Activation of 5-HT1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and alpha-methyl-5-HT (alpha-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 microM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or alpha-Me-5-HT (both 100 microM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. alpha-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT1C/2 antagonist ketanserin and the 5-HT1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic signaling inhibits hyperalgesia induced by spinal cord damage

Although dysesthesia is one of the most serious problems in patients with spinal cord injury, most of them being unresponsive to conventional treatments. In this study, we established a rat thoracic spinal cord mild-compression model that revealed thermal hyperalgesia in the hind limb. The thoracic spinal cord was compressed gently, using a 20 g weight for 20 min. The withdrawal latency of the thermal stimulation of the bilateral hind-limb was monitored using Hargreaves' Plantar test apparatus. In this model, thermal-hyperalgesia was observed for 1 week after the injury. The spinal cord injury-induced thermal-hyperalgesia was mimicked by the intrathecal application of metergoline, a non-selective 5-HT antagonist, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl]-piperazine hydrobromide (NAN190), a selective 5-HT1 antagonist, and 3-tropanyl-3,5-dichlorobenzoate (MDL72222), a selective 5-HT3 antagonist. Intraperitoneal application of fluvoxamine maleate, a selective serotonin reuptake inhibitor, reduced the intensity of hyperalgesia induced by spinal cord injury. The inhibitory effect of fluvoxamine maleate on thermal hyperalgesia was prevented by the application of the aforementioned nonselective or selective 5-HT receptor antagonists. Intrathecal application of fluvoxamine maleate and selective 5-HT receptor agonists, i.e., 8-hydroxy-2-(di-n-proplyamino)-tetralin hydrobromide (8-OH-DPAT: 5HT-1 selective) and 2-methyl-5-hydroxytryptamine maleate (2-m-5-HT: 5HT-3 selective), inhibited the spinal cord injury-induced hyperalgesia. These results suggest that the change in the descending serotonergic signal plays an important role in hyperalgesia after the spinal cord injury, and that the application of selective serotonin reuptake inhibitors will be one of the candidates for new therapeutic methods against post-spinal cord injury dysesthesia.     

Altered metabolism of excitatory amino acids, N-acetyl-aspartate and N-acetyl-aspartylglutamate in amyotrophic lateral sclerosis

Since recent studies provided evidence for abnormal glutamate metabolism in amyotrophic lateral sclerosis, we measured amino acid levels in the fasting plasma of 52 ALS patients and an equal number of controls of a similar age. In addition, the content of amino acids, N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG) were measured in spinal cord and brain tissue obtained at autopsy from patients dying of ALS. Results showed significant elevations (by about 70%) in the plasma levels of glutamate in the ALS patients as compared to controls. In contrast, glutamate levels were significantly decreased in all CNS regions studied of ALS patients (by 21–40%), with the greatest changes occurring in the spinal cord. The ratio of glutamine to glutamate was altered significantly in the spinal cord ALS tissue. In addition, reductions in the levels of aspartate (by 32–35%), NAA, and NAAG (by 40–48%) were found in the spinal cord of ALS patients. These results are consistent with a generalized defect in the metabolism of neuroexcitotoxic amino acids. An altered distribution of these compounds may occur between their intracellular and extracellular pools with resultant abnormal potentiation of excitatory transmission mediated by glutamate receptors and selective degeneration of motor neurons.     

咯利普兰对大鼠脊髓损伤后保护作用及其机制

目的探讨咯利普兰对大鼠脊髓损伤(SCI)后的保护作用及其可能机制。方法将84只SD大鼠随机分为三组:假手术组(n=4)、损伤组(n=40)和治疗组(n=40)。大鼠SCI模型采用纽约大学脊髓重物坠落伤模型。治疗组伤后即刻腹腔内注射咯利普兰,剂量为0.5 mg(/kg.d),2次/d,连续3d。损伤前后对损伤组和治疗组大鼠进行开放场地试验(BBB)评估大鼠脊髓功能。免疫组化染色分析三组大鼠损伤前后脊髓兴奋性氨基酸转运蛋白4(EAAT4)表达情况。结果损伤组和治疗组伤后即刻大鼠BBB评分均为0,处于完全瘫痪状态;然后BBB评分逐渐增高;伤后42、56和64d,治疗组BBB评分明显高于损伤组(P<0.01)。假手术组脊髓组织形态正常,EAAT4表达较少;脊髓损伤1周后,损伤中央出现较大空洞,EAAT4表达明显升高;而治疗组,组织空洞缩小,EAAT4表达较损伤组明显增加。结论咯利普兰有助于大鼠脊髓损伤后功能恢复,其机制可能与增加EAAT4表达有关。     

Participation of AMPA- and NMDA-type excitatory amino acid receptors in the spinal reflex transmission,in rat

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex.These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.     

Effects of aging on hyperalgesia and spinal dynorphin expression in rats with peripheral inflammation

The aging process is associated with various morphological and biochemical changes in the nervous system that may affect the processing of noxious inputs. This study showed greater hyperalgesia and up-regulation of spinal dynorphin (DYN) expression in aging than in young adult rats during CFA-induced peripheral inflammation. These data indicate that nociception is regulated differently in aging individuals, a fact that should be considered when selecting treatment strategies for aging populations with persistent pain.     

Trigeminal inputs to reticulospinal neurones in lampreys are mediated by excitatory and inhibitory amino acids

Reticulospinal (RS) neurones integrate sensory inputs from several modalities to generate appropriate motor commands for maintaining body orientation and initiation of locomotion in lampreys. As in other vertebrates, trigeminal afferents convey sensory inputs from the head region. The in vitro brainstem/spinal cord preparation of the lamprey was used for characterizing trigeminal inputs to RS neurones as well as the transmitter systems involved. The trigeminal nerve on each side was electrically stimulated and synaptic responses, which consisted of mixed excitation and inhibition, were recorded intracellularly in the middle and posterior rhombencephalic reticular nuclei. The EPSPs were mediated by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors. An increase in the late phase of the excitatory response occurred when Mg²⁺ ions were removed from the Ringer's solution. This effect was antagonized by 2-amino-5-phosphonopentanoate (2-AP5) or reversed by restoring Mg²⁺ ions to the perfusate suggesting the activation of N-methyl-d-aspartate (NMDA) receptors. IPSPs were mediated by glycine. These findings are similar to those reported for other types of sensory inputs conveyed to RS neurones, where excitatory and inhibitory amino acid transmission is also involved.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and α-amino 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM )-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 μM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 μM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a longlasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3± 66.1 %) and aspartate (286.3 ± 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Differential modulatory roles of cholera toxin and pertussis toxin in the regulation of pain responses induced by excitatory amino acids administered intrathecally in mice

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in excitatory amino acids induced pain response. Intrathecal (i.t.) injection of glutamate (20 microg), N-methyl-D-aspartic acid (NMDA; 60 ng), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 13 ng), and kainic acid (12 ng) showed pain response. Pretreatment with CTX (0.05 and 0.5 microg, i.t.) attenuated pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t. in a dose-dependent manner. On the other hand, i.t. pretreatment with PTX further increased the pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t., especially at the dose of 0.5 microg. Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play opposite roles in modulating the pain response induced by spinally administered. Furthermore, CTX- and PTX-sensitive G-proteins appear to modulate pain response induced by stimuli of both NMDA and non-NMDA glutamate receptors.     

Activation of locus coeruleus by prefrontal cortex is mediated by excitatory amino acid inputs

We examined the role of excitatory amino acids (EAAs) in activation of noradrenergic locus coeruleus (LC) neurons evoked by electrical stimulation of the medial prefrontal cortex (mPFC) in halothane-anesthetized rats. Microinfusion of the specific N-methyl-

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-aspartate antagonist 2-amino-5-phosphonopentanoic acid (AP5, 50 or 100 μM) into the LC significantly suppressed LC responses evoked by mPFC stimulation. Microinfusion of the selective non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 25 or 50 μM) also significantly reduced evoked LC responses. Simultaneous microinfusion of both AP5 and CNQX considerably increased the proportion of LC neurons which exhibited complete suppression of evoked responses (81%), compared to either AP5 or CNQX alone (50% each). These results indicate that LC activation by mPFC stimulation is mediated by both NMDA- and non-NMDA-type EAA channels.     

Attenuation of NMDA-induced spinal hypersensitivity by adrenal medullary transplants

Abnormal sensory hyperexcitability consequent to peripheral injury most likely involves activation of N-methyl-d-aspartate (NMDA) receptors in the spinal cord. This activation may lead to a cascade of neuroplastic events resulting in the exaggeration of sensory responses and the persistence of pathological pain states. Recent studies in our laboratory have demonstrated that the transplantation of adrenal medullary cells into the spinal subarachnoid space can alleviate pathological pain symptoms, possibly by reducing spinal hyperexcitability. The purpose of this study was to assess spinal NMDA activation-induced hypersensitivity to noxious and innocuous stimuli, and determine whether adrenal medullary transplants can intervene favorably to reduce these responses. Animals with either adrenal medullary or control transplants were injected intrathecally with several doses of NMDA, and responses to sensory stimuli were determined over time. NMDA at all doses tested (1–50 nmol) produced significant thermal and mechanical hyperalgesia and tactile allodynia in control transplanted animals, with peak severity at 30 min post-injection. In contrast, both the severity and duration of these exaggerated sensory responses were markedly reduced in animals with adrenal medullary transplants. To assess a possible contribution of released opioid peptides and catecholamines from the transplanted chromaffin cells, animals were pretreated with opiate antagonist naloxone or -adrenergic antagonist phentolamine. While naloxone was ineffective, the phentolamine partially attenuated, but did not completely abolish, the antinociceptive effects of the transplants. The results of these studies demonstrate that adrenal medullary grafts can reduce hypersensitivity responses to NMDA-mediated activation via -adrenergic modulation in addition to other neuroprotective mechanisms.     

Role of excitatory amino acids in rat vagal and sympathetic baroreflexes

Vagal baroreflexes were studied by measuring the atropine-sensitive cardioinhibition produced by raising arterial pressure with phenylephrine in anesthetized rats pretreated with the beta-adrenergic receptor antagonist nadolol. Sympathetic baroreflexes were determined in halothane-anesthetized rats by measuring the inhibition of lumbar sympathetic discharge produced by elevating arterial pressure with gradual aortic constriction. Both reflexes were drastically reduced by bilateral injections of 2.2 nmol of the glutamate receptor antagonist kynurenic acid (KYN) into either the nucleus of the solitary tract (NTS) or the ventrolateral medulla between 0 and 1 mm posterior to the level of the obex. Injections of KYN elsewhere in the medulla were generally ineffective and injections of 8-OH kynurenate (an inactive analog) into the ventrolateral medulla or NTS were also without effect. KYN injections (2.2 nmol) into the intermediate portion of the NTS produced small increases in mean arterial pressure (0-15 mm Hg) and no change in heart rate while injections of similar amounts into the ventrolateral medulla at obex level were followed by large (35-116 mm Hg) increases in pressure and bradycardia. Both types of injections produced a similar degree of blockade of vagal and sympathetic baroreflexes. These results support previous evidence that baroreceptor primary afferents may release a glutamate-like transmitter in the NTS and indicate that a similar type of excitatory transmitter is involved at the level of the ventrolateral medulla in mediating or modulating both vagal and sympathetic baroreflexes. Finally the bradycardia and hypertension produced by blocking amino acid receptors in the ventrolateral medulla appear largely unrelated to the disruption of peripheral baroreceptor inputs.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis

The spinal cord of 20 patients with amyotrophic lateral sclerosis (ALS) and 5 patients with lower motor neuron disease (LMND) were investigated immunohistochemically using anti-human excitatory amino acid transporter 1 (EAAT1) and EAAT2 antibodies which are the astrocytic transporters. The purpose of the study was to examine relationships between EAAT1 and EAAT2 immunoreactivity and degeneration of anterior horn neurons. Specimens from 20 patients without any neurological disease served as controls. In controls, spinal cord gray matter was densely immunostained by antibodies, whereas the white matter was generally not immunostained. In motor neuron disease (MND) patients, EAAT1 immunoreactivity was relatively well preserved in the gray matter despite neuronal loss of anterior horn cells. On the other hand, EAAT2 immunoreactivity in anterior horns correlated with the degree of neuronal loss of anterior horn cells: in the patients with mild neuronal depletion, anterior horns were densely immunostained by the antibody, whereas in the patients with severe neuronal loss, EAAT2 expression was markedly reduced. Degenerated anterior horn cells frequently showed a much denser EAAT1 and EAAT2 immunoreactivity around the surface of the neurons and their neuronal processes than that observed in normal-appearing neurons. There was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between LMND and ALS patients. These findings suggest that in the early stage of degeneration of anterior horn cells, EAAT1 and EAAT2 immunoreactivity is preserved in the astrocytic foot directly attached to normal-appearing neurons, whereas levels of EAAT1 and EAAT2 protein rather increase in the astrocytic foot directly attached to degenerated anterior horn neurons; the latter effect most probably reduces the elevated glutamate level, compensates for the reduced function of astroglial glutamate transporters, or represents a condensation of EAAT1 and EAAT2 immunoreactivity secondary to loss of neurites and greater condensation of astrocytic processes. Thus, we demonstrate a difference in EAAT1 and EAAT2 immunoreactivity in different stages of progression in ALS, as a feature of the pathomechanism of this disease. Received: 8 September 1999 / Revised, accepted: 28 October 1999     

Modulation of frog spinal cord interneuronal activity by activation of 5-HT3 receptors

Motoneuron membrane potentials were recorded from the ventral roots of isolated, hemisected frog spinal cords using sucrose gap techniques. The effects of the selective 5-HT₃ agonist 2-methyl-serotonin (2-Me-5HT) on the changes in motoneuron membrane potential produced by dorsal root stimulation and by superfusion of excitatory amino acid agonists were evaluated. Application of 2-Me-5HT (100 μM) did not alter motoneuron membrane potential, but did substantially reduce ( ∼ 20%) the polysynaptic ventral root potentials evoked by dorsal root stimulation. 2-Me-5HT did not change motoneuron depolarizations generated by addition to the Ringer's solution of the excitatory amino acid agonists AMPA (10–30 μM), kainate (30 μM), ort-ACPD (100 μM), but NMDA-induced motoneuron depolarizations (100 μM) were significantly and reversibly reduced ( ≈ 20%) by exposure to 2-Me-5HT (100 μM). 2-Me-5HT-evoked decreases of NMDA depolarizations were blocked by the 5-HT₃ antagonists ICS 205 930 (50–100 μM) andd-tubocurarine (3–10 μM), but not by MDL 72222 (20–100 μM), the 5-HT₂ receptor antagonist ketanserin (10 μM), or the 5-HT_1A/5-HT_2A antagonist spiperone (10 μM). Two lines of evidence support the hypothesis that the effects of 2-Me-5HT are generated by an indirect mechanism involving interneurons: (1) TTX (0.781 μM) eliminated the effect of 2-Me-5HT on NMDA-induced motoneuron depolarizations, and (2) 2-Me-5HT reduced spontaneous ventral root potentials that result from interneuronal discharges. We attempted to establish the identity of a putative transmitter released by interneurons responsible for the effects on NMDA-depolarizations produced by 2-Me-5HT, but the AMPA receptor antagonist, CNQX (10 μM), the GABA_A receptor antagonist, bicuculline (50 μM), the GABA_B receptor antagonist, saclofen (100 μM), the opioid antagonist, naloxone (100 μM), and the adenosine antagonists, CPT (20–100 μM) and CSC (10–100 μM) did not alter 2-Me-5HT-induced reductions of NMDA-depolarizations. In sum, the site of interaction between 2-Me-5HT and NMDA appears to be at interneuronal locus, but the mechanism remains unclear.     

Serotonin and/or an excitatory amino acid in the medial medulla mediates stimulation-produced antinociception from the lateral hypothalamus in the rat

Several lines of evidence have demonstrated a role for the lateral hypothalamus (LH) in an endogenous system of descending inhibition. Descending inhibition from the LH relies, at least in part, on a relay(s) in the midbrain and/or medulla. The medullary nucleus raphe magnus (NRM) serves as one such relay. The present study, in rats lightly anesthetized with pentobarbital, was undertaken to systematically examine the transmitter(s) in the medial medulla mediating descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH. The microinjection of pharmacologic receptor antagonists (5 μg) into the NRM revealed that the glutamate receptor antagonists, γ- -glutamylglycine and 2-amino-5-phosphonovalerate produced the largest increases in stimulation thresholds in the LH for inhibition of the TF reflex (107.6% and 102.6%, respectively). Methysergide, a serotonin receptor antagonist, also produced a significant increase (81.5%) in the stimulation threshold in the LH for inhibition of the TF reflex. The opioid receptor antagonist, naloxone, however, was without effect, producing only a 4.0% increase in the LH stimulation threshold. These results suggest that serotonin and/or an excitatory amino acid are transmitters at the bulbar relay in the medial medulla mediating descending inhibition of the TF reflex produced by focal electrical stimulation in the LH.     

Effects of bath-applied excitatory amino acids and their analogs on spinal interneurons of the lamprey

Depolarizations, conductance increases and time courses of the responses to bath application of glutamate, aspartate, DL-homocysteate, N-methyl-DL-aspartate (NMDLA), quisqualate and kainate were determined in interneurons of the isolated spinal cord of the lamprey, one of the most primitive vertebrates. Conductance increases produced by these excitants in perfusate containing tetrodotoxin (0.5 microgram/ml), 4-aminopyridine (1 mM) and without Ca2+ were very small in comparison with those produced by glycine or GABA. NMDLA-induced depolarizations were associated with conductance decreases and rhythmic oscillations in membrane potentials in this perfusate. Quisqualate was strongest among these amino acids in producing depolarizations and conductance increases. Responses induced by analogs were slower than those produced by glutamate and aspartate. Phylogenetic distribution of N-methyl-D-aspartate receptors on neurons and muscles is discussed.     

Nociceptive response to innocuous mechanical stimulation is mediated via myelinated afferents and NK-1 receptor activation in a rat model of neuropathic pain

Peripheral nerve injury in humans can produce a persistent pain state characterized by spontaneous pain and painful responses to normally innocuous stimuli (allodynia). Here we attempt to identify some of the neurophysiological and neurochemical mechanisms underlying neuropathic pain using an animal model of peripheral neuropathy induced in male Sprague-Dawley rats by placing a 2-mm polyethylene cuff around the left sciatic nerve according to the method of Mosconi and Kruger. von Frey hair testing confirmed tactile allodynia in all cuff-implanted rats before electrophysiological testing. Rats were anesthetized and spinalized for extracellular recording from single spinal wide dynamic range neurons (L(3-4)). In neuropathic rats (days 11-14 and 42-52 after cuff implantation), ongoing discharge was greater and hind paw receptive field size was expanded compared to control rats. Activation of low-threshold sensory afferents by innocuous mechanical stimulation (0.2 N for 3 s) in the hind paw receptive field evoked the typical brief excitation in control rats. However, in neuropathic rats, innocuous stimulation also induced a nociceptive-like afterdischarge that persisted 2-3 min. This afterdischarge was never observed in control rats, and, in this model, is the distinguishing feature of the spinal neural correlate of tactile allodynia. Electrical stimulation of the sciatic nerve at 4 and at 20 Hz each produced an initial discharge that was identical in control and in neuropathic rats. This stimulation also produced an afterdischarge that was similar at the two frequencies in control rats. However, in neuropathic rats, the afterdischarge produced by 20-Hz stimulation was greater than that produced by 4-Hz stimulation. Given that acutely spinalized rats were studied, only peripheral and/or spinal mechanisms can account for the data obtained; as synaptic responses from C fibers begin to fail above approximately 5-Hz stimulation [Pain 46 (1991) 327], the afterdischarge in response to 20-Hz stimulation suggests a change mainly in myelinated afferents and a predominant role of these fibers in eliciting this afterdischarge. These data are consistent with the suggestion that peripheral neuropathy induces phenotypic changes predominantly in myelinated afferents, the sensory neurons that normally respond to mechanical stimulation. The NK-1 receptor antagonist, CP-99,994 (0.5 mg/kg, i.v.), depressed the innocuous pressure-evoked afterdischarge but not the brief initial discharge of wide dynamic range neurons, and decreased the elevated ongoing rate of discharge in neuropathic rats. These results support the concept that following peripheral neuropathy, myelinated afferents may now synthesize and release substance P. A result of this is that tonic release of substance P from the central terminals of these phenotypically altered neurons would lead to ongoing excitation of NK-1-expressing nociceptive spinal neurons. In addition, these spinal neurons would also exhibit exaggerated responses to innocuous pressure stimulation. The data in this study put forth a possible neurophysiological and neurochemical basis of neuropathic pain and identify substance P and the NK-1 receptor as potential neurochemical targets for its management.     

Effects of kynurenate on root potentials evoked by synaptic activity and amino acids in the frog spinal cord

The effects of kynurenate (kyn) on synaptic- and excitatory amino acid-mediated responses in isolated, hemisected spinal cords of frog were examined. Kyn (0.5 mM) rapidly and reversibly blocked >90% of the synaptically mediated ventral root potential (VRP) produced by stimulation of the dorsal root. Spontaneous activities recorded from both ventral and dorsal roots were also reversibly blocked by Kyn. However, Kyn had no effect on action potentials or excitability per se, nor was it a general inhinitor of synaptic transmission since Kyn concentrations as high as 2.5 mM had no effect on synaptically mediated dorsal root potentials produced by stimulation of the ventral root. In addition, Kyn had no effect on synaptic transmission in sympathetic ganglia of frog. Although Kyn (2.5 mM) by itself produced no ventral root response in spinal cords treated with tetrodotoxin, it antagonized those induced by the excitatory amino acids N-methyl-d,l-aspartate, quisqualite, kainate, aspartate, and glutamate. The ventral responsed to all concentrations of quisqualate tested were depressed by 2.5 mM Kyn. In addition, when Kyn was washed out, the rate of recovery from Kyn block was accelerated by the presence of quisqualate. These results indicate that quisqualate and Kyn compete for common binding sites. However, low concentrations of Kyn (e.g. 0.1 mM) potentiated the peak of the response to saturating concentrations of quisqualate by as much as 30%. The durations of the potentiated quisqualate responses were significantly shorter than the control responses. Thus, Kyn does not act simply as a competitive inhibitor of quisqualate. Incontrast, Kyn appears to simply block N-methyl-d,l-aspartate responses with no signs of poptentiation or charges in kinetics.     

Long-lasting neurochemical and functional changes in rats induced by neonatal administration of substance P antiserum

Substance P (SP) antiserum was administered to rats on the second day of life. Three months later, the content of SP was significantly decreased in the dorsal part of the spinal cord and in the periaqueductal gray matter of these animals, as compared to control rats receiving a neonatal treatment of non-specific immunoglobulins. Further, the levels of Met-enkephalin and 5-hydroxyindole acetic acid (5-HIAA) were concomitantly increased in the same regions. SP receptor binding sites and opioid receptors, which appear earlier in development, were not modified in the two regions studied. On the other hand, the antinociceptive response to intracerebroventricular (i.c.v.) injection of SP or of the synthetic enkephalin analog D-Ala2,D-Leu5-enkephalin, as well as the hypertensive response to i.c.v. SP were blocked. The results suggest that, after administration to newborn rats, the antiserum is able to penetrate into SP neurons, producing a long-lasting SP suppression and a subsensitivity to the pharmacological effects of the neuropeptide. The modifications in the content of Met-enkephalin and 5-HIAA are possibly compensatory changes which subserve the functionality of central cardiovascular and pain regulatory systems after the immunolesion.