Keith T. Sillar

Development and Aminergic Neuromodulation of a Spinal Locomotor Network Controlling Swimming in Xenopus Larvaea

Abstract: In this article we review our research on the development and intrinsic neuromodulation of a spinal network controlling locomotion in a simple ventral roovertebrate. 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.

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 tadpoles. 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.

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

Amphibian metamorphosis includes a complete reorganization of an organisms 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.

The in vitro and in vivo enantioselectivity of etomidate implicates the GABAA receptor in general anaesthesia

General anaesthetics exhibiting enantioselectivity afford valuable tools to assess the fundamental mechanisms underlying anaesthesia. Here, we characterised the actions of the R-(+)- and S-(-)-enantiomers of etomidate. In mice and tadpoles, R-(+)-etomidate was more potent (approximately 10-fold) than S-(-)-etomidate in producing loss of the righting reflex. In electrophysiological and radioligand binding assays, the enantiomers of etomidate positively regulated GABAA receptor function at anaesthetic concentrations and with an enantioselectivity paralleling their in vivo activity. GABA-evoked currents mediated by human recombinant GABAA receptors were potentiated by either R-(+)- or S-(-)-etomidate in a manner dependent upon receptor subunit composition. A direct, GABA-mimetic, effect was similarly subunit dependent. Modulation of GABA receptor activity was selective; R-(+)-etomidate inhibited nicotinic acetylcholine, or 5-hydroxytryptamine3 receptor subtypes only at supra-clinical concentrations and ionotropic glutamate receptor isoforms were essentially unaffected. Acting upon reticulothalamic neurones in rat brain slices, R-(+)-etomidate prolonged the duration of miniature IPSCs and modestly enhanced their peak amplitude. S-(-)-etomidate exerted qualitatively similar, but weaker, actions. In a model of locomotor activity, fictive swimming in Xenopus laevis tadpoles, R-(+)- but not S-(-)-etomidate exerted a depressant influence via enhancement of GABAergic neurotransmission. Collectively, these observations strongly implicate the GABAA receptor as a molecular target relevant to the anaesthetic action of etomidate.

Neuromodulation of Vertebrate Locomotor Control Networks

Vertebrate locomotion must be adaptable in light of changing environmental, organismal, and developmental demands. Much of the underlying flexibility in the output of central pattern generating (CPG) networks of the spinal cord and brain stem is endowed by neuromodulation. This review provides a synthesis of current knowledge on the way that various neuromodulators modify the properties of and connections between CPG neurons to sculpt CPG network output during locomotion.

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.

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.

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.

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 μM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2–5 μM 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 5HT1a receptor agonist, 5‐carboxamidotryptamine (5CT) and both 5HT‐ and 5CT‐induced oscillations were abolished by pindobind‐5HT1, a selective 5HT1a 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 ∼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 5HT1a‐like pharmacology.

Synthesis, Conformation and Biological Evaluation of the Enantiomers of 3-Fluoro-γ-Aminobutyric Acid ((R)- and (S)-3F-GABA): An Analogue of the Neurotransmitter GABA

γ‐Aminobutyric acid or GABA (1) is one of the major inhibitory amino acid neurotransmitters of the central nervous system. This article describes the first synthesis of both the (R)‐ and (S)‐ enantiomers of 3‐fluoro‐GABA (2, 3F‐GABA). DFT calculations were carried out in a continuum solvent model (PCM–B3LYP) to estimate the preferred conformations of 3F‐GABA in aqueous solution. NMR coupling constants were calculated for each conformer and were then used to simulate the NMR spectra to evaluate the solution conformation of 3F‐GABA. A preliminary evaluation of the 3F‐GABA enantiomers shows that they act similarly as agonists of cloned GABAA receptors; however, they behave quite differently in a whole animal (Xenopus laevis tadpole model).