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Spinal dorsal roots can be affected by a wide range of lesions, leading to a significant loss of proprioceptive information transmission and greatly affecting motor behavior. In this context, the reimplantation of lesioned roots with platelet-rich plasma (PRP) may allow nerve regeneration. Therefore, the present study evaluated sensorimotor improvement following dorsal root rhizotomy and repair with PRP. For this purpose, female Lewis rats were subjected to unilateral rhizotomy (RZ) of the L4-L6 dorsal roots and divided into the following groups: (1) the unlesioned control group; (2) the group that underwent rhizotomy (RZ) without repair; and (3) the group that underwent RZ followed by root repair with PRP. PRP was obtained from human blood and characterized regarding platelet concentration, integrity, and viability. Reflex arc recovery was evaluated weekly for eight weeks by the electronic von Frey method. The spinal cords were processed 1 week postlesion to evaluate the in vivo gene expression of TNFα, TGF-β, BDNF, GDNF, VEGF, NGF, IL-4, IL-6, IL-13 by qRT-PCR and eight weeks postlesion to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. The results indicated that PRP therapy partially restores the paw withdrawal reflex over time, indicating the reentry of primary afferents from the dorsal root ganglia into the spinal cord without exacerbating glial reactivity. Additionally, the analysis of mRNA levels showed that PRP therapy has immunomodulatory properties. Overall, the present data suggest that the repair of dorsal roots with PRP may be considered a promising approach to improve sensorimotor recovery following dorsal rhizotomy., (Copyright © 2019. Published by Elsevier Inc.)

The inspiratory motor activities are greater in the intercostal muscles positioned at more rostral thoracic segments. This rostro-caudal gradient of the thoracic inspiratory motor activity is thought to be generated by the spinal interneurons. To clarify the involvement of the inhibitory thoracic interneurons in this rostro-caudal gradient, we examined the effects of 10 μM strychnine, an antagonist of glycine and GABA A receptors, applied to the neonatal rat thoracic spinal cord. The respiratory-related interneuron activities were optically recorded from thoracic segments in the isolated neonatal rat brainstem-spinal cord preparations stained with voltage-sensitive dye, and the electrical inspiratory motor activities were obtained from the third and eleventh thoracic ventral roots (T3VR, T11VR). Although strychnine caused seizure-like activities in all of the ventral roots recorded, the inspiratory motor activities continued. The inspiratory optical signals in the rostral thoracic segments (T2-T5) were significantly larger than those in the caudal thoracic segments (T9-T11) regardless of the existence of strychnine. Similarly, the percent ratio of the amplitude of the inspiratory electrical activity in the T3VR under control and strychnine was significantly larger than that in the T11VR regardless of the existence of strychnine. Strychnine significantly increased the inspiratory activity in both the T3VR and T11VR. These results suggest that the glycinergic and GABAergic inhibitory interneurons are not essential to generate the rostro-caudal gradient in the neonatal rat thoracic inspiratory motor outputs, but these interneurons are likely to play a role in the inhibitory control of inspiratory motor output., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Locomotor patterns are mainly modulated by afferent feedback, but its actual contribution to spinal network activity during continuous passive limb training is still unexplored. To unveil this issue, we devised a robotic in vitro setup (Bipedal Induced Kinetic Exercise, BIKE) to induce passive pedaling, while simultaneously recording low-noise ventral and dorsal root (VR and DR) potentials in isolated neonatal rat spinal cords with hindlimbs attached. As a result, BIKE evoked rhythmic afferent volleys from DRs, reminiscent of pedaling speed. During BIKE, spontaneous VR activity remained unchanged, while a DR rhythmic component paired the pedaling pace. Moreover, BIKE onset rarely elicited brief episodes of fictive locomotion (FL) and, when trains of electrical pulses were simultaneously applied to a DR, it increased the amplitude, but not the number, of FL cycles. When BIKE was switched off after a 30-min training, the number of electrically induced FL oscillations was transitorily facilitated, without affecting VR reflexes or DR potentials. However, 90 min of BIKE no longer facilitated FL, but strongly depressed area of VR reflexes and stably increased antidromic DR discharges. Patch clamp recordings from single motoneurons after 90-min sessions indicated an increased frequency of both fast- and slow-decaying synaptic input to motoneurons. In conclusion, hindlimb rhythmic and alternated pedaling for different durations affects distinct dorsal and ventral spinal networks by modulating excitatory and inhibitory input to motoneurons. These results suggest defining new parameters for effective neurorehabilitation that better exploits spinal circuit activity., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the present study, multi-electrode array recording was used to examine dorsal horn activity following stimulation of primary afferents in a rat dorsal root attached-spinal cord slice preparation. The multi-electrode array probe was placed under the dorsal horn slice and local field potentials evoked by stimulation on the dorsal root were analyzed. Three kinds of dorsal root-evoked responses were identified. In lamina II o , local field potentials exhibited P1 (peak latency 1.46±0.08ms), N1 (2.77±0.18ms, n=12), N2 (7.31±0.48ms), N3 (12.12±0.73ms) and P2(18.30±0.80ms) waves. In lamina II i local field potentials exhibited P (1.99±0.10ms), N1 (3.35±0.17ms) and N2 (8.58±0.44ms) waves. In laminae III-VI, local field potentials exhibited P1 (3.01±0.07ms), P2 (7.02±0.21ms) and N waves (22.57±0.79ms). Sweep spread was calculated by two dimensional current source density (2D-CSD) analysis. Both α-amino-3-hydroxy-5-methylisoxazole-4-propionic a/kainate and N-methyl-d-aspartate-type glutamate receptors participated in this neuronal circuitry. Morphine diminished local field potentials. Gabapentin diminished the negative components in lamina II and P2 component in lamina II o , but increased the positive components in lamina II i and laminae III-VI. The present study revealed that functional dorsal horn activity was preserved in the spinal cord slice preparation. Glutamatergic synapses were crucially involved in information processing. Opioid interneurons and gabapentin may play a modulatory role in regulating signal flows in the dorsal horn. Taken together, these results identify a spatio-temporal profile of dorsal horn activity evoked by dorsal root stimulation, and implicate glutamatergic and opioidergic receptors and gabapentin in this activity., (Copyright © 2017. Published by Elsevier Inc.)

Bisphenol A (BPA), a toxic chemical from plastics, is known to produce locomotor abnormalities which may imply the alteration in synaptic activity at Ia-α motoneuron synapse also. However the effect of BPA on this synapse is not known. Therefore, this study was undertaken to examine the effect of BPA on reflexes originating at Ia-α motoneuron synapse in the spinal cord. The experiments were performed on isolated hemisected spinal cords from 4 to 6d rats. Stimulation of a dorsal root evoked segmental monosynaptic (MSR) and polysynaptic (PSR) reflex potentials in the corresponding ventral root. Nitrite content (indicator of NO activity) of cords was estimated in the presence of BPA with/without antagonists. Superfusion of BPA (3-100μM) depressed the reflexes in a concentration- and time-dependent manner. The depression was ∼20, ∼50 and ∼70% at 10, 30 and 100μM of BPA, respectively. The 50% depression occurred around 15min at 30μM of BPA. Pretreatment with estrogen receptor (ERα) antagonist, tamoxifen, blocked the BPA-induced depression of reflexes, whereas, 17β-estradiol, ER agonist, did not depress the reflexes even up to 10μM. Further, pretreatment with Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or hemoglobin (Hb) blocked the BPA-induced depression of spinal reflexes. Nitric oxide (NO) donor sodium-nitroprusside depressed the MSR and PSR in a concentration-dependent manner. The nitrite concentration of the cords exposed to BPA was 733μM/gm of tissue (three times the saline group). Pretreatment with tamoxifen/l-NAME/Hb blocked the BPA-induced increase of nitrite levels. The present observations indicate that BPA depressed spinal synaptic transmission through ERα-dependent NO-mediated mechanisms. The altered synaptic activity may implicate for neurobehavioral locomotor abnormalities after exposure to BPA., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

GABAA receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain. Restoration of diminished spinal GABAA-α2 and -α3 subunit-containing receptor function is a principal contributor to this analgesia, albeit involvement of GABAA-α5-receptors has not been excluded. Thus, we compared NS11394 and TPA023 (PAMs with selectivity/efficacy at GABAA-α2/α3/α5 receptors) with TP003 (a reportedly GABAA-α3 selective PAM) against spinal sensitization. However, in-house electrophysiology studies designed to confirm the selectivity of TPA023 and TP003 for human GABAA receptors did not corroborate published data, with TP003 displaying considerable GABAA-α5 receptor efficacy. Therefore, we identified a novel PAM, NS16085, which possesses negligible efficacy at GABAA-α5 receptors, but with GABAA-α2/α3 efficacy equivalent to NS11394. At the GABAA-α1 receptor the compound gives low level of negative modulation further separating it from the other compounds. Rat pups with carrageenan-induced hindpaw inflammatory hyperalgesia were used to make ex vivo spinal dorsal root-evoked ventral root recordings. Some spontaneous activity and large numbers of spikes to repetitive stimulation of dorsal roots at C-fibre intensity, indicative of wind-up and sensitization were observed. Equimolar concentrations of NS11394, TP003 and NS16085 all attenuated wind-up to a similar degree; TPA023 was clearly less effective. In adult rats, NS16085 (3-30 mg/kg, p.o.) dose-dependently reduced formalin-induced hindpaw flinching with efficacy comparable to NS11394. Thus, potentiation of GABAA-α2 and-α3 receptors is sufficient to depress spinal sensitization and mediate analgesia after inflammatory injury. Positive modulation at GABAA-α5-receptors is apparently dispensable for this process, an important consideration given the role of this receptor subtype in cognitive function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neuropeptide Y (NPY) is present in the superficial laminae of the dorsal horn and inhibits spinal nociceptive processing, but the mechanisms underlying its anti-hyperalgesic actions are unclear. We hypothesized that NPY acts at neuropeptide Y1 receptors in the dorsal horn to decrease nociception by inhibiting substance P (SP) release, and that these effects are enhanced by inflammation. To evaluate SP release, we used microdialysis and neurokinin 1 receptor (NK1R) internalization in rat. NPY decreased capsaicin-evoked SP-like immunoreactivity in the microdialysate of the dorsal horn. NPY also decreased non-noxious stimulus (paw brush)-evoked NK1R internalization (as well as mechanical hyperalgesia and mechanical and cold allodynia) after intraplantar injection of carrageenan. Similarly, in rat spinal cord slices with dorsal root attached, [Leu(31), Pro(34)]-NPY inhibited dorsal root stimulus-evoked NK1R internalization. In rat dorsal root ganglion neurons, Y1 receptors colocalized extensively with calcitonin gene-related peptide (CGRP). In dorsal horn neurons, Y1 receptors were extensively expressed and this may have masked the detection of terminal co-localization with CGRP or SP. To determine whether the pain inhibitory actions of Y1 receptors are enhanced by inflammation, we administered [Leu(31), Pro(34)]-NPY after intraplantar injection of complete Freund's adjuvant (CFA) in rat. We found that [Leu(31), Pro(34)]-NPY reduced paw clamp-induced NK1R internalization in CFA rats but not uninjured controls. To determine the contribution of increased Y1 receptor-G protein coupling, we measured [(35)S]GTPγS binding simulated by [Leu(31), Pro(34)]-NPY in mouse dorsal horn. CFA inflammation increased the affinity of Y1 receptor G-protein coupling. We conclude that Y1 receptors contribute to the anti-hyperalgesic effects of NPY by mediating the inhibition of SP release, and that Y1 receptor signaling in the dorsal horn is enhanced during inflammatory nociception., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Compression of spinal roots is an important medical problem, which may arise from intervertebral disc herniation, tumor growth or as a result of high energy accidents. Differently from avulsion, root crushing maintains the central/peripheral nervous system (CNS/PNS) connection, although the axons are axotomized and motoneurons degenerate. Such neuronal death may decrease and delay motor function recovery. In the present study we have investigated the neuroprotective effects of mesenchymal stem cell (MSC) therapy following such proximal lesions. Motor recovery and synaptic stabilization were analyzed by the use of morphological and functional approaches. For that, crushing the ventral roots at L4, L5 and L6 was unilaterally performed in Lewis rats. Four weeks after injury, an increased motoneuron survival was observed in the MSC-treated group, coupled with a smaller decrease of inputs at the motoneuron surface and nearby neuropil, seen by synaptophysin and synapsin immunolabeling and decreased astrogliosis, seen by GFAP immunolabeling. In this sense, MSC-treated group displayed a significant preservation of GABAergic terminals, indicating a possible neuroprotection to glutamate excitotoxicity. Motor function recovery was acutely improved in MSC-treated group as compared to Dulbeco's modified eagle medium (DMEM)-treated. Overall, we provide evidence that ventral root crushing (VRC), although milder than avulsion, results in significant loss of motoneurons (~51%) that can be reduced by MSC administration within the spinal cord. Such treatment also improves the number of synapses immunoreactive against molecules present in inhibitory inputs. Also, an increased number of regenerated axons was obtained in the MSC-treated group, in comparison to the DMEM-treated control. Overall, MSC therapy acutely improved limb strength and gait coordination, indicating a possible clinical application of such treatment following proximal lesions at the CNS/PNS interface., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Most investigations into the role of the body axis in vertebrate locomotion have focused on the trunk, although in most tetrapods, the tail also plays an active role. In salamanders, the tail contributes to propulsion during swimming and to dynamic balance and maneuverability during terrestrial locomotion. The aim of the present study was to obtain information concerning the neural mechanisms that produce tail muscle contractions during locomotion in the salamander Pleurodeles waltlii. We recorded the ventral root activities in in vitro spinal cord preparations in which locomotor-like activity was induced via bath application of N-methyl-d-aspartate (20μM) and d-serine (10μM). Recordings showed that the tail spinal cord is capable of producing propagated waves of motor activity that alternate between the left and right sides. Lesion experiments further revealed that the tail rhythmogenic network is composed of a double chain of identical hemisegmental oscillators. Finally, using spinal cord preparations bathed in a chamber partitioned into two pools, we revealed efficient short-distance coupling between the trunk and tail networks. Together, our results demonstrate the existence of a pattern generator for rhythmic tail movements in the salamander and show that the global architecture of the tail network is similar to that previously proposed for the mid-trunk locomotor network in the salamander. Our findings further support the view that salamanders can control their trunk and tail independently during stepping movements. The relevance of our results in relation to the generation of tail muscle contractions in freely moving salamanders is discussed., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

It is not well-studied how the ubiquitous neuromodulator adenosine (ADO) affects mammalian locomotor network activities. We analyzed this here with focus on roles of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)-sensitive A(1)-type ADO receptors. For this, we recorded field potentials from ventral lumbar nerve roots and electrically stimulated dorsal roots in isolated newborn rat spinal cords. At ≥ 25μM, bath-applied ADO slowed synchronous bursting upon blockade of anion-channel-mediated synaptic inhibition by bicuculline (20 μM) plus strychnine (1 μM) and this depression was countered by DPCPX (1 μM) as tested at 100 μM ADO. ADO abolished this disinhibited rhythm at ≥ 500 μM. Contrary, the single electrical pulse-evoked dorsal root reflex, which was enhanced in bicuculline/strychnine-containing solution, persisted at all ADO doses (5 μM-2 mM). In control solution, ≥ 500 μM ADO depressed this reflex and pulse train-evoked bouts of alternating fictive locomotion; this inhibition was reversed by 1 μM DPCPX. ADO (5 μM-2 mM) did not depress, but stabilize alternating fictive locomotion evoked by serotonin (10 μM) plus N-methyl-d-aspartate (4-5 μM). Addition of DPCPX (1μM) to control solution did not change either the dorsal root reflex or rhythmic activities indicating lack of endogenous A(1) receptor activity. Our findings show A(1) receptor involvement in ADO depression of the dorsal root reflex, electrically evoked fictive locomotion and spontaneous disinhibited lumbar motor bursting. Contrary, chemically evoked fictive locomotion and the enhanced dorsal root reflex in disinhibited lumbar locomotor networks are resistant to ADO. Because ADO effects in standard solution occurred at doses that are notably higher than those occurring in vivo, we hypothesize that newborn rat locomotor networks are rather insensitive to this neuromodulator., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Increases in lung volume inhibit the inspiratory output from the medulla, but the effect of lung inflation on the voluntary control of breathing in humans is not known. We tested corticospinal excitability using transcranial magnetic stimulation (TMS) to evoke a response in the scalene muscles. TMS was delivered at rest at three different lung volumes between functional residual capacity (FRC) and total lung capacity (TLC) during incremental inspiratory and incremental expiratory manoeuvres. Motor evoked potentials (MEPs) in scalenes were ∼50% larger at a high lung volume (FRC+∼90% inspiratory capacity [IC]) compared to lower lung volumes (FRC and FRC+∼40% IC) in both inspiratory and expiratory manoeuvres (p<0.001). The change in MEP size was not due to differences in pre-stimulus EMG amplitude (p=0.29). The results suggest a differential effect of lung inflation on the automatic and voluntary control of breathing in humans., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Delta opioid receptor (DOR) activation protects the adult mammalian brain during oxygen-glucose deprivation (OGD), but it is not known whether neonatal spinal motor circuits are also protected. Also, it is unclear whether the timing of spinal DOR activation relative to spinal OGD is important for neuroprotection. Thus, a split-bath in vitro neonatal rat brainstem/spinal cord preparation was used to record spontaneous respiratory motor output from cervical (C4-C5) and thoracic (T5-T6) ventral spinal roots while exposing only the spinal cord to OGD solution (0 mM glucose, bubbled with 95% N(2)/5% CO(2)) or DOR agonist drugs (DADLE, DPDPE). Spinal OGD solution application caused respiratory motor output frequency and amplitude to decrease until all activity was abolished (i.e. end-point times) after 25.9±1.4 min (cervical) and 25.2±1.4 min (thoracic). Spinal DOR activation via DPDPE (1.0 μM) prior-to and during spinal OGD increased cervical and thoracic end-point times to 35-48 min. Spinal DADLE or DPDPE (1.0 μM) application 15 min following spinal OGD onset increased cervical and thoracic end-point times to 36-45 min. Brief spinal DPDPE (1.0 μM) application for 10 min at 25 min before spinal OGD onset increased cervical and thoracic end-point times to 41-46 min. Overall, the selective DOR agonist, DPDPE, was more effective at increasing end-point times than DADLE. Naltrindole (DOR antagonist; 10 μM) pretreatment blocked DPDPE-dependent increase in end-point times, suggesting that DOR activation was required. Spinal naloxone (1.0 μM) application before and during spinal OGD also increased end-point times to 31-33 min, but end-point times were not altered by Mu opioid receptor (MOR) activation or DOR activation/MOR blockade, indicating that there are complex interactions between OGD and opioid signaling pathways. These data suggest DOR activation before, during, and after spinal OGD protects central motor networks and may provide neuroprotection during unpredictable perinatal ischemic events., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Phosphodiesterase type 5 inhibitors (PDE5-Is) improve storage symptoms in benign prostatic hyperplasia patients, despite a lack of effect on peak urinary flow rate. Moreover, vardenafil improves urodynamic parameters in spinal cord-injured (SCI) patients with neurogenic detrusor overactivity (NDO). SCI rats also display NDO characterized by nonvoiding contractions (NVCs) during bladder filling, resulting in an increased bladder afferent nerve firing (BANF)., Objective: We postulated that vardenafil could improve urodynamic parameters by reducing BANF. The effect of vardenafil has been investigated on intravesical pressure by cystometry experiments while recording BANF in response to bladder filling., Design, Setting, and Participants: Complete T7-T8 spinalization was performed in 15 female adult Sprague-Dawley rats (250-275 g)., Measurements: At 21-29 d postspinalization, fine filaments were dissected from the L6 dorsal roots and placed across a bipolar electrode. Bladder afferent nerve fibers were identified by electrical stimulation of the pelvic nerve and bladder distension. SCI rats were decerebrated before cystometry experiments. Bladders were filled to determine the maximal bladder filling volume (BFV) for each rat. Then, after bladder stabilization at 75% of maximal BFV, saline (n=7) or vardenafil 1 mg/kg (n=8) was delivered intravenously. NVCs and BANF were recorded for 45 min., Results and Limitations: In all SCI rats, BANF was already present and regular at resting conditions (26.2±4.1 spikes per second). During bladder filling, intravesical pressure (IVP) slowly increased with transient NVCs superimposed. Concomitantly, BANF progressively increased up to 2.4-fold at maximal BFV (2.08±0.24 ml). After stabilization at submaximal BFV, BANF was increased by 186±37%. Vardenafil injection induced an immediate decrease in NVCs compared to saline (p<0.001) and BANF (52% decrease vs 28% in saline after 45 min; p<0.001)., Conclusions: Systemic vardenafil reduced both NVCs and BANF in unanesthetized, decerebrate, SCI rats. These findings provide new insights into the mechanism of action by which PDE5-Is improve storage symptoms in SCI patients., (Copyright © 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

To investigate the susceptibility of the group II metabotropic glutamate receptor mGlu2 to agonist-induced desensitization, the receptor was stably expressed in Chinese hamster ovary (CHO-mGlu2) or C6 glioma cells (C6-mGlu2). Exposure of CHO-mGlu2 cells to the group II mGlu receptor agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG-1; 10 μM) for up to 15 h did not affect the subsequent ability of LCCG-1 to inhibit forskolin-stimulated cAMP accumulation. Similarly, in C6-mGlu2 cells, prolonged exposure to LCCG-1 also did not affect the subsequent ability of LCCG-1 to inhibit cAMP formation. In contrast, exposure of CHO-mGlu2 cells to the protein kinase C activator phorbol myristate acetate (PMA) suppressed the ability of LCCG-1 to inhibit cAMP formation. Using an in vitro model of group II mGlu receptor activity, the hemisected neonatal rat spinal cord preparation, the ability of the selective group II agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC) to depress the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) did not desensitize when applied for up to 2 h. Together these results indicate that in contrast to most G protein-coupled receptors, the mGlu2 receptor is resistant to agonist-induced homologous desensitization, and that in vitro data suggests that resistance to desensitization is a physiologically relevant property of this mGlu receptor subtype., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Synapse specific differences in NR2 subunit expression exist in several systems within the mammalian CNS. Here we have studied such differences on motoneurons in the neonatal rat cord using ifenprodil known to inhibit voltage-, use- and glycine-independent responses mediated by NR2B-containing N-methyl-d-aspartate receptors (NMDARs) with high specificity. In neonatal rats (P1-P9), the synapses made by the dorsal root (DR) fibres were more sensitive to ifenprodil than ventrolateral funiculus (VLF) connections on the same motoneuron. DR connections exhibited very little additional blockade to bath-applied MK-801 whereas VLF connections displayed a further decrease in amplitude. This suggests that at this immediate postnatal age, DR synapses on motoneurons contain a higher proportion of ifenprodil-sensitive diheteromeric NR1/NR2B receptors than VLF synapses. Since DR synapses have been shown in other studies to be less mature than VLF synapses on the same motoneuron at this developmental stage, these data are interpreted as indicating that less mature NMDA receptors feature a higher proportion of NR2B subunits which declines as the synapse matures. This novel finding of staggered development of NMDA receptors from different synaptic inputs on the same motoneuron is discussed in the context of its developmental and functional implications., (Published by Elsevier Ltd.)

Both the firing frequency of primary afferents and neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons increase with the intensity of noxious stimulus. Accordingly, we studied how the pattern of firing of primary afferent influences NK1R internalization. In rat spinal cord slices, electrical stimulation of the dorsal root evoked NK1R internalization in lamina I neurons by inducing substance P release from primary afferents. The stimulation frequency had pronounced effects on NK1R internalization, which increased up to 100 Hz and then diminished abruptly at 200 Hz. Peptidase inhibitors increased NK1R internalization at frequencies below 30 Hz, indicating that peptidases limit the access of substance P to the receptor at moderate firing rates. NK1R internalization increased with number of pulses at all frequencies, but maximal internalization was substantially lower at 1-10 Hz than at 30 Hz. Pulses organized into bursts produced the same NK1R internalization as sustained 30 Hz stimulation. To determine whether substance P release induced at high stimulation frequencies was from C-fibers, we recorded compound action potentials in the sciatic nerve of anesthetized rats. We observed substantial NK1R internalization when stimulating at intensities evoking a C-elevation, but not at intensities evoking only an Adelta-elevation. Each pulse in trains at frequencies up to 100 Hz evoked a C-elevation, demonstrating that C-fibers can follow these high frequencies. C-elevation amplitudes declined progressively with increasing stimulation frequency, which was likely caused by a combination of factors including temporal dispersion. In conclusion, the instantaneous firing frequency in C-fibers determines the amount of substance P released by noxious stimuli.

Effect of 3-nitropropionic acid (3-NPA) and ischemia (glucose- and O(2)-free solution) on synaptic transmission in hemisected spinal cord from 4 to 8 day old rats was examined in vitro. Stimulation of a dorsal root (L3-5 segments) evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. Superfusion of 3-NPA (0.17-3.4 mM) depressed the reflexes in a concentration- and time-dependent manner. At 3.4 mM of 3-NPA, the reflexes were abolished by 35 min. Time required to produce 50% depression (T-50) was around 170, 80, 40 and 17 min for MSR and 110, 70, 25 and 16 min for PSR at 0.17, 0.51, 1.7 and 3.4mM of 3-NPA, respectively. Ischemia also produced a time-dependent depression of reflexes and abolished them by 35 min and the T-50 values were around 18 min. Presence of creatine phosphate (10mM) in the superfusing medium did not alter the time course of 3-NPA-induced depression of reflexes but prolonged the ischemia-induced depression. dl-2-amino-5-phosphonovaleric acid (NMDA receptor antagonist; 10 microM) failed to block the 3-NPA (3.4 mM)-induced depression of reflexes, but blocked the ischemia-induced depression. The results indicate that 3-NPA-induced depression of spinal reflexes does not involve NMDA receptors and is different from ischemia-induced depression.

L1 is a cell adhesion molecule associated with axonal outgrowth and fasciculation during spinal cord development and may reiterate its developmental role in adults following injury; L1 is upregulated on certain sprouting and regenerating axons in adults, but it is unclear if L1 expression is necessary for, or contributes to, regrowth of axons. This study asks if L1 is required for small-diameter primary afferents to sprout by conducting unilateral dorsal rhizotomies (six segments; T10-L2) on both wild-type and L1 mutant mice. First we determined that L1 co-localizes substantially with the peptidergic (calcitonin gene-related peptide; CGRP) but minimally with the nonpeptidergic (isolectin B4; IB4) primary afferents in intact wild-type and L1 mutant mice. However, we encountered a complication using IB4 to identify primary afferents post-rhizotomy; we detected extensive abnormal IB4 expression in the dorsal horn and dorsal columns. Much of this aberrant IB4 labeling is associated with fibrous astrocytes and microglia. Five days after dorsal rhizotomy a large decrease in peptidergic and nonpeptidergic afferents is evident on the deafferented side in both wild-type and L1 mutants. Three months after surgery the peptidergic primary afferents sprouted into the center of the denervated dorsal horn in both wild-type and mutant mice, and quantitative analyses confirmed a sprouting density of similar magnitude in both genotypes. In contrast, we did not detect sprouting in the nonpeptidergic primary afferents in either genotype. These results suggest that the absence of L1 neither diminishes nor enhances sprouting of peptidergic small-diameter primary afferent axons following a dorsal rhizotomy.

Recent renewed interest in the study of rhythmic behaviors and pattern-generating circuits has been inspired by the currently well-established role of oscillating neuronal networks in all aspects of the function of our nervous system: from sensory integration to central processing, and of course motor control. An integrative rather than reductionist approach in the study of pattern-generating circuits is in accordance with current developments. The lamprey spinal cord, a relatively simple and much-studied preparation, is a useful model for such a study. It is an example of a chain of coupled oscillatory units that is characterized by its ability to demonstrate robust coordinated rhythmic output when isolated in vitro. The preparation allows maximum control over the chemical (neuromodulators and hormones) as well as neuronal environment (sensory and descending inputs) of the single oscillatory unit: the pattern-generating circuit. The current study made use of recently developed tools for nonlinear analysis of time-series, specifically neurophysiological signals. These tools allow us to reveal and characterize biological-functional complexity and information capacity of the neuronal output recorded from the lamprey model network. We focused on the importance of different types of inputs to an oscillatory network and their effect on the network's functional output. We show that the basic circuit, when isolated from short- and long-range neuronal inputs, demonstrates its full potential of information capacity: maximal variation quantities and elevated functional complexity. Morphological and functional constraints result in the network exhibiting only a limited range of the above. This constitutes an important substrate for plasticity in neuronal network function.

The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking glutamate decarboxylase 67. Eur J Neurosci 21:173-178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem-spinal cord blocks prepared from GAD65-/-:67-/- and vesicular GABA transporter (VGAT)-/-mice on embryonic day 14 (E14)-E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65-/-:67-/- preparations but not in VGAT-/- preparations. C4 discharges were observed in the knockout mice on E14-E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a "blind" patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65-/-:67-/- mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT-/- mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age.

Trauma to the conus medullaris and cauda equina may result in autonomic, sensory, and motor dysfunctions. We have previously developed a rat model of cauda equina injury, where a lumbosacral ventral root avulsion resulted in a progressive and parallel death of motoneurons and preganglionic parasympathetic neurons, which are important for i.e. bladder control. Here, we report that an acute implantation of an avulsed ventral root into the rat conus medullaris protects preganglionic parasympathetic neurons and motoneurons from cell death as well as promotes axonal regeneration into the implanted root at 6 weeks post-implantation. Implantation resulted in survival of 44+/-4% of preganglionic parasympathetic neurons and 44+/-4% of motoneurons compared with 22% of preganglionic parasympathetic neurons and 16% of motoneurons after avulsion alone. Retrograde labeling from the implanted root at 6 weeks showed that 53+/-13% of surviving preganglionic parasympathetic neurons and 64+/-14% of surviving motoneurons reinnervated the graft. Implantation prevented injury-induced atrophy of preganglionic parasympathetic neurons and reduced atrophy of motoneurons. Light and electron microscopic studies of the implanted ventral roots demonstrated a large number of both myelinated axons (79+/-13% of the number of myelinated axons in corresponding control ventral roots) and unmyelinated axons. Although the diameter of myelinated axons in the implanted roots was significantly smaller than that of control roots, the degree of myelination was appropriate for the axonal size, suggesting normal conduction properties. Our results show that preganglionic parasympathetic neurons have the same ability as motoneurons to survive and reinnervate implanted roots, a prerequisite for successful therapeutic strategies for autonomic control in selected patients with acute conus medullaris and cauda equina injuries.

Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Female rats typically display lower thresholds to painful stimuli and display more robust responses following nerve injury as compared with males. There is a body of evidence implicating the sex hormones in mediating this sex difference. In the present study, we sought to determine which gonadal hormones were involved in mediating the observed female hypersensitivity in female rats both prior to and following experimental nerve root injury using a chronic hormone replacement paradigm. Female rats were ovariectomized and hormone pellets containing 17beta-estradiol, progesterone (P), 17beta-estradiol+progesterone or placebo were implanted s.c. Our results demonstrate that only the group of female rats that received progesterone alone maintained the hypersensitive phenotype following ovariectomy, compared with gonadally intact male rats. This result was observed both in response to thermal stimuli in non-injured female rats and to thermal and tactile stimuli following L5 nerve root ligation, a model of low back pain associated with lumbar radiculopathy. Postmortem analysis of serum gonadal hormone concentrations demonstrates that the hormonal manipulations were successful and the exogenous hormones were similar to physiological levels observed in the sham-ovariectomized controls. Taken together, these results demonstrate the critical role for progesterone in mediating enhanced female tactile and thermal hypersensitivity following L5 nerve root ligation.

Intrinsic spinal networks generate a locomotor rhythm characterized by alternating electrical discharges from flexor and extensor motor pools. Because this process is preserved in the rat isolated spinal cord, this preparation in vitro may be a useful model to explore methods to reactivate locomotor networks damaged by spinal injury. The present electrophysiological investigation examined whether the broad spectrum potassium channel blocker tetraethylammonium could generate locomotor-like patterns. Low (50-500 microM) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1-10 mM) evoked alternating discharges between flexor and extensor motor pools, plus large depolarization of motoneurons with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABA(A) and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I metabotropic glutamate receptors. Furthermore, tetraethylammonium also evoked irregular discharges on dorsal roots. Rhythmic alternating patterns elicited by tetraethylammonium on ventral roots were relatively stereotypic, had limited synergy with fictive locomotion induced by dorsal root stimuli, and were not accelerated by 4-aminopyridine. Horizontal section of the spinal cord preserved irregular ventral root discharges and dorsal root discharges, demonstrating that the action of tetraethylammonium on spinal networks was fundamentally different from that of 4-aminopyridine. These results show that a potassium channel blocker such as tetraethylammonium could activate fictive locomotion in the rat isolated spinal cord, although the pattern quality lacked certain features like frequency modulation and strong synergy with other inputs to locomotor networks.

An understanding of the biomechanical and physiological properties of spinal nerve roots, particularly in response to tension, is critical in understanding the pathomechanisms of pain and nerve root injury and subsequent management of related injuries. Biomechanical properties of dorsal nerve roots at the lumbar and sacral levels were evaluated at various strain rates. Nerve roots were stretched at two different rates, 0.01 mm/s (Group A, quasistatic) and 15 mm/s (Group B, dynamic). Load, displacement and digital video data were obtained as the nerve roots were stretched until failure. Maximum stress, strain at maximum stress and modulus of elasticity (E) were calculated from the load-displacement measurements. Comparison of mechanical properties and failure patterns of nerve roots at two different rates revealed significant differences. Maximum load, maximum stress and E values of 5.7+/-2.7 gm, 257.9+/-111.3 kPa and 1.3+/-0.8 MPa were observed for Group A and 13.9+/-7.5 gm, 624.9+/-306.8 kPa and 2.9+/-1.5 MPa were observed for Group B, respectively. Higher maximum load, maximum stress and E values occurred at the dynamic stretch rate as compared to the quasistatic stretch rate, illustrating the strain-rate dependency of spinal nerve roots. No differences were observed in the strain values. Differences in mechanical behavior of nerve roots were also observed among the four root levels (L4-S1). A significant interaction effect was observed between nerve root diameter and stretch rates. Overall, results from the present study demonstrate viscoelastic material properties of spinal nerve roots and provide better insight on the tensile properties of nerve roots at different strain rates.

The purpose of this study was to evaluate the glottis constriction response induced by a sudden and involuntary increase in gastric and oesophageal pressures by Tll-Ll intervertebral magnetic stimulation of the abdominal muscle roots in nine healthy subjects. Twitch flow, twitch gastric, and oesophageal pressures were measured after abdominal muscle root stimulation, which allowed pharyngo-laryngeal muscle activation to be characterized. Pharyngeal endoscopies were performed on five subjects to assess vocal cord movements. All stimulations induced positive gastric and oesophageal pressures and expiratory flow, which increased with stimulation intensity (flow: R=0.32; p<0.0001; oesophageal pressure: R=0.26; p=0.001; gastric pressure: R=0.37; p<0.0001). Twitch gastric pressure and twitch oesophageal pressure were negatively correlated with twitch flow (respectively, R=-0.183, p<0.05; R=-0.35, p<0.0001). Upper airway resistance was higher at peak oesophageal pressure than at peak flow (p<0.001). Peak twitch gastric and twitch oesophageal pressure latencies were similar (133+/-4ms and 122+/-4ms) but longer than peak twitch flow and EMG latencies (62+/-2ms and 73+/-4ms, p<0.0001). Glottis constriction following magnetic abdominal muscle root stimulation was seen in all subjects during endoscopy, with a latency estimated at between 80 and 100ms. This method could be a new, simple tool for assessing the upper airway constriction protective reflex.

C fiber dorsal root reflexes (DRR) contribute to neurogenic inflammation and possibly also to touch-evoked pain (allodynia) induced by intradermal capsaicin. The responses of C fibers in the sural nerve to graded mechanical stimuli before and following intradermal capsaicin were studied in 39 adult male rats. Two-thirds of 111 fibers were without spontaneous activity, while the remaining fibers averaged 1.41+/-0.25 spontaneous antidromic spikes per second. Among the quiescent C fibers only two had excitatory receptive fields, whereas the active C fibers showed three patterns of activity, an excitatory response, an inhibitory response, or no response to mechanical stimulation. The excitatory responses were to high intensity mechanical stimuli alone, while inhibitory responses were evoked in a graded fashion by both noxious and innocuous mechanical stimuli. Intradermal injection of capsaicin increased spontaneous and evoked DRRs in all C fibers with excitatory responses to mechanical stimuli, but none acquired responses to innocuous stimuli. Capsaicin initially produced inhibition of spontaneous activity in C fibers with inhibitory or no receptive fields, but this later resumed and achieved a rate higher than baseline. Mechanical stimuli re-applied following the resumption of spontaneous discharges failed to produce any response. Spontaneous DRRs were increased by topical application of 1 mM beta-alanine (a competitive antagonist for GABA transporters) and abolished by ipsilateral spinal nerve L5 lesion, verifying antidromic origin. The role of C fiber DRRs in normal sensory transmission and during hyperalgesia is discussed.

The primary objective of our study was to examine the role of the inhibitory neurotransmitters glycine and GABA in modulating spontaneous activity and coordinating neurochemically induced locomotor-like rhythms in the mouse spinal cord. Motor outputs were recorded in lumbar ventral roots of 1-4-day old neonatal mice, and the function of glycinergic and GABAergic synapses in regulating spontaneous and induced activities was examined by suppressing synaptic inhibition using selective glycine or GABAA receptor antagonists. Strychnine (0.5 microM), a glycine receptor antagonist, did not change the pattern of spontaneous activity that consisted of random single spikes and discharges of variable durations and intervals. In contrast, blocking GABAA receptors with either picrotoxin (10 microM) or bicuculline (5 microM) triggered bilaterally synchronous, non-rhythmic discharges. These findings suggested that GABAergic synapses suppressed excitatory synapses, and their disinhibition synchronized spontaneous discharges between the two sides of the spinal cord. Locomotor-like rhythms alternating between the two sides of the spinal cord were triggered by the neurotransmitter agonists 5-HT, N-methyl-D,L-aspartic acid and dopamine. Blocking glycine receptors increased tonic discharges, and in most preparations it reduced the phase correlation between the alternating rhythms. Inhibiting GABAA receptor-mediated synapses synchronized the onset and prolonged the duration of rhythmic discharges. Intraburst alternating peaks were evident and those were suppressed by strychnine, suggesting that they were mediated via glycinergic synapses. Our findings indicated that GABAergic and glycinergic synapses played different roles in modulating neurochemically induced locomotion rhythms. GABAergic inhibition regulated the onset and duration of neurochemically induced locomotor-like rhythms, and glycinergic inhibition stabilized the pattern of the alternating rhythms.

Encoding of sensory information by nucleus of the solitary tract (NTS) neurons is incompletely understood. Using extracellular single-unit recording in alpha-chloralose-urethane anesthetized cats, we have examined the discharge characteristics of NTS neurons to activation of somatic Adelta and C fiber afferents by skeletal muscle contraction evoked by electrical stimulation of lower lumbar/upper sacral ventral roots. Generally, somatic afferent stimulation evoked two distinct firing patterns. The first population (36/43 cells) increased their firing rate to brief somatic stimuli. A subset (21/27 cells) exhibited a rapid decay of their firing rate during sustained somatic stimulation. Peak instantaneous firing frequency (F(p)) increased proportionally with the intensity of somatic stimulation (105+/-4 vs. 119+/-4 vs. 139+/-4 Hz, 10, 20 and 40 Hz, respectively, P<0.0001), whereas steady-state firing frequency (F(ss)) was not altered (25+/-2 vs. 27+/-2 vs. 27+/-2 Hz, 10, 20 and 40 Hz, respectively, P=0.72). Two indices were derived to quantify the decay properties. The decay rate constant (obtained from exponential curve fitting) was not altered by stimulation frequency (461+/-10 vs. 442+/-14 vs. 429+/-26 ms, 10, 20 and 40 Hz, respectively, P=0.415), nor was the decay index (derived to express the percent reduction in firing rate with respect to the initial peak firing rate; 76+/-2 vs. 77+/-2 vs. 81+/-2%, 10, 20 and 40 Hz, respectively, P=0.187). In contrast, the second population (seven of 43 cells) decreased their firing rate to stimulation. Of the NTS neurons tested for barosensitivity (29/36), none responded to pressure stimulation. These results have identified a population of somatosensitive NTS neurons that exhibit rapid firing rate decay properties during sustained stimulation. However, this population could faithfully encode phasic excitation during rhythmic somatosensory input. These results are discussed in relation to the role of somatosensory input on baroreflex function.

4-Aminopyridine (4-AP) is suggested to improve symptomatology of spinal injury patients because it may facilitate neuromuscular transmission, spinal impulse flow and the operation of the locomotor central pattern generator (CPG). Since 4-AP can also induce repetitive discharges from dorsal root afferents, this phenomenon might interfere with sensory signals necessary to modulate CPG activity. Using electrophysiological recording from dorsal and ventral roots of the rat isolated spinal cord, we investigated 4-AP-evoked discharges and their relation with fictive locomotor patterns. On dorsal roots 4-AP (5-10 microM) induced sustained synchronous oscillations (3.3+/-0.8 s period) smaller than electrically evoked synaptic potentials, persistent after sectioning off the ventral region and preserved in an isolated dorsal quadrant, indicating their dorsal horn origin. 4-AP oscillations were blocked by tetrodotoxin, or 6-cyano-7-nitroquinoxaline-2,3-dione and d-amino-phosphonovalerate, or strychnine and bicuculline, suggesting they were network mediated via glutamatergic, glycinergic and GABAergic transmission. Isolated ventral horn areas could not generated 4-AP oscillations, although their intrinsic disinhibited bursting was accelerated by 4-AP. Thus, ventral horn areas contained 4-AP sensitive sites, yet lacked the network for 4-AP induced oscillations. Activation of fictive locomotion by either application of N-methyl-D-aspartate and serotonin or stimulus trains to a single dorsal root reversibly suppressed dorsal root oscillations induced by 4-AP. This suppression was due to depression of dorsal network activity rather than simple block of root discharges. Since dorsal root oscillations evoked by 4-AP were turned off when the fictive locomotor program was initiated, these discharges are unlikely to interfere with proprioceptive signals during locomotor training in spinal patients.

The corticospinal tract is widely used to study regeneration and is essential for voluntary movements in humans. In young rats, corticospinal axons on the uninjured side sprout and grow into the denervated side. Neurotrophin-3 (NT-3) induces such crossed collateral sprouting in adults. We investigated whether local intraspinal NT-3 infusions would promote collateral sprouting of spared corticospinal terminals from within a partially denervated side, as this would be more appropriate for enhancing function of unilateral and specific movements. Adult rats received a partial bilateral transection of the pyramids, leaving approximately 40% of each tract intact. Vehicle or vehicle plus NT-3 (3 or 10 microg/day) was infused for 14 days into the left side of the cervical (C5/6) or lumbar (L2) cord. The corticospinal processes on the left side were anterogradely traced with cholera toxin B (CTB; which labeled gray matter processes more robustly than biotinylated dextran amine) injected into the front or hind limb area of the right sensorimotor cortex, respectively, 3 days before analysis. Unexpectedly, approximately 40% fewer CTB-labeled corticospinal processes were detectable in the cervical or lumbar gray matter of NT-3-treated rats than in vehicle-infused ones. Vehicle-infused injured rats had more corticospinal processes in the center of the cord than normal rats, evidence for lesion-induced collateral sprouting. NT-3 caused sprouting of local calcitonin gene-related peptide-positive fibers. These results suggest that NT-3 reduces collateral sprouting of spared corticospinal axons from within the denervated regions, possibly because of the injury environment or by increasing sprouting of local afferents. They identify an unexpected context-dependent outgrowth inhibitory effect of NT-3.

Lack of regeneration in the CNS has been attributed to many causes, including the presence of inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs). However, little is known about the contribution of CSPGs to regeneration failure in vivo, in particular at the dorsal root entry zone (DREZ), a unique CNS region that blocks regeneration of sensory fibers following dorsal root injury without glial scar formation. The goal of the present study was to evaluate the presence, regulation, and cellular identity of the proteoglycans Brevican, Neurocan, Versican V1 and Versican V2 in the DREZ using CSPG-specific antibodies and nucleic acid probes. Brevican and Versican V2 synthesized before the lesion were still present at high levels in the extracellular matrix of the DREZ several weeks after injury. In addition, Brevican was transiently expressed by reactive oligodendrocytes, and by a subset of astrocytes thereafter. Versican V2 mRNA appeared in NG2-positive cells with the morphology of oligodendrocyte precursor cells. Neurocan and Versican V1 levels were low before injury, and appeared in nestin-positive astrocytes and in NG2-positive cells, respectively, following lesion. Versican V1, but not V2, was also transiently increased in the peripheral dorsal root post-lesion. This is the first thorough description of the expression and cell association of individual proteoglycans following dorsal root lesion. It demonstrates that the proteoglycans Brevican, Neurocan, Versican V1, and Versican V2 are abundant in the DREZ at the time regenerating sensory fibers reach the PNS/CNS border and may therefore participate in growth-inhibition in this region.

This study examined the effect of hypothermia on respiratory neural output from brainstem-spinal cord preparations of a cold tolerant rodent, the Syrian hamster. Brainstem-spinal cords from neonatal hamsters (0-6 days) were placed in a recording dish and respiratory-like neural activity was recorded from roots of the first cervical nerve. The preparations were cooled and warmed in a continuous or stepwise fashion. Inputs from the pons completely inhibited neural activity under steady state conditions. With the pons removed, fictive breathing was robust. Cooling caused respiratory arrest, followed by spontaneous resumption of activity on re-warming. Preparations from older hamsters (4-6 days old) were more cold tolerant than younger preparations (0-3 days old). Motor discharge was episodic during continuous cooling, and seizure-like discharge was observed during continuous warming. These phenomena were not observed with stepwise temperature changes suggesting that transient temperature effects on membrane properties may be involved. These preparations were not as cold tolerant as hamster pups in vivo but they retained the ability to autoresuscitate at all ages studied.

Windup, the frequency dependent build-up of spinal neuronal responses is an electrophysiological model of the development of the central sensitization in the chronic pain states. NR2B subunit containing NMDA-type glutamate receptors are implicated in the windup of dorsal horn neurons, while their role at the motoneuronal level is controversial. The cumulative motoneuronal depolarization in hemisected rat spinal cord preparation is an in vitro model of windup. The role of NR2B receptors in this process, and in the mediation of dorsal root stimulation evoked ventral root reflex potentials was elucidated. Three selective NR2B antagonists; CP-101,606; CI-1041 and Co-101244 (1 microM) were used. They had only weak, but statistically significant inhibitory effect on the early part of ventral root response, and did not influence the cumulative depolarization. On the contrary, non-selective NMDA antagonist APV (40 microM) decreased both responses markedly. We conclude that the pharmacological sensitivities of windup at the sensory and motor levels are different. NR2B containing NMDA receptors have major role in the mediation of the windup of dorsal horn neurons, but their contribution to this phenomenon at the motor level is negligible.

Although analgesic action of xenon has been reported, little is known about the effect of xenon at the spinal cord, which plays a crucial role in nociceptive transmission. We studied the effect of xenon on nociceptive reflex (the slow ventral root potential) and the monosynaptic reflex in neonatal rat spinal cord in vitro in comparison with nitrous oxide. Xenon (30%) and nitrous oxide (30%) were applied for 17 min through superfusing artificial cerebrospinal fluid. Xenon and nitrous oxide significantly reduced the amplitude of nociceptive reflex by approximately 70% and approximately 25%, respectively. Xenon and nitrous oxide also significantly reduced the amplitude of the monosynaptic reflex by approximately 35% and approximately 15%, respectively. These results indicate that xenon suppressed the synaptic transmission at the spinal cord, especially those of the slow ventral root potential, which reflect nociceptive transmission.

In the isolated brain of the fire-bellied toad, Bombina orientalis, the spatial distribution of vestibular and somatosensory responses in thalamic nuclei was studied following electrical activation of the Vth nerve, the ramus anterior of the VIIIth nerve and of the dorsal roots of spinal nerves 3 and 8. Responses were systematically mapped in frontal planes through the diencephalon at four rostro-caudal levels. The calculated activity maps were superimposed on the outlines of diencephalic nuclei, and those nuclei that received particularly large inputs from the stimulated sensory nerve roots were indicated. Maximal response amplitudes coincided with ventral, central, and posterior thalamic areas and exhibited a topography that differed for each sensory nerve root. Maximal responses evoked from the Vth nerve were largely separated from those from spinal dorsal roots 3 and 8, whereas maximal vestibular responses partly overlapped with those from the other somatosensory nerve roots. Our findings indicate that within the amphibian thalamus sensory signals originating from different nerve roots are largely represented in separate areas as is the case in the thalamus of amniotes. However, the anterior dorsal thalamus which is the only origin of ascending pathways to the medial and dorsal pallium (assumed homologues of the mammalian hippocampus and neocortex, respectively) receives only minor vestibular and somatosensory input. This corroborates the view that amphibians lack a direct sensory thalamo-cortical, or "lemnothalamic," pathway typical of mammals and birds.

The maturation of dorsal root reflexes (DRRs) in lumbar roots was characterized in neonatal rats at 1, 2 and 3 weeks after birth using an in vitro isolated spinal cord preparation with attached dorsal roots and dorsal root ganglia (DRG). Changes of DRRs in rats of increasing age were also tested by administration of capsaicin to the DRG and related to spinal mechanisms of hyperalgesia by defining the behavioral responses of neonatal rats to intradermal capsaicin. DRRs evoked by stimulating the adjacent root in 1 week old rats are characterized by highly desynchronized waveforms with power spectra concentrated at frequencies greater than 200 Hz. DRRs in 1 week old rats show very little change in amplitude or area with increasing afferent stimulation strength. In contrast DRRs in 2 and 3 weeks old rats are highly synchronized with power concentrated at frequencies less than 100 Hz and show a graded increase in amplitude and area with increasing stimulus strength. The recovery of DRR amplitude in a paired pulse stimulus protocol is faster in 1 week rats than in 2 or 3 weeks old rats. Finally, DRRs in 2 and 3 week old rats show increased amplitude and area following application of capsaicin to the DRG of the stimulating root whereas those in 1 week old rats do not. These changes parallel the behavioral responses of neonatal rats as 2 and 3 weeks old rats show secondary mechanical hyperalgesia following intradermal capsaicin, but 1 week old rats do not. Our data indicate that the spinal circuitry for DRRs in the neonatal period undergoes rapidly dynamic development in the rat. This development is sufficiently rapid that mechanisms of spinal sensitization induced by capsaicin can be studied in rats 2 weeks old and older.

Objectives: To investigate, in an experimental study, the efficacy of a different stimulation signal in a porcine model to suppress formalin-induced unstable detrusor contractions. The current mode of stimulation in peripheral nerve evaluation tests and sacral neuromodulation is based on rectangular signal shapes. Published reports, however, have revealed that up to 80% of patients do not respond to peripheral nerve evaluation stimulation., Methods: After placement of electrodes at S3 in 12 anesthetized Göttinger minipigs, unstable contractions were induced by intravesical instillation of formalin. Ten-minute stimulation phases with a quasi-trapezoidal signal and a rectangular signal followed. An interval of 30 minutes elapsed between the two series of stimulations. The pressure values were registered on a urodynamic unit and evaluated as contractions and amplitudes per minute. Six minipigs were treated in the same way but were not stimulated and served as the control group., Results: After formalin instillation, the average number of involuntary detrusor contractions was 3.5/min (+/- 0.8) and the sum of amplitudes was 7.2/min (+/- 1.1). Subsequent NaCl instillation and quasi-trapezoidal stimulation reduced the contractions to 0.3/min (+/- 0.3) and the sum of amplitudes to 0.8/min (+/- 0.4). A contraction rate of 1.1/min (+/- 0.1) and a sum of amplitudes of 5.1/min (+/- 2.4) were recorded under stimulation with a rectangular signal. In the control group, no significant reduction was recorded., Conclusions: The acquired data demonstrate that quasi-trapezoidal stimulation suppresses unstable detrusor contractions in the minipig more effectively than does conventional rectangular stimulation as presently applied in sacral neuromodulation.

We examined the effect of iontophoretic application of bicuculline methiodide and strychnine hydrochloride on the mesencephalic locomotor region (MLR)-induced inhibition of dorsal horn cells in paralyzed cats. The activity of 60 dorsal horn cells was recorded extracellularly in laminae I, II, V-VII of spinal segments L7-S1. Each of the cells was shown to receive group III muscle afferent input as demonstrated by their responses to electrical stimulation of the tibial nerve (mean latency and threshold of activation: 20.1+/-6.4 ms and 15.2+/-1.4 times motor threshold, respectively). Electrical stimulation of the MLR suppressed transmission in group III muscle afferent pathways to dorsal horn cells. Specifically the average number of impulses generated by the dorsal horn neurons in response to a single pulse applied to the tibial nerve was decreased by 78+/-2.8% (n=60) during the MLR stimulation. Iontophoretic application (10-50 nA) of bicuculline and strychnine (5-10 mM) suppressed the MLR-induced inhibition of transmission of group III afferent input to laminae I and II cells by 69+/-5% (n=10) and 29+/-7% (n=7), respectively. Likewise, bicuculline and strychnine suppressed the MLR-induced inhibition of transmission of group III afferent input to lamina V cells by 59+/-13% (n=14) and 39+/-11% (n=10), respectively. Our findings raise the possibility that GABA and glycine release onto dorsal horn neurons in the spinal cord may play an important role in the suppression by central motor command of thin fiber muscle afferent-reflex pathways.

The effects of Ptychodiscus brevis toxin (PbTx) on the Ia-alpha motoneuron synaptic transmission in neonatal rat spinal cord in vitro was examined. The stimulation of a dorsal root evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root in Mg2+-free medium. Superfusion with PbTx (2.8-84 microM) depressed the MSR and the PSR in a concentration-dependent manner. At 2.8 microM of PbTx, the depression of MSR and PSR was 24+/-8.3% and 37+/-9.7%, respectively. The maximal depression was seen at 84 microM of the toxin (78% for MSR and 96% for PSR). The concentration of toxin required to produce 50% depression was 28.3+/-6.4 microM for MSR and 5.5+/-1.1 microM for PSR. The PbTx (28 microM) did not alter the magnitude of the dorsal root or the ventral root potentials. Addition of MgSO4 (1.3 mM) or DL-2-amino-5-phosphonovaleric acid (APV; 10 microM) to the physiological solution abolished the PSR totally and decreased the MSR by about 30%. In both the conditions, the PbTx-induced depression of the MSR was attenuated significantly. The PbTx-induced depression was blocked completely in the presence of APV+6-cyano-7-nitroquinoxaline-2,3-dione (0.1 microM). NMDA (1 microM) by itself did not alter the magnitude of MSR or PSR but enhanced the PbTx-induced depression (28 microM) of PSR significantly. 7-Chlorokynurenic acid (3 microM; glycine(B) antagonist) did not block the PbTx-induced depression of MSR. D-serine (glycine(B) agonist) did not reverse the PbTx-induced depression of reflexes although it reversed the 7-chlorokynurenic acid-induced depression of PSR. The results indicate that the PbTx depressed the spinal reflexes without altering the magnitude of dorsal root or ventral root activity. The depression of the PSR involved NMDA receptors while that of the MSR involved NMDA and non-NMDA receptors. The PbTx actions did not involve the glycine(B) site of the NMDA receptor.

The present study investigated the modulatory actions of nociceptin/orphanin FQ on excitatory glutamatergic transmission in spinal dorsal horn. In transverse spinal cord slices with an attached dorsal root, mono- and polysynaptic A delta-fibre-evoked extracellular field potentials were recorded from superficial dorsal horn. Nociceptin/orphanin FQ showed bidirectional effects on monosynaptic transmission with a potentiation at lower concentrations (100-300 nM) and a dose-dependent depression at higher concentrations (1-3 microM). The polysynaptic field potential was dose-dependently depressed by nociceptin/orphanin FQ (100 nM-3 microM). None of the actions of nociceptin/orphanin FQ was reversed by the non-specific opioid receptor antagonist naloxone, the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovaleric acid or the peptide nocistatin. The bidirectional actions of nociceptin/orphanin FQ on the monosynaptic field potential may provide an in vitro model for the bidirectional actions of nociceptin/orphanin FQ in behavioural studies showing hyperalgesia at low doses of intrathecal nociceptin/orphanin FQ and analgesia at higher doses.

Effects of low- and high-frequency fatigue were studied on muscle dynamics in isometric conditions of the cat gastrocnemius. Fatiguing sessions consisted of 25-28 repetitions of the standard tests that included an 18-s interval of continuous frequency-modulated stimulation preceded and followed by single stimuli evoking twitch contractions. The rate of the continuous part was changed in accordance with a symmetrical double-trapezoidal signal, including three successive phases of constant rate at 10, 40 and 10s(-1); between these phases, each lasting for 4s, the rate changed linearly within a 2-s interval. The following modes of muscle activation were applied: (i) stimulation of single filaments constituting approximately one-fifth to one-seventh of the total cross-section of the L(7) and S(1) ventral roots; (ii) the distributed stimulation of five similar filaments; and (iii) direct stimulation of muscle through bipolar wire electrodes. A relative drop in tension, the fatigue index, expressed as the ratio at the end of a fatigue session over its value at the beginning of the test, was used to quantify fatigue effects. The fatigue indices during low-rate stimulation were 0.56+/-0.03 (mean+/-S.D.) at the first phase and 0. 64+/-0.02 at the third phase, while during high-rate stimulation this parameter was only 0.32+/-0.02. The high-rate stimulation noticeably increased the mean tension during low-rate stimulation; the ratio between the reactions at the third and the first phases could be as much as two to three times greater than that at the beginning of the fatigue session. It was demonstrated that the potentiation was connected with after-effects of the rate-tension hysteresis. The hysteresis decreased with fatigue, the fatigue index for the rate-tension loop areas ranging from 0.39 to 0.52 (0.45+/-0. 05, mean+/-S.D.). The fatigue processes developed more quickly and intensively in the previously fatigued muscles: the obtained fatigue indices were 0.73+/-0.05 and 0.70+/-0.10 at the first and third phases, and 0.62+/-0.06 (mean+/-S.D.) at the second phase of stimulation, respectively. In the cases of distributed and direct stimulation applied to muscles in a fresh state, fatigue dynamics did not differ significantly from those observed during single-filament stimulation. In experiments with distributed stimulation applied to previously fatigued muscles, a powerful depression of the high-rate components was registered in several cases, which seemed to be connected with depressive effects at the level of nerve-muscle synaptic transmission. The effects of low- and high-frequency fatigue were studied in isometric conditions of muscle contraction. In addition to the well-known differentiation between low- and high-frequency fatigue effects, the complex pattern of efferent stimulation used allowed us to identify additional fatigue-related changes in the rate-tension hysteresis. This hysteresis seems to be one of the possible mechanisms directed to compensate for low-frequency fatigue in the muscle contraction.

The cyclobutylglycine (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1alpha and 5alpha, with IC50 values of 1.0+/-0.4 microM and 1.6+/-1.4 microM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent Kd values were estimated to be 0.3 microM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 microM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root-ventral root potential elicited at 8 x threshold was depressed by LY393053 with IC50 values of 9.0+/-0.7 microM and 12.7+/-1.7 microM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1-10 mg/kg intraperitoneally. An ED50 value of 6.0 mg/kg was obtained in this test. By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.

The response to dorsal root stimulation, at one to two times threshold, was investigated in the isolated cervical enlargement of the turtle spinal cord. At frequencies near 10 Hz the synaptic response in motoneurons and the cord dorsum potential, after an initial lag time, oscillated in amplitude with a period of more than 1 s. The mono- and polysynaptyic postsynaptic response in motoneurons, the pre- and postsynaptic component of the cord dorsum potential and the dorsal root potential oscillated in synchrony. These oscillations were only observed with stimulus frequencies in the range 9-11 Hz. The oscillating response could only be evoked from stimulus sites to which dorsal root potentials were conducted from the spinal cord (2-3 mm). At more distant stimulus sites cyclic variations in amplitude of the cord dorsum potential and the synaptic response in motoneurons were not observed. During an oscillating spinal response to a stimulus train in one dorsal root filament, the response evoked by a stimulus in another short filament (2-3 mm) from the same root varied in amplitude with the induced oscillation. The spinal response to a stimulus in a longer filament (i.e. more than 3 mm) did not oscillate. It is argued that the oscillating responses described rely on interactions between distributed elements rather than on unit oscillators. We also show that primary afferent transmission is unaffected by the substantial variations in dorsal root potentials during oscillations.

The in vitro brainstem-spinal cord preparation of newborn rats (0 to six-days-old) was used to investigate the development of pathways descending ventrally from the brainstem, which are important for the control of posture and locomotion. The ventral funiculus of the spinal cord was stimulated at the cervical (C1) level. Responses were recorded at the lumbar level from either motoneurons or ventral roots using intracellular microelectrodes or suction electrodes, respectively. Responses consisted of a pure excitation lasting 15 ms, followed by mixed excitatory/inhibitory responses. The inhibition was, at least partly, mediated by glycine. Excitatory amino acid transmission appears to be responsible for the excitation. The characteristics of the ventral funiculus-evoked postsynaptic potentials and ventral root potentials changed significantly with age. Their latency decreased whereas the slope and the area, measured over the first 15 ms, increased. The increase of the ventral funiculus input to motoneurons was slightly more pronounced than that of the monosynaptic dorsal root-evoked potentials from day 0 to day 4. These data suggest a gradual arrival of ventral descending axons in the lumbar enlargement which may be responsible for the gradual acquisition of postural control that takes place during the first days after birth. This is a prerequisite for the development of the adult pattern of quadrupedal locomotion, with elevated trunk.

In vitro brainstem spinal cord preparations isolated from newborn rats were used to separately test the effects of modifications of FCO2 and pH of artificial cerebrospinal fluid on the frequency and amplitude of spinal respiratory activity recorded from C2-C8 ventral roots. Different substances such as L-glutamic acid (3 x 10[-3] M), N-methyl-D-aspartic acid (5 x 5 x 10[-6] M), amphetamine (6 mg/100 ml), 5-hydroxytryptophane (10[-3] M), or modified K+ (10[-3] M) were tested for their capacity to elicit stable changes in spinal respiratory activity over a long time period (more than 30 min) and with high frequency of occurrence, i.e., in at least 50% of the cases. None of the above drugs were found to be suitable for the investigation of the chemosensitivity of the spinal respiratory generator (sRG) because they were only able to maintain spinal respiratory activity for around 15 min. Given these data, the previously used procedure of activation through initial deep diethyl ether anaesthesia of newborn rats was employed [3] to test the chemosensitivity of the sRG because this treatment resulted in the maintenance of spinal respiratory activity with a regular pattern for 30 min, even if it occurred in only 25% of the preparations. After an increase in FCO2 from 5 to 7% (at constant pH 7.4), a significant (p < 0.05) enhancement of the mean frequency was observed on spinal respiratory bursting in both brainstem spinal cord and isolated spinal cord preparations. The changes in burst amplitude, however, were quite variable from one experiment to the other. At constant FCO2 (5%), a decrease in pH from 7.4 to 7.2 enhanced spinal respiratory frequency on brainstem spinal cord or isolated spinal cord preparations, while an increase in pH from 7.4 to 7.6 decreased it. Under these pH conditions, we did not observe any reproducible variations in spinal burst amplitude. From these results, we conclude that this spinal generator is chemosensitive to both CO2 and [H+], suggesting that it belongs to the respiratory system. Our data provide evidence for the existence of spinal CO2 and/or H+ chemoreceptors.

Sucrose gap recordings from the dorsal roots of isolated, hemisected frog spinal cords were used to determine the effects of metabotropic L-glutamate receptor activation on primary afferent terminals by (+/-)-1-amino-trans-1,3-cyclopentane-dicarboxylic acid (t-ACPD). Dorsal root potentials evoked by ventral root volleys were significantly reduced by t-ACPD (30 microM), as were GABA- and muscimol-induced afferent terminal depolarizations. The effects of t-ACPD on GABA-depolarizations depended upon activation of group I metabotropic glutamate receptors, i.e. the effects were blocked by the group I/II antagonist (RS)-alpha-methyl-4-carboxyphenylglycine, but not by the group II antagonist alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine or the group III antagonist alpha-methyl-(S)-2-amino-4-phosphonobutyrate and were mimicked by the group I agonist 3,5-dihydroxyphenylglycine but were not mimicked by the group III agonist (S)-2-amino-4-phosphonobutyrate. Increasing the intracellular concentration of 3'-5'-cyclic adenosine monophosphate with 8-bromo-cAMP, forskolin, and 3-isobutyl-1-methylxanthine significantly reduced GABA depolarizations, but the protein kinase inhibitors Rp-adenosine 3,5-cyclic monophosphothioate triethylamine and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide did not alter t-ACPD's depression of GABA depolarizations. The actions of t-ACPD on GABA depolarizations were neither mimicked nor blocked by phorbol-12-myristate 13-acetate, thapsigargin, staurosporine, or arachidonic acid, presumptive indications that the effects of t-ACPD did not involve phosphoinositide hydrolysis, the release of Ca2+ from intracellular stores, or the formation of arachidonate. t-ACPD's effects on GABA depolarizations were blocked by 20 mM Mg2+, the broad spectrum L-glutamate antagonist kynurenate, and the selective N-methyl-D-aspartate antagonist D(-)-2-amino-5-phosphonovaleric acid, but not by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Low concentrations of N-methyl-D-aspartate (10 microM) mimicked the effect of t-ACPD on GABA responses. These results suggest that t-ACPD's depression of GABA depolarizations involves an indirect, three-stage mechanism that includes activation of Group I metabotropic glutamate receptors on interneurons and/or on afferent terminals, the release of L-glutamate from the latter structures, and the activation of N-methyl-D-aspartate receptors on primary afferent terminals. The depression of GABA depolarizations caused by the release of L-glutamate from afferent terminal and/or interneurons leads to a block of presynaptic inhibition (produced in the frog spinal cord by GABA) resulting in a positive feed-forward amplification of reflex transmission.

Nociceptin, also referred to as orphanin FQ, is believed to be the endogenous ligand for the ORL1. Nociceptin, when injected intracerebroventricularly to mice, produced hyperalgesia in behavioral tests. Recent studies have demonstrated the presence of ORL1 transcript in the spinal cord, and ORL1-like immunoreactivity has been localized to nerve fibers and somata throughout the spinal cord. Here, we report the localization of nociceptin-like immunoreactivity to fiber-like elements of the superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell recordings from substantia gelatinosa neurons in transverse lumbar spinal cord slices of 22-26-day-old rats showed that exogenous nociceptin at low concentrations (100-300 nM) depressed excitatory postsynaptic potentials evoked by stimulation of dorsal rootlets without causing an appreciable change of resting membrane potentials and glutamate-evoked depolarizations. At a concentration of 1 microM, nociceptin hyperpolarized substantia gelatinosa neurons and suppressed spike discharges. The hyperpolarizing and synaptic depressant action of nociceptin was not reversed by the known opioid receptor antagonist naloxone (1 microM). Our result provides evidence that nociceptin-like peptide is concentrated in nerve fibers of the rat dorsal horn and that it may serve as an inhibitory transmitter within the substantia gelatinosa.

(-)-(R)-2-methyl-3-(1-pyrrolidinyl)-4'-trifluoromethylpropiophenone++ + monohydrochloride, lanperisone hydrochloride (NK433) administered intravenously or orally depressed the mono- and polysynaptic reflex potential, dorsal root reflex potential, flexor reflex mediated by group II afferent fibers, patellar and flexor reflexes. These effects were reduced by spinal transection. NK433 inhibited the facilitation of the flexor reflex mediated by group II afferent fibers that was induced by intrathecal administration of noradrenaline-HCl. (+)-(1R,2R)-2-methyl-3-(1-pyrrolidinyl)-1-(4-trifluoromethylphenyl)-1-pr opanol (LPS-9)-HCl, a metabolite of NK433, also inhibited the spinal reflexes. Given orally, NK433 had effects more than three times stronger and tending to be longer-lasting than those of eperisone-HCl. These results suggest that NK433 exerts a non-selective inhibition on spinal reflexes and that inhibition of the descending noradrenergic tonic facilitation within the spinal cord is involved in the mechanism of spinal reflex depression by NK433. LPS-9 could contribute to the potent activity of NK433 after oral administration.

1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg(2+)-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists--that is, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine > CPP > APV = 7-Clkyn > HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro.