mechanical stimulation of the skin. Responses to innocuous brushing of the skin were facilitated by GLUT and NMDA (and in some neurons by QUIS) and sometimes reduced by AP-7. GLUT also facilitated the responses to noxious mechanical stimuli. The results are consistent with anatomical evidence for the presence of synapses that contain EAAs on primate STT cells and with the proposal that the co-release of EAAs and neuropeptides may contribute to hyperalgesia.     

Excitability changes in lumbosacral spinothalamic neurons produced by non-noxious mechanical stimuli and by graded electrical stimuli applied to the face

The changes in the excitability of lumbosacral spinothalamic neurons produced by activating afferents in the trigeminal nerve using electrical or mechanical stimuli was investigated in cats anesthetized with alpha-chloralose. In most spinothalamic neurons, weak electrical stimuli or step indentations of the skin of the face produced an increase followed by decrease in the excitability of these cells. In experiments in which the effect of activating specific groups of trigeminal afferent fibers on these excitability changes was evaluated, the suppression could be produced by activating only the fastest conducting cutaneous afferent fibers. Step indentations of the facial skin affected the excitability of spinothalamic neurons in a manner similar to electrical stimuli. The duration of the suppression phase appeared to be largely independent of the duration of the step indentation of the facial skin. It was concluded that the descending system mediating the suppression phase is activated largely by cutaneous afferents from rapidly adapting receptors. The effects of subtotal spinal cord lesions on the excitation and suppression phases produced by facial stimulation indicate that the pathways mediating the supppression descend bilaterally in the dorsal part of the lateral fasciculus. The excitation phase appears to be mediated largely by pathways in the dorsal part of the ipsilateral lateral fasciculus.     

Enhanced responses of spinothalamic tract neurons to excitatory amino acids accompany capsaicin-induced sensitization in the monkey.

Sensitization of the responses of dorsal horn neurons to mechanical stimulation may play a role in the generation of hyperalgesia. Intradermal injection of capsaicin (CAP) provides a model of experimental hyperalgesia that possesses a component of allodynia. This hyperalgesia is produced by chemical stimulation of C-fibers, leading to sensitization of dorsal horn neurons, including spinothalamic tract (STT) cells. The changes in the physiological responses of STT neurons following intradermal CAP in monkeys parallel the acute pain and hyperalgesia produced by intradermal CAP in humans. The present study addresses the role that excitatory amino acids (EAAs) may play in the sensitization of STT neurons by intradermal CAP. Our results show that the background discharge rate and the responses of STT cells to mechanical stimulation increase following intradermal CAP. In addition, the responses of the sensitized cells to one or more iontophoretically released EAA agonists, including NMDA, glutamate, aspartate, kainate, DL-alpha-amino-3-hydroxy-5-methyl-isoxazoleproprionic acid, and/or quisqualate, increase following intradermal CAP. It is proposed that an increase in the responses of STT neurons to EAAs contributes to the hyperalgesia produced by this noxious chemical stimulus.     

A role of excitatory amino acids in the activation of locus coeruleus neurons following cutaneous thermal stimuli

Previous electrophysiological experiments have shown that brain noradrenaline neurons in the locus ceruleus are activated by thermal cutaneous stimuli. In the present study a putative involvement of excitatory amino acids (EAA) in cutaneous LC activation was analyzed. Intraventricular administration of kynurenic acid (1 mumol), a broad spectrum EAA antagonist, or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 0.1 mumol) as well as subcutaneous administration of the specific NMDA antagonist MK 801 (2 mg/kg) almost totally abolished the response of LC neurons to both non-noxious and noxious cutaneous sensory stimuli. We propose that the activation of LC neurons following thermal cutaneous stimuli is mediated via release of EAA from nerve terminals emanating from nucleus paragigantocellularis (PGi).     

The cells of origin of the primate spinothalamic tract.

Spinothalamic tract cells in the lumbar, sacral and caudal segments of the primate spinal cord were labelled by the retrograde transport of horseradish peroxidase (HRP) injected into the thalamus. The laminar distribution of stained spinothalamic cells in the lumbosacral enlargement differed according to whether the HRP was injected into the lateral or the medial thalamus. Lateral injections labelled cells in most laminae, but the largest numbers of cells were in laminae I and V. The highest concentrations of cells labelled from the medial thalamus were in laminae VI-VIII. Ninety percent or more of the stained spinothalamic cells in the lumbosacral enlargement were contralateral to the injection site. In the conus medullaris stained spinothalamic cells were most numerous in laminae I, V and VI following lateral thalamic injections of HRP. Many of the cells of the conus were in Stilling's nucleus. Twenty-three percent of the cells in the conus were ipsilateral to the injection site in the lateral thalamus. Only a few cells in the conus were labelled by medial thalamic injections. The total number of spinothalamic cells from L5 caudally was estimated to be at least 1,200-2,500. An injection of HRP into the midbrain resulted in laminar distribution of labelled cells much like that produced by a lateral thalamic injection. The types of spinothalamic tract cells and the sizes of their somata were determined for different laminae. The cell types resemble those already described from Golgi and other studies of the spinal cord gray matter. The spinothalamic tract cells in lamina I included Waldeyer cells and numerous small fusiform, pyriform or triangular cells. Those in lamina II included limitrophe and central cells. Spinothalamic cells in lamina III were central cells. Most of the labelled cells in laminae IV-X were polygonal, although there were also flattened cells in these layers. The smallest spinothalamic cells were in laminae I-III, while the largest were in laminae V and VII-IX. Spinothalamic cells in the conus medullaris included cells like those in the lumbosacral enlargement, but also a special cell type in Stilling's nucleus. Some cells in the conus had dendrites that crossed the midline. Spinothalamic axons could sometimes be traced to the ventral white commissure within one or a few sections. In longitudinal sections, most labelled axons were in the ventral part of the lateral funiculus on the side of the injection, although a few were in the ventral funiculus or on the contralateral side. The axons were widely dispersed, and a few were located adjacent to the pia-glial membrane.     

Retinogeniculate transmission by NMDA and non-NMDA receptors in the cat.

The contributions of N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid (EAA) receptors to retinogeniculate transmission were investigated in the cat. The EAA antagonists 2-amino-5-phosphonovaleric acid (APV) and kynurenic acid (KYN) were used to block the NMDA receptors and all EAA receptors, respectively. Antagonistic effects on the visual response were assessed with single On/Off stimuli of 2 s duration or repetitive flicker stimulation (5 Hz) with a light spot projected onto the receptive field center. With APV, the NMDA response could be almost completely abolished but the visual response to repetitive stimulation was reduced on average by only 34%. Initial (transient) components of the single flash response were attenuated on average by 23%, the residual (sustained) component by 48%. With KYN the responses to NMDA, quisqualate (QUIS) and glutamate (GLU) were abolished or strongly reduced as was the visual response to flicker (mean 58%) and single flash stimulation (mean transient 73%, sustained 90%). Prolonged iontophoretic applications of the agonists GLU, QUIS and NMDA revealed receptor desensitization or competitive interactions with the naturally released transmitter in a dose-dependent manner. When the responses to any of the 3 agonists declined during continuous application, superimposed visual responses were clearly reduced in amplitude. Visual response amplitudes were also reduced when superimposed on steady state QUIS responses but unchanged in amplitude when superimposed on steady state NMDA responses. In conclusion, non-NMDA as well as NMDA receptors seem to participate in cat retinogeniculate transmission. Non-NMDA receptors appear to be most important for the initial component but can also maintain the visual response, while the NMDA receptors seem to be more effective during the later component of the response.     

A review of in vivo modulation of cerebellar cGMP levels by excitatory amino acid receptors: role of NMDA, quisqualate and kainate subtypes.

1. Agonists of all three EAA receptor subtypes augment cerebellar cGMP levels in vivo. 2. Agonists of the NMDA-associated glycine receptor also increase cerebellar cGMP levels but with lesser efficacy than EAA agonists. 3. Pharmacological agents, such as harmaline and pentylenetetrazol, which enhance endogenous EAA transmission also increase cerebellar cGMP levels in vivo. 4. Increases in cerebellar cGMP elicited by EAA receptor agonists are blocked by inhibition of nitric oxide synthase with N-monomethyl-L-arginine. 5. Basal cerebellar cGMP levels are decreased in a dose-dependent manner by competitive and non-competitive NMDA receptor antagonists but not by blockade of the NMDA-associated glycine receptor. 6. Selective alpha-1 blockade also antagonizes the actions of NMDA-dependent increases in cerebellar cGMP, suggesting NMDA receptor modulation of NE release from noradrenergic mossy fibers. 7. Quisqualate-dependent increases in cerebellar cGMP were blocked by the nonselective EAA antagonist, DNQX, but not by glycine antagonists or noncompetitive NMDA antagonists. 8. The sigma ligands, ifenprodil and BMY 14802, which did not alter or increased basal cerebellar cGMP levels, respectively, antagonized NMDA-dependent increases in cGMP levels. 9. The polyamines, spermine and spermidine, also did not change basal cGMP levels but nonselectively antagonized EAA-mediated increases in cGMP. 10. In summary, all 3 major EAA subtypes appear to modulate cerebellar cGMP levels in vivo. These actions also involve the intermediate generation of nitric oxide. The NMDA receptor population also appears to reside mainly on noradrenergic nerve endings in the cerebellum.     

A1 neurons and excitatory amino acid receptors in rat caudal medulla mediate vagal excitation of supraoptic vasopressin cells

Extracellular recordings from the supraoptic nucleus of the rat established that vasopressinergic neurosecretory cells were excited by stimulation of cervical but not abdominal vagal afferents. This response was absent or significantly attenuated after microinjection of gamma-aminobutyric acid into a region of the caudal medulla known to contain the A1 noradrenaline cell group. Consistent with the possible involvement of the A1 group, vagal stimulation approximately doubled the frequency of proto-oncogene expression in A1 noradrenaline neurons, as indicated by the occurrence of nuclear Fos-like immunoreactivity in tyrosine hydroxylase-positive neurons of the caudal ventrolateral medulla. Finally, A1 region microinjection of either the N-methyl-D-aspartic acid (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly reduced vasopressin cell responses to vagal stimulation. These findings suggest that: (i) the A1 group is an essential component in a pathway which relays facilitatory vagal input of cardiopulmonary origin to neurosecretory vasopressin cells, and (ii) the activation of A1 neurons in this pathway involves both NMDA and non-NMDA excitatory amino acid receptors, an observation consistent with an input to A1 cells which generates 'mixed' excitatory postsynaptic potentials.     

Excitatory amino acid neurotoxicity in cultured retinal neurons: involvement of N-methyl-D-aspartate (NMDA) and non-NMDA receptors and effect of ganglioside GM1

Cultures of chicken day 8 embryo retinal cells, essentially free of contaminating non-neuronal elements, were used to examine the neurotoxicity of various excitatory amino acid transmitter receptor agonists. At 7 days in vitro, N-methyl-D-aspartate (NMDA), following 24 hr exposure to 0.1-1.0 mM, destroyed 60-70% of the multipolar neurons, but apparently spared photoreceptors. The cytotoxic effect of NMDA was prevented by extracellular Mg2+ or phencyclidine, suggesting a role for the NMDA ion channel; competitive NMDA antagonists were also neuroprotective. The mixed excitatory amino acid receptor agonist glutamate (0.1-1.0 mM) was also neurotoxic (approximately 70% loss of multipolar neurons) and strongly blocked by NMDA (but weakly by non-NMDA) antagonists and Mg2+, indicating a major action at NMDA receptors. As with NMDA, glutamate did not appear to affect photoreceptors. The neurotoxic action of kainate against multipolar retinal neurons, as reported by others, was confirmed here. Kainate neuronal injury was sensitive to the quinoxalinedione non-NMDA antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyanoquinoxaline-2,3-dione (CNQX), but not to Mg2+ or phencyclidine. Ibotenate and quisqualate, even at millimolar concentrations, were not neurotoxic. The monosialoganglioside GM1 was also effective in reducing NMDA and non-NMDA agonist neurotoxicity to retinal neurons. Maximal ganglioside benefit required 1-2 hr of pretreatment with 100-200 microM GM1. The percentage of multipolar neurons remaining after the neurotoxin insult approximately doubled with GM1 treatment. Gangliosides may thus have a therapeutic potential in excitatory amino acid-initiated neuropathologies.     

Paroxysmal long-lasting depolarizations in cultured hippocampal neurons are generated by activation of NMDA and non-NMDA receptors

In primary cultures of hippocampal neurons from the embryonic rat, spontaneous depolarizations lasting up to 6 sec adn resembling paroxysmal depolarization shifts (PDAs) appeared after 11 day7s in vitro. These depolarizations are presumably generated by sy6naptic events, because: (1) both their appearance and duration are independent of membrane potential, (2) the amplitudes of the underlying currents depend monotonically on membrane potential, and (1) they are reversed at the reversal potential of the excitatory postsynaptic potentials (EPSPs). In addition, PDAs disappeared reversiblyy when sodium-dependent action potentials were blocked by tetrodotoxin (10μM) and when synaptic transmission was reduced by elevated Mg²⁺ (5mM). Further, the fact that these depolarizations can apppear simultaneously in two neurons in paired recordings also points to a synaptic origin. Inhibition of glutaminergic synaptic transmission by kynurenic acid (50μM) and the NMDA-antagonist D-2-amino-5-phosphonovaleric acid (APV; 50μM) led to a marked shortening of the depolarizations. This blocking effect of kynurenic acid and APV and comparison with the currents elicited by locally applied glutamate or NMDA provide evidence for an activation of both types of glutamate receptors to induce PDSs. The role of alteration of glutaminergic synaptic transmission in the induction and maintenance of these depolarizations is discussed in the context of results from the literature on the appearrance of PDSs in cultures grown under chronic blockade of glutamate receptors. © 1993 Wiley-Liss, Inc.     

Implication of non-NMDA and NMDA receptors in cochlear ischemia.

We have investigated the hypothesis that the acute ischemic swelling of the radial dendrites connected to the inner hair cells (IHCs) is mediated by glutamatergic receptors. In control cochleas, after 20 min ischemia all the dendrites were dramatically swollen. Conversely, after a perfusion of 50 microM 6-7-dinitroquinoxaline-2,3-dione (DNQX) before ischemia, most dendrites were protected although those contacting the IHCs on their modiolar side frequently swelled. After 50 microM of D-2-amino-5-phosphonopentanoate (D-AP5), no dendrite protection could be obtained. Finally, after DNQX and D-AP5, no dendrite swelling occurred. These results suggest that, in the cochlea, the acute ischemic swelling of dendrites primarily occurs via non-NMDA receptors. However, in radial dendrites contacting the IHCs on their modiolar side, NMDA receptors may contribute to excitotoxicity.     

Temporomandibular-evoked jaw muscle reflex: role of brain stem NMDA and non-NMDA receptors.

This study investigated the possible involvement of brain stem excitatory amino acid receptor mechanisms and the trigeminal subnucleus caudalis (Vc) in temporomandibular joint (TMJ)-evoked reflex jaw muscle activity. Glutamate injected into the TMJ of anesthetized rats reflexly evoked activity in the jaw muscles. Application of lidocaine, but not saline, to the surface of the caudal brainstem overlying Vc significantly suppressed TMJ-evoked jaw muscle activity, while application of NMDA or non-NMDA receptor antagonists also significantly attenuated jaw muscle activity. These results provide evidence that Vc is a critical relay in the TMJ-evoked reflex activation of the jaw muscles, and that both NMDA and non-NMDA receptor mechanisms may contribute to these effects.     

兴奋性氨基酸在缺血性海马神经元损害中的作用的研究

采用大鼠全脑缺血模型,研究脑缺血再灌流海马氨基酸含量的动态变化及相应病理改变,观察NMDA(N-甲基-D-门冬氮酸)受体拮抗剂MK-801的疗效,提示兴奋性氨基酸(Glu,Asp)可能参与海马神经元损害,MK-801能有效防止海马CA_1区迟发性神经元坏死。兴奋性氨基酸受体拮抗剂的研究,将为临床缺血性中风治疗提供新的途径。     

Stimulation of NTS activates NMDA and non-NMDA receptors in rat cardiac vagal neurons in the nucleus ambiguus

While it is widely accepted that tonic and reflex changes in cardiac vagal activity play significant roles in cardiovascular function, little is known about the synaptic pathways in the brainstem responsible for the control of cardiac vagal neurons in the nucleus ambiguus (NA). In this study, we identified the principal post-synaptic receptors activated in cardiac vagal neurons upon stimulation of the nucleus tractus solitarius (NTS). Cardiac vagal neurons were identified by the presence of a retrograde fluorescent tracer and were visualized in rat brainstem slices. Perforated patch clamp techniques were used to record post-synaptic currents. NTS stimulation activated glutamatergic currents in cardiac vagal neurons with a typical delay of 8–18 ms. Post-synaptic responses were separated into NMDA and non-NMDA components using

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-2-amino-5-phophonovalerate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. In conclusion, this study characterizes a monosynaptic glutamatergic pathway from NTS that activates NMDA and kainate/AMPA post-synaptic receptors in cardiac vagal neurons.     

The role of putative excitatory amino acid neurotransmitters in the initiation of locomotion in the lamprey spinal cord. I. The effects of excitatory amino acid antagonists

The activation of N-methyl-D-aspartate (NMDA) and kainate receptors will evoke fictive locomotion in the appropriate motor pattern for locomotion in the isolated lamprey spinal cord, but not a selective activation of quisqualate receptors. The present experiments test whether the initiation of locomotion in response to sensory stimulation depends on these types of receptors. An in vitro preparation of the lamprey spinal cord with part of its tailfin left innervated has been used. In this preparation a sequence of fictive locomotion (i.e. alternating bursts in the segmental ventral roots with a rostrocaudal phase lag) can be elicited by continual sensory stimulation of the tailfin. The effects of excitatory amino acid antagonists were studied by recordings from ventral roots (extracellularly) and motoneurones (intracellularly). It was found that the strong initial bursts of each swimming sequence induced by sensory stimulation were depressed by combined NMDA/kainate antagonists (cis-2,3-piperidine dicarboxylate (PDA) and gamma-D-glutamylglycine (gamma-DGG] whereas the less intense burst activity, occurring particularly towards the end of each swimming sequence, was depressed by a selective NMDA antagonist, 2-amino-5-phosphonovalerate (2-APV). This condition could be mimicked in an isolated spinal cord preparation by an application of L-glutamate; the low-level fictive locomotion induced by low doses of L-Glu (less than 100 microM) was depressed by a NMDA antagonist (2-APV), and, if higher doses were applied, the activity was only depressed by PDA/gamma-DGG. The mode and time course of the depression (by excitatory amino acid antagonists) of fictive locomotion, induced by sensory stimulation, shows that the putative excitatory amino acid neurotransmitter directly or indirectly acts at the pattern generating circuitry within the spinal cord.     

Dual-component synaptic potentials in the lamprey mediated by excitatory amino acid receptors

The synaptic mechanisms underlying amino acid-mediated excitation in the lamprey spinal cord have been investigated. Fine stimulating electrodes were used to stimulate single axons in the spinal cord and evoke unitary EPSPs in lamprey motoneurons and one type of premotor interneuron, the CC interneuron. Three types of EPSP, distinguished by their time course and sensitivity to amino acid antagonists, were seen. Fast EPSPs had a fast rise time (mean, 6.5 msec) and a short half-decay time (mean, 22.5 msec). Slow EPSPs lasted at least 200 msec, had a slow rise time (mean, 28 msec), and a long half-decay time (mean, 109 msec). The third type of unitary potential, called "mixed" EPSP, also lasted at least 200 msec, had a fast rise time (mean, 12 msec), and a long half-decay time (mean, 105 msec). Lamprey neurons were found to possess 3 types of excitatory amino acid receptor: N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. 2-Amino-5-phosphonovaleric acid (APV) or Mg2+ blocked the depolarizations caused by N-methyl-D,L-aspartate (NMA) but not those of kainate or quisqualate. Cis-2, 3-piperidine dicarboxylic acid (PDA) blocked the depolarizations caused by NMA and kainate but not those of quisqualate. Fast EPSPs were unaffected by the bath application of APV or Mg2+ but were greatly reduced by PDA, suggesting that these EPSPs were mediated by non-NMDA, possibly kainate receptors. Both APV and Mg2+ blocked the slow EPSPs, suggesting that they were mediated by NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological evidence for the existence of NMDA and non-NMDA receptors on rat ventral tegmental dopamine neurons

In vitro extracellular single-unit recordings from rat midbrain slices were used to assess the effects of excitatory amino acid agonists on the activity of A10 dopamine neurons. N-methyl-D-aspartic acid (NMDA), kainic acid (KA), and β-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) elicited dose-dependent increases in firing rates. The relative potencies for the 3 compounds was AMPA > KA > NMDA. None of the excitations was accompained by burst firing, but frequently periods of nonrecordable activity occurred following pronounced stimulation. Concurrent application of the excitatory amino acid antagonist CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylate) selectively blocked the excitations elicited by NMDA but not by KA or AMPA. Likewise the selective non-NMDA antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline] blocked only the excitatory effects of AMPA and KA but not those elicited by NMDA. NBQX appeared to be less optent at antagonizing KA than AMPA. These results suggest that mesolimbic-mesocortical dopamine neurons possess both NMDA and non-NMDA receptors, and possibly distinct AMPA and KA recognition sites. © 1993 Wiley-Liss, Inc.