Neuronal bursting induced by NK3 receptor activation in the neonatal rat spinal cord in vitro.

Intracellular recording from lumbar motoneurons and extracellular recording from ventral roots of the neonatal rat isolated spinal cord were used to study the mechanisms responsible for the excitation mediated by NK3 tachykinin receptors. The selective NK3 agonists senktide or [MePhe7]neurokinin B induced a slow depolarization with superimposed oscillations (mean period +/- SD was 2.8 +/- 0.8 s) that, in the majority of cases, showed left-right alternation at segmental level and were synchronous between L2 and L5 of the same side. During agonist wash out (5-20 min) a delayed form of hyperexcitability emerged consisting of bursts lasting 8 +/- 2 s (average interburst interval 55 +/- 21 s) with superimposed oscillations usually with homosegmental alternation and heterosegmental synchronicity. Such bursting was accompanied by depression of GABAergic dorsal root potentials evoked by dorsal root stimulation and of the recurrent inhibitory postsynaptic potential recorded from motoneurons. Despite bursting, motoneuron membrane potential returned to baseline while input resistance was increased. Bursts were a network-dependent phenomenon triggered by previous NK3 receptor activation because bursting was suppressed by glutamate receptor antagonists and was insensitive to motoneuron membrane potential or subsequent application of an NK3 receptor antagonist. NK3 receptors operated synergistically with N-methyl-D-aspartate (NMDA) and 5-hydroxytryptamine (5-HT) to trigger fully alternating locomotor-like rhythms while NK3 receptor antagonism disrupted the same rhythm. In summary, in the neonatal rat spinal cord NK3 receptors could trigger rhythmic activity predominantly with alternation at segmental level but with synchronous coupling between ipsilateral motor pools. NK3 receptor activation could also facilitate fictive locomotor patterns induced by NMDA and 5-HT.     

NK1, NK2 and NK3 tachykinin receptor localization and tachykinin distribution in the ileum of the mouse

Tachykinin receptors NK1r, NK2r and NK3r bind tachykinins with different affinities and share pharmacological and molecular differences among animal species. NK1r, NK2r, NK3r and tachykinin (SP/NKA) distribution was studied by immunohistochemistry in the ileum of mouse since no data are available for this species. The results were then compared to those obtained in the rat and guinea pig either by us or by others to ascertain interspecies similarities and/or differences. NK1r- and NK3r-immunoreactivity (IR) were detected in neurons and NK1r-IR in the interstitial cells of Cajal at the deep muscular plexus. At variance with rat and guinea pig, NK1r-IR was also found in the myoid cells of the villi, while NK2r-IR was never detected in nerve varicosities. This latter datum suggests that the NK2r does not play a presynaptic role in the mouse. Unexpectedly, a high NK2r-IR and the presence of NK3r-IR were observed at the inner portion of the circular muscle layer in the mouse as well as in the rat and guinea pig, demonstrating a subregional distribution of these receptors. Tachykinin distribution did not show noticeable species-related differences. The present findings show species-related differences in the tachykinin receptor distribution that might be related to a different tachykinin control of intestinal motility.     

Activity-related extracellular potassium transients in the neonatal rat spinal cord: an in vitro study

Transient increases and decreases in extracellular potassium (delta[K+]o) were recorded from the gray matter of hemisected, neonatal rat spinal cords isolated from 3, 4, 9- and 10-day-old pups. delta[K+]o were evoked in both the ventral and dorsal regions of the gray matter by electrical stimulation. In the ventral horn, repetitive stimulation of the ventral root was required to elicit detectable delta[K+]o. By contrast, single dorsal root stimuli evoked clear delta[K+]o. In the dorsal horn, single orthodromic stimuli elicited delta[K+]o as large as 4-5 mM from a baseline of 4.5 mM. With repetitive stimulation the [K+]o reached, but never exceeded, a ceiling of 10-11 mM. Undershoots were seen only after repetitive stimulation. Spontaneous delta[K+]o were observed in the ventral horn and were well correlated with ventral root activity. Spontaneous delta[K+]o were rare in the dorsal cord, but were recorded after bath application of apamin or tetraethylammonium. The magnitude and distribution of evoked K+ transients and postsynaptic components of the evoked field potential were well correlated in both the dorsal and the ventral gray matter. delta[K+]o were reversibly blocked by 1 mM CdCl2 in the bath and diminished by 1 mM BaCl2. Bath application of mephenesin, apamin or tetraethylammonium diminished evoked delta[K+]o in a stimulus-dependent manner. In apamin and tetraethylammonium, decreases from control responses were largest with high intensity stimulation, the opposite was the case with mephenesin. These results are interpreted in terms of the spinal circuits activated by high- and low-intensity electrical stimulation. We conclude that activity-related delta[K+]o in neonatal spinal cord are large enough to modulate neuronal electrical activity and the [K+]o is well regulated compared to other immature CNS regions studied. Thus, local increases in [K+]o may, by modulating neuronal activity, play a role in neonatal spinal cord developmental processes.     

Effect of metabotropic glutamate receptor activity on rhythmic discharges of the neonatal rat spinal cord in vitro

To extend our understanding of the network-based properties which enable a neuronal circuit to produce sustained electrical oscillations, we explored the potential contribution of metabotropic glutamate receptors (mGluRs) to generation of rhythmic discharges. The in vitro spinal cord of the neonatal rat was used as a model to find out if electrical patterns characterized by either alternating or synchronous motor pool discharges (recorded from lumbar ventral roots) required mGluR activation or were modulated by it. Alternating patterns of fictive locomotion (induced by NMDA and 5HT) were slowed down and blocked by the broad spectrum mGluR agonist (±)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD; 5–50 M) and unaffected by the broad spectrum mGluR antagonist (RS)--methyl-4-carboxyphenylglycine (MCPG; 1 mM). The regular, synchronous bursting emerging in the presence of strychnine and bicuculline was accelerated by t-ACPD with a commensurate decrease in single burst length, an effect antagonized by MCPG which per se did not affect bursting. The action of t-ACPD was selectively inhibited by the L-type Ca²⁺ blocker nifedipine which, however, did not change rhythm acceleration evoked by NMDA. These data suggest that neither alternating nor synchronous oscillatory discharges were apparently dependent on mGluR activation via endogenously released glutamate. However, mGluR activation by the agonist t-ACPD modulated rhythmic patterns, indicating that such receptors are a potential target for pharmacological up- or downregulation of spinal rhythmicity.The first two authors contributed equally to this workDue to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Fictive locomotor patterns generated by tetraethylammonium application to the neonatal rat spinal cord in vitro

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.     

Norepinephrine depolarizes lateral horn cells of neonatal rat spinal cord in vitro

Superfusion of norepinephrine (NE) (1-50 microM) onto lateral horn cells, including antidromically identified sympathetic preganglionic neurons (SPNs), situated in thin transverse neonatal rat thoracolumbar spinal cord slices caused a membrane depolarization and repetitive cell discharges. The NE depolarization was associated with an increase in membrane resistance, and the response became smaller upon conditioning hyperpolarization; a clear reversal of polarity, however, was not observed. Pretreating the slices with phentolamine and prazosin but not yohimbine or propranolol prevented the depolarizing effect of NE. This finding, in conjunction with the evidence of the presence of noradrenergic fibers in the spinal cord, suggests that NE may serve as an excitatory neurotransmitter to neurons of the lateral horn.     

Voltage-sensitivity of motoneuron NMDA receptor channels is modulated by serotonin in the neonatal rat spinal cord

Both N-methyl-D-aspartate (NMDA) and serotonin (5-HT) receptors contribute to the generation of rhythmic motor patterns in the rat spinal cord. Co-application of these chemicals is more effective at producing locomotor-like activity than either neurochemical alone. In addition, NMDA application to rat spinal motoneurons, synaptically isolated in tetrodotoxin, induces nonlinear membrane behavior that results in voltage oscillations which can be blocked by 5-HT antagonists. However, the mechanisms underlying NMDA and 5-HT receptor interactions pertinent to motor rhythm production remain to be determined. In the present study, an in vitro neonatal rat spinal cord preparation was used to examine whether NMDA receptor-mediated nonlinear membrane voltage is modulated by 5-HT. Whole-cell recordings of spinal motoneurons demonstrated that 5-HT shifts the region of NMDA receptor-dependent negative slope conductance (RNSC) of the current-voltage relationship to more hyperpolarized potentials and enhances whole-cell inward current. The influence of 5-HT on the RNSC was similar to the effect on the RNSC of decreasing the extracellular Mg(2+)concentration. The results suggest that 5-HT may modulate this form of membrane voltage nonlinearity by regulating Mg(2+) blockade of the NMDA ionophore.     

Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord

We used anatomical and physiological approaches to characterize nicotinic receptors (AChRs) on Renshaw cells of the neonatal rat spinal cord. Confocal imaging of Renshaw cells, identified by their characteristic pattern of gephyrin immunoreactivity, revealed that these neurons are immuno-positive for the alpha4 and beta2 AChR subunits but not for the alpha7 subunit. We used whole cell recording in spinal cord slices to characterize synaptic transmission from alpha-motor neurons to Renshaw cells, which could be identified pharmacologically by the sensitivity of transmission to d-tubocurarine. alpha-Motor neuron-to-Renshaw cell synapses were blocked by 10 microM dihydro-beta-erythroidine (dHbetaE), but not 50 nM methyllycaconitine (MLA), a selective alpha7 antagonist. These findings support a role for alpha4beta2-like AChRs, but not alpha7 AChRs, in rapid excitatory transmission between alpha-motor neurons and Renshaw cells in rat spinal cord.     

神经营养素-3能够促进体外受损伤大鼠脊髓神经元的存活及其神经突起生长

目的探讨神经营养素-3(NT-3)对体外机械性损伤的大鼠脊髓神经元存活及其神经突起生长影响。方法将体外培养的大鼠脊髓神经元分为4组:正常组、对照组、20 ng/ml NT-3组和40 ng/ml NT-3组。培养4 d后,除正常组外,其余3组建立划痕损伤模型。在划痕损伤后,对照组不做处理,另外2组分别在培养液中加入20 ng/mlNT-3和40 ng/ml NT-3继续培养。直至培养第6 d,应用4%多聚甲醛固定4组细胞。固定后的细胞分别做转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)、微管相关蛋白2(MAP2)和生长相关蛋白-43(GAP43)免疫荧光染色,检测划痕损伤的神经元凋亡及其神经突起生长情况。结果免疫荧光化学染色显示,与对照组相比,应用NT-3处理的2组可以显著降低划痕损伤后的脊髓神经元凋亡率,并促进其神经突起生长。尤其是40 ng/mlNT-3组的神经元凋亡率最低,其神经突起可穿过划痕损伤边界。结论 NT-3能够促进体外机械性损伤的大鼠脊髓神经元存活及其神经突起生长。     

The tachykinin NK1 receptor antagonist GR205171 abolishes the retching activity of neurons comprising the central pattern generator for vomiting in dogs

Tachykinin NK1 receptor antagonists are known to act centrally and to have broad-spectrum antiemetic effects, but their precise site of action has not yet been defined. To identify this site, the effects of the NK1 receptor antagonist GR205171 on the activities of neurons comprising the central pattern generator (CPG) for vomiting were observed in decerebrate paralyzed dogs. A non-respiratory neuron in each of nine dogs was considered to be a CPG neuron based on its response to abdominal vagal stimulation, its location in the CPG area in the reticular formation dorsomedial to the retrofacial nucleus, its firing patterns in prodromal and retching phases and its response to apomorphine. In response to vagal stimulation at 3-10 Hz, the firing of these neurons transiently increased at the onset of stimulation (fast component), gradually increased again (slow component), and finally developed into rhythmic bursts synchronous with retching bursts of the phrenic and abdominal muscle nerves. GR205171 (25-50 microg/kg, i.v.) abolished the slow component and retching bursts in the neurons, and the retching activities of both nerves, but did not change the fast component. The responses of these neurons to repetitive pulse-train vagal stimulation exhibited a vigorous 'wind-up' and finally developed into retching bursts. Both the 'wind-up' phenomenon and retching bursts disappeared after the application of GR205171. These results suggest that the site of the antiemetic action of NK1 receptor antagonists is located in the CPG or in the pathway connecting the solitary nucleus to the CPG.     

Two types of motor rhythm induced by NMDA and amines in an in vitro spinal cord preparation of neonatal rat

Rhythmic motor activities were studied in two rat spinal cord preparations; in the first one, the spinal cord was completely isolated and the ventral roots activity was recorded; in the other, spinal cord and hindlimbs were kept in order to record muscle activities. Motor patterns were therefore recorded in ventral roots and/or hindlimb muscles. Two kinds of specific patterns were elicited by neurochemicals. The first which was induced by N-methyl-D,L-aspartate (NMDA) and serotonin (5-HT) was a slow rhythm (0.5-0.2 Hz) of left and right alternating bursts of spikes. The second one which was induced by NMDA, 5-HT and norepinephrine (NE), was a high frequency rhythm (5-10 Hz). One particularity observed was that these two rhythms could occur simultaneously. The relationship between the two in vitro rhythms is discussed and they are compared with those reported in other rhythmic systems.     

Differential modulation by tetraethylammonium of the processes underlying network bursting in the neonatal rat spinal cord in vitro

In the rat spinal cord in vitro, block of synaptic inhibition evokes persistent, regular disinhibited bursting which is a manifestation of the intrinsic network rhythmicity and is readily recorded from ventral roots. This model is advantageous to explore the network mechanisms controlling burst periodicity, and duration. We questioned the relative contribution of K+ conductances to spontaneous rhythmicity by investigating the effects of the broad K+ channel blocker tetraethylammonium (TEA). In TEA (10 mM) solution, bursts occurred at the same rate but became substantially longer, thus showing an unusual dissociation between mechanisms of burst periodicity and duration. In the presence of TEA, electrical stimulation of a single dorsal root or N-methyl-D-aspartate application (5 microM) could, however, fasten bursting associated with immediate decrease in burst length, thus demonstrating maintenance of short-term plasticity. Either riluzole (1 microM) or surgical sectioning that isolated a single spinal segment strongly depressed bursting which could, however, be revived by TEA. In the presence of TEA, the L-type channel blocker nifedipine (20 microM) made bursting faster and shorter. Our data are best explained by assuming that TEA increased network excitability to generate rhythmic bursting, an effect that was counteracted by intrinsic mechanisms, partly dependent on L-type channel activity, to retain standard periodicity. TEA-sensitive mechanisms were, nevertheless, an important process to regulate burst duration. Our results are consistent with the proposal of a hierarchical structural of the central pattern generator in which the circuits responsible for rhythmicity (the clock) drive the discharges of those creating the motor commands (pattern).     

Role of tachykinin NK1, NK2 and NK3 receptors in the modulation of visceral hypersensitivity in the rat

Tachykinins are known to be involved in the processing of information leading to central sensitization and nociception. Using an animal model of repetitive colorectal distensions (CRD), we investigated the effect of spinal administration of tachykinin receptor antagonists in the mediation of visceral hypersensitivity. Intrathecal administration of the NK(1) receptor antagonist RP-67,580 (6.5 nmol) and the NK(3) receptor antagonist R-820 (6.5 nmol) completely blocked the CRD-induced hyperalgesia for both noxious and innocuous stimuli. The intrathecal administration of SR-48,968, a tachykinin NK(2) receptor antagonist, did not affect the visceral pain threshold of hypersensitive animals. Thus, the results from the present experiment support the concept that tachykinins with actions at spinal NK(1) and NK(3) but not NK(2) receptor sites are involved in visceral hypersensitivity mediated by nociceptive and non-nociceptive afferent inputs.     

Enzymatic inactivation of tachykinin neurotransmitters in the isolated spinal cord of the newborn rat.

A mixture of peptidase inhibitors increased the magnitude of the saphenous nerve-evoked slow depolarization of a lumbar ventral root and prolonged the similarly evoked inhibition of monosynaptic reflex (MSR) in the isolated spinal cord of the newborn rat in the presence of naloxone. The saphenous nerve-evoked MSR inhibition was curtailed by a tachykinin antagonist, GR71251, and after the treatment with GR71251, the peptidase inhibitor mixture no more prolonged the MSR inhibition. The present results suggest that enzymatic degradation plays a role in the termination of action of tachykinins released from primary afferents in the newborn rat spinal cord. The results provide a further support for the notion that tachykinins serve as neurotransmitters in the spinal cord of the newborn rat.     

Propriospinal neurons are sufficient for bulbospinal transmission of the locomotor command signal in the neonatal rat spinal cord

We recently showed that propriospinal neurons contribute to bulbospinal activation of locomotor networks in the in vitro neonatal rat brainstem–spinal cord preparation. In the present study, we examined whether propriospinal neurons alone, in the absence of long direct bulbospinal transmission to the lumbar cord, can successfully mediate brainstem activation of the locomotor network. In the presence of staggered bilateral spinal cord hemisections, the brainstem was stimulated electrically while recording from lumbar ventral roots. The rostral hemisection was located between C1 and T3 and the contralateral caudal hemisection was located between T5 and mid-L1. Locomotor-like activity was evoked in 27% of the preparations, which included experiments with staggered hemisections placed only two segments apart. There was no relation between the likelihood of developing locomotor-like activity and the distance separating the two hemisections or specific level of the hemisections. In some experiments, where brainstem stimulation alone was ineffective, neurochemical excitation of propriospinal neurons (using 5-HT and NMDA) at concentrations subthreshold for producing locomotor-like activity, promoted locomotor-like activity in conjunction with brainstem stimulation. In other experiments, involving neither brainstem stimulation nor cord hemisections, the excitability of propriospinal neurons in the cervical and/or thoracic region was selectively enhanced by bath application of 5-HT and NMDA or elevation of bath K⁺ concentration. These manipulations produced locomotor-like activity in the lumbar region. In total, the results suggest that propriospinal neurons are sufficient for transmission of descending locomotor command signals. This observation has implications for regeneration strategies aimed at restoration of locomotor function after spinal cord injury.     

Depression by nicotine of pain-related nociceptive activity in the rat thalamus and spinal cord

Summary To assess the possible role of nicotinergic control in nociception and pain, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurones in the thalamus and from ascending axons in the spinal cord. Intravenous administration of nicotine (0.01–0.5 mg/kg) depressed the nociceptive activity evoked in the thalamus and the spinal cord in a dose-dependent way. The maximum depression in thalamus and spinal cord was 40% of control activity and obtained at a dose of 0.025 mg/kg. Likewise, local administration of nicotine to the spinal cord by intrathecal injection (5, 10, and 30 g) reduced the nociceptive activity evoked in neurones of the thalamus and in ascending axons of the spinal cord, the maximum of the depression being 40% of control activity. The depressant effect of nicotine (0.05 mg/kg) was reduced by mecamylamine (1 mg/kg) but not by atropine (0.5 mg/kg). It is concluded that the antinociceptive effect of nicotine is due to a specific action of the alcaloid at the spinal level.