Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis.

Amphibian metamorphosis includes a complete reorganization of an organism's locomotory system from axial-based swimming in larvae to limbed propulsion in the young adult. At critical stages during this behavioural switch, larval and adult motor systems operate in the same animal, commensurate with a gradual and dynamic reconfiguration of spinal locomotor circuitry. To study this plasticity, we have developed isolated preparations of the spinal cord and brainstem from pre- to post-metamorphic stages of the amphibian Xenopus laevis, in which spinal motor output patterns expressed spontaneously or in the presence of NMDA correlate with locomotor behaviour in the freely swimming animal. Extracellular ventral root recordings along the spinal cord of pre-metamorphic tadpoles revealed motor output corresponding to larval axial swimming, whereas postmetamorphic animals expressed motor patterns appropriate for bilaterally synchronous hindlimb flexion-extension kicks. However, in vitro recordings from metamorphic climax stages, with the tail and the limbs both functional, revealed two distinct motor patterns that could occur either independently or simultaneously, albeit at very different frequencies. Activity at 0.5-1 Hz in lumbar ventral roots corresponded to bipedal extension-flexion cycles, while the second, faster pattern (2-5 Hz) recorded from tail ventral roots corresponded to larval-like swimming. These data indicate that at intermediate stages during metamorphosis separate networks, one responsible for segmentally organized axial locomotion and another for more localized appendicular rhythm generation, coexist in the spinal cord and remain functional after isolation in vitro. These preparations now afford the opportunity to explore the cellular basis of locomotor network plasticity and reconfiguration necessary for behavioural changes during development.

Divergent actions of serotonin receptor activation during fictive swimming in frog embryos.

We have investigated the pharmacology underlying locomotor system responses to serotonin (5-HT) in embryos of the frog, Rana temporaria, to provide a comparison to studies in embryos of its close relative, Xenopus laevis. Our findings suggest that two divergent mechanisms underlie the modulation of locomotion by 5-HT in Rana. Bath-applied 5-HT or 5-carboxamidotyptamine, a 5-HT(1,5A,7) receptor agonist, can modulate fictive swimming in a dose-dependent manner, increasing burst durations and cycle periods. However, activation of 5-HT(1,7) receptors with R8-OHDPAT or 8-OHDPAT fails to mimic 5-HT, and in some cases exerts exactly the opposite response; decreasing burst durations and cycle periods. Elevating endogenous 5-HT levels by blocking re-uptake with clomipramine transiently increases burst durations. The receptors involved in this endogenous response include 5-HT(1A) receptors, as in Xenopus, but also 5-HT(7) receptors. However, like the 8-OHDPAT enantiomers, prolonged re-uptake inhibition can result in a motor response in the opposite direction to exogenous 5-HT. This effect is not reversed by 5-HT(1A) and/or 5-HT(7) receptor antagonism, implicating 5-HT(1B/1D) receptors. Remarkably, antagonism of these receptors using methiothepin unmasks a dose-dependent response to clomipramine, reminiscent of exogenous 5-HT. Our data suggest that 5-HT(1A,7) and 5-HT(1B/1D) receptors act as gain-setters of burst durations, whilst 5-HT(5A) receptors are involved in the effects of bath-applied 5-HT on locomotion.

Spatiotemporal pattern of nicotinamide adenine dinucleotide phosphate-diaphorase reactivity in the developing central nervous system of premetamorphic Xenopus laevis tadpoles.

We have catalogued the progressive appearance of putative nitric oxide synthase (NOS)-containing neurons in the developing central nervous system (CNS) of Xenopus laevis. Xenopus embryos and larvae were processed in wholemount and in cross section using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry as a marker for NOS within the CNS. The temporal sequence of NADPH-d reactivity identified discrete groups and subgroups of neurons in the forebrain, midbrain, and hindbrain on the basis of their morphology, location, and order of appearance during development. A proportion of these groups of neurons appeared to be important in sensory processing and motor control. Staining also appeared at specific stages in the spinal cord, the retina, and the skin. After the appearance of labelling, NADPH-d reactivity continued in each of the cell groups throughout the stages examined. We found no evidence for staining that subsequently disappeared at later stages in any cell group, indicating a persistent rather than transient role for NO in the Xenopus tadpole CNS. These results are discussed in light of recent findings on possible roles for NADPH-d-positive cell groups within the developing motor circuitry.

Adrenoreceptor-mediated modulation of the spinal locomotor pattern during swimming in Xenopus laevis tadpoles.

This study focused on the contribution of different adrenoreceptor subtypes to the modulation of fictive swimming activity in a relatively simple, yet intact, lower vertebrate system, the immobilized Xenopus laevis tadpole and explored their possible role in mediating the noradrenergic modulation of spinal motor networks. In Xenopus embryos, near the time of hatching, activation of alpha(1) adrenoreceptors increased the duration of episodes of fictive swimming, whilst in larvae, 24 h after hatching, they were decreased. Activation of alpha(2) adrenoreceptors, however, markedly reduced episode duration at both developmental stages. Cycle periods in both stages were increased by the activation of alpha(1) and/or alpha(2) receptor subclasses, whereas beta adrenoreceptors were not apparently involved in the modulation of cycle periods or the duration of swim episodes. However, both beta and alpha(1) receptor activation decreased the intersegmental delay in the head-to-tail propagation of swimming activity, while alpha(2) receptors did not influence these rostro-caudal delays. Activation of neither alpha, nor beta, receptor subclasses had any consistent effect on the duration of ventral motor bursts. Our findings suggest that noradrenergic modulation of the swim-pattern generator in Xenopus tadpoles is mediated through the activation of alpha and beta adrenoreceptors. In addition, activation of particular receptor subclasses might enable the selective modulation of either the segmental rhythm generating networks, the intersegmental coordination of those networks or control at both levels simultaneously.

Induction of a non-rhythmic motor pattern by nitric oxide in hatchling Rana temporaria embryos.

Nitric oxide (NO) is a ubiquitous neuromodulator with a diverse array of functions in a variety of brain regions, but a role for NO in the generation of locomotor activity has yet to be demonstrated. The possibility that NO is involved in the generation of motor activity in embryos of the frog Rana temporaria was investigated using the NO donors S-nitroso-n-acetylpenicillamine (SNAP; 100--500 micromol l(-1)) and diethylamine nitric oxide complex sodium (DEANO; 25--100 micromol l(-1)). Immobilised Rana temporaria embryos generate a non-rhythmic 'lashing' motor pattern either spontaneously or in response to dimming of the experimental bath illumination. Bath-applied NO donors triggered a qualitatively similar motor pattern in which non-rhythmic motor bursts were generated contra- and ipsilaterally down the length of the body. The inactive precursor of SNAP, n-acetyl-penicillamine (NAP), at equivalent concentrations did not trigger motor activity. NO donors failed to initiate swimming and had no measurable effects on the parameters of swimming induced by electrical stimulation. Intracellular recordings with potassium-acetate-filled electrodes revealed that the bursts of ventral root discharge induced by NO donors were accompanied by phasic depolarisations in motor neurons. During the inter-burst intervals, periods of substantial membrane hyperpolarization below the normal resting potential were observed, presumably coincident with contralateral ventral root activity. With KCl-filled electrodes, inhibitory potentials were strongly depolarising, suggesting that inhibition was Cl(-)-dependent. The synaptic drive seen in motor neurons after dimming of the illumination was very similar to that induced by the NO donors. NADPH-diaphorase histochemistry identified putative endogenous sources of NO in the central nervous system and the skin. Three populations of bilaterally symmetrical neurons were identified within the brainstem. Some of these neurons had contralateral projections and many had axonal processes that projected to and entered the marginal zones of the spinal cord, suggesting that they were reticulospinal.

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 micromol 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.

The development of neuromodulatory systems and the maturation of motor patterns in amphibian tadpoles.

The relative simplicity of the amphibian tadpole nervous system has been utilised as a model for the mechanisms underlying the generation and development of vertebrate locomotion. In this paper, we review evidence on the role of descending brainstem projections in the maturation and intrinsic modulation of tadpole spinal motor networks. Three transmitter systems that have been investigated utilise the biogenic amines serotonin (5HT) and noradrenaline (NA) and the inhibitory amino acid gamma-aminobutyric acid (GABA). The distribution, development and spinal targets of these systems will be reviewed. More recent data on the role of nitric oxide (NO) will also be discussed. This ubiquitous gaseous signalling molecule is known to play a crucial role in the developing nervous system, but until recently, had not been directly implicated in the brain regions involved in motor control. NO appears to be produced by three homologous brainstem clusters in the developing motor networks of two closely related amphibian species, Xenopus laevis and Rana temporaria but, surprisingly, it plays contrasting roles in these species. Given the presumed co-localisation and interaction of nitric oxide with conventional neurotransmitters, we discuss the potential relationship of nitrergic neurons with 5HT, NA and GABA in these amphibian models.

Development and role of GABA(A) receptor-mediated synaptic potentials during swimming in postembryonic Xenopus laevis tadpoles.

We have investigated the contribution of GABA(A) receptor activation to swimming in Xenopus tadpoles during the first day of postembryonic development. Around the time of hatching stage (37/8), bicuculline (10-50 microM) causes a decrease in swim episode duration and cycle period, suggesting that GABA(A) receptor activation influences embryonic swimming. Twenty-four hours later, at stage 42, GABA(A) receptor activation plays a more pronounced role in modulating larval swimming activity. Bicuculline causes short, intense swim episodes with increased burst durations and decreased cycle periods and rostrocaudal delays. Conversely, the allosteric agonist, 5beta-pregnan-3alpha-ol-20-one (1-10 microM) or the uptake inhibitor, nipecotic acid (200 microM) cause slow swimming with reduced burst durations and increased cycle periods. These effects appear to be mainly the result of GABA release from the spinal terminals of midhindbrain reticulospinal neurons but may also involve spinal GABAergic neurons. Intracellular recordings were made using KCl electrodes to reverse the sign and enhance the amplitude of chloride-dependent inhibitory postsynaptic potentials (IPSPs). Recordings from larval motoneurons in the presence of strychnine (1-5 microM), to block glycinergic IPSPs, provided no evidence for any GABAergic component to midcycle inhibition. GABA potentials were observed during episodes, but they were not phase-locked to the swimming rhythm. Bicuculline (10-50 microM) abolished these sporadic potentials and caused an apparent decrease in the level of tonic depolarization during swimming activity and an increase in spike height. Finally, in most larval preparations, GABA potentials were observed at the termination of swimming. In combination with the other evidence, our data suggest that midhindbrain reticulospinal neurons become involved in an intrinsic pathway that can prematurely terminate swim episodes. Thus during the first day of larval development, endogenous activation of GABA(A) receptors plays an increasingly important role in modulating locomotion, and GABAergic neurons become involved in an intrinsic descending pathway for terminating swim episodes.

Effects of noradrenaline on locomotor rhythm-generating networks in the isolated neonatal rat spinal cord.

We have studied the effects of the biogenic amine noradrenaline (NA) on motor activity in the isolated neonatal rat spinal cord. The motor output was recorded with suction electrodes from the lumbar ventral roots. When applied on its own, NA (0.5-50 microM) elicited either no measurable root activity, or activity of a highly variable nature. When present, the NA-induced activity consisted of either low levels of unpatterned tonic discharges, or an often irregular, slow rhythm that displayed a high degree of synchrony between antagonistic motor pools. Finally, in a few cases, NA induced a slow locomotor-like rhythm, in which activity alternated between the left and right sides, and between rostral and caudal roots on the same side. As shown previously, stable locomotor activity could be induced by bath application of N-methyl-D-aspartate (NMDA; 4-8.5 microM) and/or serotonin (5-HT; 4-20 microM). NA modulated this activity by decreasing the cycle frequency and increasing the ventral root burst duration. These effects were dose dependent in the concentration range 1-5 microM. In contrast, at no concentration tested did NA have consistent effects on burst amplitudes or on the background activity of the ongoing rhythm. Moreover, NA did not obviously affect the left/right and rostrocaudal alternation of the NMDA/5-HT rhythm. The NMDA/5-HT locomotor rhythm sometimes displayed a time-dependent breakdown in coordination, ultimately resulting in tonic ventral root activity. However, the addition of NA to the NMDA/5-HT saline could reinstate a well-coordinated locomotor rhythm. We conclude that exogenously applied NA can elicit tonic activity or can trigger a slow, irregular and often synchronous motor pattern. When NA is applied during ongoing locomotor activity, the amine has a distinct slowing effect on the rhythm while preserving the normal coordination between flexors and extensors. The ability of NA to "rescue" rhythmic locomotor activity after its time-dependent deterioration suggests that the amine may be important in the maintenance of rhythmic motor activity.

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 (approximately 0.5 Hz) compared with swimming (approximately 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.

Development and aminergic neuromodulation of a spinal locomotor network controlling swimming in Xenopus larvae.

In this article we review our research on the development and intrinsic neuromodulation of a spinal network controlling locomotion in a simple vertebrate. Swimming in hatchling Xenopus embryos is generated by a restricted network of well-characterized spinal neurons. This network produces a stereotyped motor pattern which, like real swimming, involves rhythmic activity that alternates across the body and progresses rostrocaudally with a brief delay between muscle segments. The stereotypy results from motoneurons discharging a single impulse in each cycle; because all motoneurons appear to behave similarly there is little scope for altering the output to the myotomes from one cycle to the next. Just one day later, however, Xenopus larvae generate a more complex and flexible motor pattern in which motoneurons can discharge a variable number of impulses which contribute to ventral root bursts in each cycle. This maturation of swimming is due, in part, to the influence of serotonin released from brain-stem raphespinal interneurons whose axonal projections innervate the cord early in larval life. Larval swimming is differentially modulated by both serotonin and by noradrenaline: serotonin leads to relatively fast, intense swimming whereas noradrenaline favors slower, weaker activity. Thus, these two biogenic amines select opposite extremes from the spectrum of possible output patterns that the swimming network can produce. Our studies on the cellular and synaptic effects of the amines indicate that they can control the strength of reciprocal glycinergic inhibition in the spinal cord. Serotonin and noradrenaline act presynaptically on the terminals of glycinergic commissural interneurons to weaken and strengthen, respectively, crossed glycinergic inhibition during swimming. As a result, serotonin reduces and noradrenaline increases interburst intervals. The membrane properties of spinal neurons are also affected by the amines. In particular, serotonin can induce intrinsic oscillatory membrane properties in the presence of NMDA. These depolarizations are slow compared to the cycle periods during swimming and so may contribute to enhancement of swimming over several consecutive cycles of activity.

Aminergic modulation of glycine release in a spinal network controlling swimming in Xenopus laevis.

1. Neuromodulators can effect changes in neural network function by strengthening or weakening synapses between neurons via presynaptic control of transmitter release. We have examined the effects of two biogenic amines on inhibitory connections of a spinal rhythm generator in Xenopus tad poles. 2. Glycinergic inhibitory potentials occurring mid-cycle in motoneurons during swimming activity are reduced by 5-hydroxytryptamine (5-HT; serotonin) and enhanced by noradrenaline (NA). These opposing effects on inhibitory synaptic strength are mediated presynaptically where 5-HT decreases and NA increases the probability of glycine release from inhibitory terminals. 3. The amines also have contrasting effects on swimming: 5-HT increased motor burst durations while NA reduced swimming frequency. Aminergic modulation of glycinergic transmission may thus control fundamental parameters of swimming and force the spinal network to generate opposite extremes of its spectrum of possible outputs.

Voltage oscillations in Xenopus spinal cord neurons: developmental onset and dependence on co-activation of NMDA and 5HT receptors.

The development of intrinsic, N-methyl-D-aspartate (NMDA) receptor-mediated voltage oscillations and their dependence on co-activation of 5-hydroxytryptamine (5HT) receptors was explored in motor neurons of late embryonic and early larval Xenopus laevis. Under tetrodotoxin, 100 microM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2-5 microM 5HT, oscillations were observed in 12% of embryonic and 70% of larval motor neurons. The voltage oscillations depended upon co-activation of NMDA and 5HT receptors since they were curtailed by selectively blocking NMDA receptors with D-2-amino-5-phosphonovaleric acid (APV) or by excluding Mg2+ from the experimental saline. 5HT applied in the absence of NMDA also failed to elicit oscillations. Oscillations could be induced by the non-selective 5HT1alpha receptor agonist, 5-carboxamidotryptamine (5CT) and both 5HT- and 5CT-induced oscillations were abolished by pindobind-5HT1, a selective 5HT1alpha receptor antagonist. To test whether 5HT enables voltage oscillations by modulating the voltage-dependent block of NMDA channels by Mg2+, membrane conductance was monitored under tetrodotoxin. Although 5HT caused membrane hyperpolarization of 4-8 mV, there was little detectable change in conductance. NMDA application caused an approximate 20 mV depolarization and an 'apparent' decrease in conductance, presumably due to the conductance pulse bringing the membrane into a voltage region where Mg2+ blocks the NMDA ionophore. 5HT further decreased conductance, which we propose is due to its enhancement of the voltage-dependent Mg2+ block. When the membrane potential was depolarized by approximately 20 mV via depolarizing current injection (to mimic the NMDA-induced depolarization), 5HT increased rather than decreased membrane conductance. Furthermore, 5HT did not affect the increase in membrane conductance following NMDA applications in zero Mg2+ saline. The results suggest that intrinsic, NMDA receptor-mediated voltage oscillations develop in a brief period after hatching, and that they depend upon the co-activation of 5HT and NMDA receptors. The enabling function of 5HT may involve the facilitation of the voltage-dependent block of the NMDA ionophore by Mg2+ through activation of receptors with 5HT1alpha-like pharmacology.

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

The neuroactive steroid 5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha) modulates GABAA 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 microM) 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.

Involvement of brainstem serotonergic interneurons in the development of a vertebrate spinal locomotor circuit.

Brainstem neurons modulate the rhythmic output of spinal locomotor circuitry in adult vertebrates, but how these influences develop is largely unknown. We demonstrate that the ingrowth of serotonergic axons to the spinal cord of Xenopus tadpoles plays a critical role in locomotor burst development by transforming the output of embryonic amphibian swimming circuitry into a more mature and flexible form. Our experiments show that exposure to a monoamine neurotoxin (5,7 dihydroxytryptamine) deletes serotonergic raphespinal projections and prevents the normal maturation of larval swimming. Furthermore, the mature larval rhythm resumes an embryo-like form following either a pharmacological blockade of serotonin receptors or when receptor activation is prevented by acute spinalization.

Oscillatory membrane properties of spinal cord neurons that are active during fictive swimming in Rana temporaria embryos.

We have sought evidence for intrinsic oscillatory membrane properties in spinal cord neurons which participate in fictive locomotion in Rana temporaria embryos. In the presence of tetrodotoxin to synaptically isolate intracellularly recorded neurons, the bath application of N-methyl-d-aspartate (NMDA) depolarizes neurons by 20 to 30 mV. It does not, however, elicit continuous membrane potential oscillations, as have been found in homologous spinal neurons of the lamprey. However, addition of the neuromodulatory amine 5-HT to the bathing medium rapidly induces repetitive large scale (up to 40mV) oscillations in membrane potential. These oscillations depend upon the presence of magnesium ions and are abolished by the NMDA antagonist, APV. They do not occur when 5-HT is applied in the absence of NMDA. Expression of oscillatory membrane behaviour thus appears to rely upon an interaction between 5-HT and NMDA receptors. Neuroanatomical evidence shows that endogenous 5-HT-containing descending projections are present in the ventrolateral margins of the spinal cord at this stage in development and could therefore trigger these oscillations during normal motor behaviour. Preliminary pharmacological studies support the conclusion that activation of 5-HT1a receptors is involved in the induction process.

Electrical coupling and intrinsic neuronal oscillations in Rana temporaria spinal cord.

In the presence of TTX and NMDA, spinal cord neurons in Rana temporaria embryos generate membrane potential oscillations, but only when 5-HT is added to the perfusate. These oscillations are voltage-dependent due to magnesium block of the NMDA receptor ionophore and can vary in amplitude between 0 and 40 mV with imposed membrane polarization. In contrast, the intrinsic frequency of the oscillations is unaffected by changes in membrane potential. This could result from electrical coupling amongst homonymous motoneurons. Here we present initial evidence for such connections and discuss their implications for the segmental control of rhythmic motor behaviour.

Modulation of rhythmic swimming activity in post-embryonic Xenopus laevis tadpoles by 5-hydroxytryptamine acting at 5HT1a receptors.

5HT modulates the rhythmic locomotor output of most vertebrates by enhancing the duration and intensity of motor bursts in each cycle, but there is little clear evidence on the pharmacological profile of the 5HT receptor subtype(s) involved. In this study we extend our previous work on the role of 5HT in the development and modulation of locomotor behaviour in newly hatched Xenopus tadpoles by examining the 5HT receptor type responsible for enhancing the swimming activity in immobilized preparations. By applying a range of agonists and antagonists against different 5HT receptor subtypes, we conclude that serotonergic modulation of swimming activity is accomplished via the activation of just one receptor type with a pharmacological profile similar to the mammalian 5HT1a receptor. The effects of 5HT on burst duration (an increase) and on episode length (a decrease) are mimicked by the 5HT1a receptor agonists, 5-carboxamidotryptamine (5CT) and R(+)-8-OH-DPAT, and reversed by the 5HT1a receptor antagonist NAN-190. Agents acting at other 5HT1, as well as 5HT2 and 5HT3, receptor subtypes were without noticeable effect on the 5HT-enhanced swimming rhythm.

Presynaptic inhibition of primary afferent transmitter release by 5-hydroxytryptamine at a mechanosensory synapse in the vertebrate spinal cord.

The effects of the neuromodulatory monoamine 5-HT (serotonin) on a cutaneous mechanosensory (Rohon-Beard, R-B neuron) pathway in the spinal cord of postembryonic Xenopus laevis tadpoles have been examined. In paralyzed animals, exogenous 5-HT at 1-10 microM reversibly inhibits (within 1-2 min) the activation of fictive swimming in response to electrical stimulation of R-B free nerve endings in the skin. At threshold stimulus intensities for swimming under control conditions, intracellularly recorded EPSPs in contralateral motoneurons are completely abolished by 5-HT without any obvious change in neuronal conductance or membrane potential. However, increasing the stimulus voltage can activate swimming with enhanced motor burst discharge on each cycle (Sillar et al., 1992). This suggested that 5-HT inhibits the swim-initiating pathway rather than the motor rhythm-generating circuitry itself. Extracellular recordings from the central projections of R-B neurons indicated that the amine does not impair the generation of mechanoafferent impulses or their propagation into the spinal cord. However, 5-HT application blocks impulse activity in dorsolaterally positioned sensory interneurons (DLis) that are contacted by R-B neurons, suggesting that 5-HT acts at R-B to DLi synapses in the dorsal cord. By recording with microelectrodes from DLis, we find that skin stimulus-evoked EPSPs at this first-order synapse in the swim-initiating pathway are reversibly suppressed by 5-HT. No obvious change in DLi membrane potential or conductance could be detected during the inhibition, suggesting a presynaptic site of action for 5-HT. To investigate this suggestion further, the effects of 5-HT on the spontaneous release of R-B sensory transmitter (excitatory amino acid, EAA) were examined, again by recording postsynaptically from DLis. In quiescent preparations, DLis receive spontaneous glycinergic, GABAergic, and EAA receptor-mediated PSPs. The inhibitory potentials are abolished by strychnine and curare, respectively. The excitatory potentials that remain are not blocked by application of the calcium channel blocker cadmium chloride at 1 mM, but are suppressed by the EAA receptor antagonist kynurenic acid. They therefore resemble the TTX-resistant EPSPs described previously in Xenopus DLis (Sillar and Roberts, 1991), which are presumed to arise from the spontaneous liberation of EAA transmitter from R-B terminals. Bath application of 5-HT dramatically reduces the rate of occurrence of these spontaneous EPSPs consistent with a presynaptic locus for the inhibitory effects of 5-HT.(ABSTRACT TRUNCATED AT 400 WORDS)

5HT induces NMDA receptor-mediated intrinsic oscillations in embryonic amphibian spinal neurons.

The existence and possible contribution of intrinsic membrane potential oscillations to the generation of locomotor rhythmicity was investigated in spinal cord neurons of newly hatched Rana temporaria tadpoles, by intracellular recording from immobilized animals. The bath application of 100 microM N-methyl-D-aspartate (NMDA) evoked continuous swimming-like activity in ventral motor roots and rhythmic synaptic drive to ventrally located spinal neurons, presumed to be motoneurons. In 0.5 microM tetrodotoxin-treated preparations, similar applications of NMDA depolarized neurons by ca. 20 mV, but did not lead to intrinsic oscillatory activity, although some evidence for voltage-dependent membrane bi-stability was obtained. However, bath application of the neuromodulatory amine, serotonin (5HT; 5 microM), in the presence of NMDA and TTX, reversibly induced sustained membrane potential oscillations (up to 40 mV in amplitude) that were similar in waveform to those already described in other adult vertebrate motor systems. The TTX-resistant oscillations were dependent upon the presence of magnesium ions in the bathing solution and were abolished by the NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV). The results suggest that in this simple, developing vertebrate locomotor system, the activation of 5HT receptors on spinal cord neurons in turn modulates NMDA receptor activation to enable the expression of intrinsic oscillatory membrane properties which could contribute to the generation of locomotor behaviour.