Metamodulation of a spinal locomotor network by nitric oxide

Flexibility in the output of spinal networks can be accomplished by the actions of neuromodulators; however, little is known about how the process of neuromodulation itself may be modulated. Here we investigate the potential "meta"-modulatory hierarchy between nitric oxide (NO) and noradrenaline (NA) in Xenopus laevis tadpoles. NO and NA have similar effects on fictive swimming; both potentiate glycinergic inhibition to slow swimming frequency and GABAergic inhibition to reduce episode durations. In addition, both modulators have direct effects on the membrane properties of motor neurons. Here we report that antagonism of noradrenergic pathways with phentolamine dramatically influences the effect of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swimming frequency, but not its effect on episode durations. In contrast, scavenging extracellular NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(PTIO) does not influence any of the effects of NA on fictive swimming. These data place NO above NA in the metamodulatory hierarchy, strongly suggesting that NO works via a noradrenergic pathway to control glycine release but directly promotes GABA release. We confirmed this possibility using intracellular recordings from motor neurons. In support of a natural role for NO in the Xenopus locomotor network, PTIO not only antagonized all of the effects of SNAP on swimming but also, when applied on its own, modulated both swimming frequency and episode durations in addition to the underlying glycinergic and GABAergic pathways. Collectively, our results illustrate that NO and NA have parallel effects on motor neuron membrane properties and GABAergic inhibition, but that NO serially metamodulates glycinergic inhibition via NA.Publisher PDFPeer reviewe

Neural events underlying escape swimming behaviour in the squat lobster 'Galathea strigosa' (Crustacea, Anomura)

1. The anatomy and physiology of escape swimming behaviour in the squat lobster, Galathea strigosa, have been investigated and the results discussed in the context of comparative mechanisms of escape in rela~ed species. 2. In contrast to many other decapods, G. strigosa, does not possess a giant-fibre system which underlies escape. 3. In terms of the number, size and position of neuronal somata, the fast flexor motorneuron pools in Galathea and crayfish are homologous. 4. A neuron, homologous with the crayfish MeG, has been studied. Unlike the crayfish neuron, MoGH is a typical, unspecialized fast flexor motorneuron. 5. The anatomy of afferent and efferent neurons involved in abdominal extension has been investigated. The extensor motorneuron and accessory neuron pools in crayfish and Galathea are largely homologous. 6. A small degree of intersegmental and interspecific variation in abdominal flexor and extensor motorneuron pools is reported. 7. The anatomy and physiology of the abdominal MRO's has been examined. These are found to be homologous in structure and function with other decapod MRO's. 8. The considerable differences between the phasic and tonic MRO sensory dendrites may account for their different response characteristics. 9. The MRO's excite both extensor motorneurons and the flexor inhibitor motorneuron via an apparently monosynaptic pathway. Similar input properties have been described for the crayfish MHO's . 10. The MRO's, which are shown to fire in response to abdominal flexion, produce EPSP's in extensor motorneurons which both summate and facilitate. This feature has not been decribed previously and may be important in the reflex function of the MHO's during escape swimming behaviour. 11. The relative roles of proprioceptive and exteroceptive feedback on the generation of the swimming rhythm have been studied using a variety of preparations involving restraint and deafferenta- tion. 12. Sensory feedback both excites and inhibits swimming. It is deduced that proprioceptive feedback has excitatory effects and extero- ceptive feedback inhibits swimming behaviour. 13. It is suggested that the MHO's may playa role in exciting the neural circuits underlying swimming bewvbur via both direct connections with the thoracic nervous system and a restricted portion of the abdominal motorneuron pool. 14. A deafferented preparation has been used to analyse the motor programme underlying swimming behaviour. The ability to record swimming activity, identical with that recorded in the intact animal, in the absence of sensory feedback from the abdomen, suggests that swimming behaviour is controlled by a central pattern generator (CPG). 15. A method of inducing swimming activity by high frequency electrical- stimulation of abdominal sensori-motor roots is described. 16. The CPG for swimming behaviour is shown to be most likely to reside in the suboesophageal or thoracic ganglion. 17. The activity of flexor and extensor motorneurons in abdominal ganglia has been analysed at the cellular level using both extra- cellular and intracellular recording techniques. 18. Fast flexor motorneurons are driven by a combination of brief unitary synaptic potentials and a large underlying oscillatory slow wave depolarization. 19. Current injection into the somata of fast flexor motorneurons during swimming has dramatic effects on slow wave amplitude and suggests that motorneuron drive results from powerful periodic excitation via chemical synapses. 20. In contrast to the fast flexor motorneurons, fast extensor motorneurons are driven by only brief unitary synaptic potentials and not by an underlying slow wave depolarization. The contrasting mechanisms for excitation in antagonistic sets of motorneurons is documented and a possible explanation presented. 21. Among the fast extensor motorneurons there is an apparent gradation in spike thresholds which can be correlated with a gradation in soma diameter. The largest of the available pool of extensor motorneurons has the highest spike threshold. 22. The activity of the phasic inhibitors of the extensor and flexor muscles has been analysed. The extensor inhibitor, which fires in antiphase with other extensor motorneurons during the flexion phase of the swim cycle, appears to receive the same slow wave depolarization as fast flexor motorneurons. The extensor inibitor motorneuron burst is terminated by a high frequency barrage of IPSP's superimposed upon the membrane slow wave. The flexor inhibitor motorneuron receives complex excitation and inhibition during swimming, involving both unitary events and membrane waves. 23. The coordination of segmented limb structures during swimming has been investigated. The walking legs are physically protracted during flexion while the unmodified male swimmerets are flicked posteriorly. 24. Swimmeret retraction during swimming is controlled by the activity of a single swimmeret motorneuron which appears to be part of the swimming circuit and which may also be a primitive homologue of the Segmental Giant neuron in crayfish. 25. It is concluded that escape swimming behaviour is homologous with non-giant backwards swimming in crayfish and may also be homologous with swimming in certain sand crab species. The evolutionary relationships of a number of decapods is discussed on the basis of escape circuitry and it is suggested that Galathea may represent an ancestral type of swimming decapod

Mechanisms underlying the endogenous dopaminergic inhibition of spinal locomotor circuit function in Xenopus tadpoles

This work was supported by the Biotechnology and Biological Science Research Council (BBSRC) [grant number BB/J01446X/1].Dopamine plays important roles in the development and modulation of motor control circuits. Here we show that dopamine exerts potent effects on the central pattern generator circuit controlling locomotory swimming in post-embryonic Xenopus tadpoles. Dopamine (0.5–100 μM) reduced fictive swim bout occurrence and caused both spontaneous and evoked episodes to become shorter, slower and weaker. The D2-like receptor agonist quinpirole mimicked this repertoire of inhibitory effects on swimming, whilst the D4 receptor antagonist, L745,870, had the opposite effects. The dopamine reuptake inhibitor bupropion potently inhibited fictive swimming, demonstrating that dopamine constitutes an endogenous modulatory system. Both dopamine and quinpirole also inhibited swimming in spinalised preparations, suggesting spinally located dopamine receptors. Dopamine and quinpirole hyperpolarised identified rhythmically active spinal neurons, increased rheobase and reduced spike probability both during swimming and in response to current injection. The hyperpolarisation was TTX-resistant and was accompanied by decreased input resistance, suggesting that dopamine opens a K+ channel. The K+ channel blocker barium chloride (but not TEA, glybenclamide or tertiapin-Q) significantly occluded the hyperpolarisation. Overall, we show that endogenously released dopamine acts upon spinally located D2-like receptors, leading to a rapid inhibitory modulation of swimming via the opening of a K+ channel.Publisher PDFPeer reviewe

Sodium pumps mediate activity-dependent changes in mammalian motor networks

Funding: Laurence Picton [grant number BB/JO1446X/1] and Matthew Broadhead [grant number BB/M021793/1] were supported by the Biotechnology and Biological Science Research Council (BBSRC). Filipe Nascimento was supported by The Alfred Dunhill Links Foundation.Ubiquitously expressed sodium pumps are best known for maintaining the ionic gradients and resting membrane potential required for generating action potentials. However, activity- and state-dependent changes in pump activity can also influence neuronal firing and regulate rhythmic network output. Here we demonstrate that changes in sodium pump activity regulate locomotor networks in the spinal cord of neonatal mice. The sodium pump inhibitor, ouabain, increased the frequency and decreased the amplitude of drug-induced locomotor bursting, effects that were dependent on the presence of the neuromodulator dopamine. Conversely, activating the pump with the sodium ionophore monensin decreased burst frequency. When more "natural" locomotor output was evoked using dorsal-root stimulation, ouabain increased burst frequency and extended locomotor episode duration, whereas monensin slowed and shortened episodes. Decreasing the time between dorsal-root stimulation, and therefore interepisode interval, also shortened and slowed activity, suggesting that pump activity encodes information about past network output and contributes to feedforward control of subsequent locomotor bouts. Using whole-cell patch-clamp recordings from spinal motoneurons and interneurons, we describe a long-duration (∼60 s), activity-dependent, TTX- and ouabain-sensitive, hyperpolarization (∼5 mV), which is mediated by spike-dependent increases in pump activity. The duration of this dynamic pump potential is enhanced by dopamine. Our results therefore reveal sodium pumps as dynamic regulators of mammalian spinal motor networks that can also be affected by neuromodulatory systems. Given the involvement of sodium pumps in movement disorders, such as amyotrophic lateral sclerosis and rapid-onset dystonia parkinsonism, knowledge of their contribution to motor network regulation also has considerable clinical importance.Publisher PDFPeer reviewe

The distribution of NADPH-diaphorase-labelled interneurons and the role of nitric oxide in the swimming system of Xenopus laevis larvae.

The possible involvement of the free radical gas nitric oxide (NO) in the modulation of spinal rhythm-generating networks has been studied using Xenopus laevis larvae. Using NADPH-diaphorase histochemistry, three putative populations of nitric oxide synthase (NOS)-containing cells were identified in the brainstem. The position and morphology of the largest and most caudal population suggested that a proportion of these neurons is reticulospinal. The possible contribution of nitrergic neurons to the control of swimming activity was examined by manipulating exogenous and endogenous NO concentrations in vivo with an NO donor (SNAP, 100-500 mu mol l(-1)) and NOS inhibitors (L-NAME and L-NNA, 0.5-5 mmol l(-1)), respectively. In the presence of SNAP, swim episode duration decreased and cycle period increased, whereas the NOS inhibitors had the opposite effects. We conclude from these data that the endogenous release of NO from brainstem neurons extrinsic to the spinal cord of Xenopus laevis larvae exerts a continuous modulatory influence on swimming activity, functioning like a 'brake'. Although the exact level at which NO impinges upon the swimming rhythm generator has yet to be determined, the predominantly inhibitory effect of NO suggests that the underlying mechanisms of NO action could involve modulation of synaptic transmission and/or direct effects on neuronal membrane properties.</p

A role for slow NMDA receptor-mediated, intrinsic neuronal oscillations in the control of fast fictive swimming in Xenopus laevis larvae.

In larvae of the amphibian, Xenopus laevis, spinal neurons which are active during fictive swimming also display tetrodotoxin-resistant membrane potential oscillations following the coactivation of N-methyl-DL-aspartate (NMDA) and 5-hydroxytryptamine (serotonin or 5-HT) receptors (Scrymgeour-Wedderburn et al., 1997; Eur: J. Neurosci., 9, 1473-1482). The oscillations are slow (approximate to 0.5 Hz) compared with swimming (approximate to 7-35 Hz) raising doubt over their contribution to the cycle by cycle depolarizations occurring during swimming. We investigated an alternative: that the intrinsic oscillations modulate swimming activity over many consecutive cycles. Bath application of NMDA induced continuous fictive swimming that differed between embryonic and larval preparations. In 81% of larval preparations (n = 36), there was a slow (approximately every 2 s) rhythmic modulation of ventral root activity in which burst durations and intensities increased as cycle periods decreased. This pattern of activity was enhanced rather than abolished following blockade of glycine and gamma-aminobutyric acid (GABA) A receptors and presumably therefore resulted from a periodic increase in the excitation of motor neurons. To determine whether this slow rhythm resulted from intrinsic, 5-HT-dependent membrane potential oscillations, larvae were spinalized to prevent the release of 5-HT from brainstem raphe neurons. The resulting pattern of NMDA-induced activity lacked any slow modulation. The slow modulation could also be enhanced by the bath application of a 5-HT receptor agonist (5-carboxamidotryptamine) and abolished either by the addition of an antagonist (pindobind-5-HT1A) or by removal of magnesium ions, providing more direct evidence for a contribution of intrinsic oscillations. Thus, the 5-HT-dependent intrinsic oscillations modulate NMDA-induced swimming activity over several consecutive cycles.</p

Pre- and postsynaptic modulation of spinal GABAergic neurotransmission by the neurosteroid, 5beta-pregnan-3alpha-ol-20-one.

The neuroactive steroid 5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha) modulates GABA(A) receptor function by potentiating postsynaptic GABA currents. While much is now known about the postsynaptic action of neurosteroids, far less is known about how they affect neurotransmission. We have investigated the synaptic actions of 5 beta 3 alpha in a simple vertebrate model, the embryo of the clawed toad, Xenopus laevis, in which a known GABAergic pathway, activated by the rostral cement gland, terminates swimming when the animal contacts an obstruction. Cement gland stimulation evokes bicuculline-sensitive inhibitory postsynaptic potentials (IPSPs) in motorneurones that terminate swimming and which are greatly enhanced by the presence of (1-5 mu M) 5 beta 3 alpha. In the presence of TTX, depolarising inhibitory potentials are recorded with KCl-filled microelectrodes reflecting the spontaneous release of transmitter. The majority are glycinergic with durations of 20-80 ms and are blocked by strychnine while the remainder are GABAergic with durations of 90-200 ms and are abolished by bicuculline. We show here that, in the presence of 5 beta 3 alpha, the spontaneous GABA IPSPs lengthen dramatically in some cases to over 500 ms, but the glycine potentials are unaffected. The steroid has no other detectable postsynaptic effects in that the range of amplitudes of GABA potentials is unaffected and there is no change in the resting membrane potential. However, 5 beta 3 alpha also caused a marked increase in the rate of occurrence of spontaneous GABA potentials. This suggests a novel presynaptic site of action in which the steroid enhances the probability of vesicular GABA release from GABA terminals. (C) 1997 Elsevier Science B.V.</p

Metamodulation of a spinal locomotor network by nitric oxide

Flexibility in the output of spinal networks can be accomplished by the actions of neuromodulators; however, little is known about how the process of neuromodulation itself may be modulated. Here we investigate the potential "meta"-modulatory hierarchy between nitric oxide (NO) and noradrenaline (NA) in Xenopus laevis tadpoles. NO and NA have similar effects on fictive swimming; both potentiate glycinergic inhibition to slow swimming frequency and GABAergic inhibition to reduce episode durations. In addition, both modulators have direct effects on the membrane properties of motor neurons. Here we report that antagonism of noradrenergic pathways with phentolamine dramatically influences the effect of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swimming frequency, but not its effect on episode durations. In contrast, scavenging extracellular NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(PTIO) does not influence any of the effects of NA on fictive swimming. These data place NO above NA in the metamodulatory hierarchy, strongly suggesting that NO works via a noradrenergic pathway to control glycine release but directly promotes GABA release. We confirmed this possibility using intracellular recordings from motor neurons. In support of a natural role for NO in the Xenopus locomotor network, PTIO not only antagonized all of the effects of SNAP on swimming but also, when applied on its own, modulated both swimming frequency and episode durations in addition to the underlying glycinergic and GABAergic pathways. Collectively, our results illustrate that NO and NA have parallel effects on motor neuron membrane properties and GABAergic inhibition, but that NO serially metamodulates glycinergic inhibition via NA.</p