Longitudinal neuronal organization and coordination in a simple vertebrate: a continuous, semi-quantitative computer model of the central pattern generator for swimming in young frog tadpoles.

When frog tadpoles hatch their swimming requires co-ordinated contractions of trunk muscles, driven by motoneurons and controlled by a Central Pattern Generator (CPG). To study this co-ordination we used a 3.5 mm long population model of the young tadpole CPG with continuous distributions of neurons and axon lengths as estimated anatomically. We found that: (1) alternating swimming-type activity fails to self-sustain unless some excitatory interneurons have ascending axons, (2) a rostro-caudal (R-C) gradient in the distribution of excitatory premotor interneurons with short axons is required to obtain the R-C gradient in excitation and resulting progression of motoneuron firing necessary for forward swimming, (3) R-C delays in motoneuron firing decrease if excitatory motoneuron to premotor interneuron synapses are present, (4) these feedback connections and the electrical synapses between motoneurons synchronise motoneuron discharges locally, (5) the above findings are independent of the detailed membrane properties of neurons.

Roles for inhibition: studies on networks controlling swimming in young frog tadpoles.

The hatchling frog tadpole provides a simple preparation where the fundamental roles for inhibition in the central nervous networks controlling behaviour can be examined. Antibody staining reveals the distribution of at least ten different populations of glycinergic and GABAergic neurons in the CNS. Single neuron recording and marker injections have been used to study the roles and anatomy of three types of inhibitory neuron in the swimming behaviour of the tadpole. Spinal commissural interneurons control alternation of the two sides by producing glycinergic reciprocal inhibition. By interacting with the special membrane properties of excitatory interneurons they also contribute to rhythm generation through post-inhibitory rebound. Spinal ascending interneurons produce recurrent glycinergic inhibition of sensory pathways that gates reflex responses during swimming. In addition their inhibition also limits firing in CPG neurons during swimming. Midhindbrain reticulospinal neurons are excited by pressure to the head and produce powerful GABAergic inhibition that stops swimming when the tadpole swims into solid objects. They may also produce tonic inhibition while the tadpole is at rest that reduces spontaneous swimming and responsiveness of the tadpole, keeping it still so it is not noticed by predators.

Origin of excitatory drive to a spinal locomotor network.

A long-standing hypotheses is that locomotion is turned on by descending excitatory synaptic drive. In young frog tadpoles, we show that prolonged swimming in response to a brief stimulus can be generated by a small region of caudal hindbrain and rostral spinal cord. Whole-cell patch recordings in this region identify hindbrain neurons that excite spinal neurons to drive swimming. Some of these hindbrain reticulospinal neurons excite each other. We consider how feedback excitation within the hindbrain may provide a mechanism to drive spinal locomotor networks.

Glutamate and acetylcholine corelease at developing synapses.

Most neurons release a single fast-acting low-molecular-weight transmitter at synapses to activate and open postsynaptic ion channels. We challenge this principle with evidence for corelease of the two major excitatory transmitters, glutamate and acetylcholine (ACh), from single identified neurons in the developing frog tadpole spinal cord. Whole-cell patch electrodes were used to record from single spinal neurons. When action potentials and inhibition were blocked, spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded. These were fully blocked only by joint application of glutamate [alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR)] and nicotinic ACh receptor (nAChR) antagonists. Fast nAChR and slow NMDAR mEPSCs were isolated pharmacologically. We then show that some mEPSCs have both the fast nAChR rise and slow NMDAR decay and conclude that some individual synaptic vesicles corelease glutamate and ACh. Whole-cell recordings from pairs of neurons were then used to identify the spinal interneurons coreleasing the two excitatory transmitters. One anatomical class of interneuron with descending axons was found to excite other spinal neurons by activating nAChR, AMPAR, and NMDAR simultaneously at its synapses. Although Jonas, Bischofberger, and Sandkuhler [Jonas, P., Bischofberger, J. & Sandkuhler, J. (1998) Science 281, 419-424] showed that the inhibitory transmitters GABA and glycine can be coreleased at spinal synapses, the Xenopus tadpole provides a case where the two main CNS excitatory transmitters are released from single vesicles, and where the presynaptic neuron coreleasing two transmitters has been identified.

Primitive roles for inhibitory interneurons in developing frog spinal cord.

Understanding the neuronal networks in the mammal spinal cord is hampered by the diversity of neurons and their connections. The simpler networks in developing lower vertebrates may offer insights into basic organization. To investigate the function of spinal inhibitory interneurons in Xenopus tadpoles, paired whole-cell recordings were used. We show directly that one class of interneuron, with distinctive anatomy, produces glycinergic, negative feedback inhibition that can limit firing in motoneurons and interneurons of the central pattern generator during swimming. These same neurons also produce inhibitory gating of sensory pathways during swimming. This discovery raises the possibility that some classes of interneuron, with distinct functions later in development, may differentiate from an earlier class in which these functions are shared. Preliminary evidence suggests that these inhibitory interneurons express the transcription factor engrailed, supporting a probable homology with interneurons in developing zebrafish that also express engrailed and have very similar anatomy and functions.

Brainstem control of activity and responsiveness in resting frog tadpoles: tonic inhibition.

The hatchling Xenopus laevis tadpole was used to study the brain neurons controlling responsiveness. Tadpoles have reduced motor activity and responsiveness when they hang at rest, attached by cement gland mucus. Afferent input from cement gland mechanosensory neurons has both a phasic role in stopping swimming and a tonic role in reducing responsiveness while tadpoles hang attached. Both these roles depend on GABA(A)-mediated inhibition. We provide evidence supporting the hypothesis that long-term reduced responsiveness in attached tadpoles results from tonic activity in the reticulospinal GABAergic pathway mediating the stopping response. Two groups of putative stopping pathway interneurons were recorded in the caudal and rostral hindbrain of immobilised tadpoles. Both groups showed a sustained increase in activity during simulated attachment. This attached activity was irregular and unstructured. We consider whether low-level firing in cement gland afferents (at approximately 1 Hz) during simulated attachment is sufficient to explain the low-level firing (at approximately 0.5 Hz) in reticulospinal neurons. We then ask if a small population of these neurons (approximately 20) could produce sufficient inhibition of spinal neurons to reduce the whole tadpole's responsiveness. We conclude that for most of their 1st day of life GABAergic brainstem neurons could produce inhibition continuously while the tadpole is at rest.

Dorsal spinal interneurons forming a primitive, cutaneous sensory pathway.

In mammals, sensory projection pathways are provided by just three classes of spinal interneuron that develop from the roof-plate. We asked whether similar sensory projection interneurons are present primitively in a developing lower vertebrate where function can be more readily studied. Using an immobilized Xenopus tadpole spinal cord preparation, we define the properties and connections of spinal sensory projection interneurons using whole cell patch recordings from single neurons or pairs, identified by dye filling. Dorsolateral interneurons lie in the tadpole equivalent of the spinal dorsal horn, and have dorsally located dendrites and an ipsilateral ascending axon that projects into the midbrain. These neurons receive direct, mainly AMPA receptor (AMPAR)-mediated, excitation from skin touch sensory neurons. They in turn produce AMPAR and N-methyl-d-aspartate receptor (NMDAR)-mediated excitation of spinal locomotor pattern generator neurons rostrally on the same side of the cord. During swimming, they receive glycinergic modulatory inhibition from ascending interneurons in the spinal locomotor central pattern generator. We conclude that spinal dorsolateral interneurons are a primitive class of excitatory sensory projection neurons activated by ipsilateral cutaneous afferents and carrying excitation ipsilaterally and rostrally as far as the midbrain to initiate or accelerate swimming.

A direct comparison of whole cell patch and sharp electrodes by simultaneous recording from single spinal neurons in frog tadpoles.

High-impedance, sharp intracellular electrodes were compared with whole cell patch electrodes by recording from single spinal neurons in immobilized frog tadpoles. A range of neuron properties were examined using sharp or patch test electrodes while making simultaneous recordings with a second control patch electrode. Overall, test patch electrodes did not significantly alter the activity recorded by the control electrode, and recordings from the two electrodes were essentially identical. In contrast, sharp electrode recordings differed from initial control patch recordings. In some cases, differences were due to real changes in neuron properties: the resting membrane potential became less negative and the neuron input resistance (R(i)) fell; this fall was larger for neurons with a higher R(i). In other cases, the control patch electrode revealed that differences were due to the recording properties of the sharp electrode: tip potentials were larger and more variable; resting potentials appeared to be more negative; and spike amplitude was attenuated. However, sharp electrode penetration did not, in most cases, significantly alter the pattern of neuron firing in response to injected current or the normal pattern of activity following sensory stimulation or during fictive swimming. We conclude that sharp electrodes introduce a significant leak to the membrane of tadpole spinal neurons compared with patch electrodes but that this does not change the fundamental firing characteristics or activity of the neurons.

Spinal inhibitory neurons that modulate cutaneous sensory pathways during locomotion in a simple vertebrate.

During locomotion, reflex responses to sensory stimulation are usually modulated and may even be reversed. This is thought to be the result of phased inhibition, but the neurons responsible are usually not known. When the hatchling Xenopus tadpole swims, responses to cutaneous stimulation are modulated. This occurs because sensory pathway interneurons receive rhythmic glycinergic inhibition broadly in phase with the motor discharge on the same side of the trunk. We now describe a new whole-cell recording preparation of the Xenopus tadpole CNS. This has been used with neurobiotin injection to define the passive and firing properties of spinal ascending interneurons and their detailed anatomy. Paired recordings show that they make direct, glycinergic synapses onto spinal sensory pathway interneurons, and the site of contact can be seen anatomically. During swimming, ascending interneurons fire rhythmically. Analysis shows that their firing is more variable and not as reliable as other interneurons, but the temporal pattern of their impulse activity is suitable to produce the main peak of gating inhibition in sensory pathway interneurons. Ascending interneurons are not excited at short latency after skin stimulation but are strongly active after repetitive skin stimulation, which evokes vigorous and slower struggling movements. We conclude that ascending interneurons are a major class of modulatory neurons producing inhibitory gating of cutaneous sensory pathways during swimming and struggling.

Modelling inter-segmental coordination of neuronal oscillators: synaptic mechanisms for uni-directional coupling during swimming in Xenopus tadpoles.

Locomotion requires longitudinal co-ordination. We have examined uni-directional synaptic coupling processes between two classes of neuronal network oscillators: autonomously active "intrinsic" oscillators, and "potential" oscillators that lack sufficient excitatory drive for autonomous activity. We model such oscillator networks in the bilaterally-symmetrical, Xenopus tadpole spinal cord circuits that co-ordinate swimming. "Glutamate" coupling EPSPs can entrain a second oscillator of lower frequency provided their strength is sufficient. Fast (AMPA) EPSPs advance spiking on each cycle, while slow (NMDA) EPSPs increase frequency over many cycles. EPSPs can also enable rhythmicity in "potential" oscillators and entrain them. IPSPs operate primarily on a cycle-by-cycle basis. They can advance or delay spiking to entrain a second "intrinsic" oscillator with higher, equal or lower frequency. Bilaterally symmetrical coupling connections operate twice per cycle: once in each half-cycle, on each side of the receiving oscillator. Excitatory and inhibitory coupling allow entrainment in complimentary areas of parameter space.

Defining classes of spinal interneuron and their axonal projections in hatchling Xenopus laevis tadpoles.

Neurobiotin was injected into individual spinal interneurons in the Xenopus tadpole to discern their anatomical features and complete axonal projection patterns. Four classes of interneuron are described, with names defining their primary axon projection: Dorsolateral ascending and commissural interneurons are predominantly multipolar cells with somata and dendrites exclusively in the dorsal half of the spinal cord. Ascending interneurons have unipolar somata located in the dorsal half, but their main dendrites are located in the ventral half of the spinal cord. Descending interneurons show bigger variance in their anatomy, but the majority are unipolar, and they all have a descending primary axon. Dorsolateral commissural interneurons are clearly defined using established criteria, but the others are not, so cluster analysis was used. Clear discriminations can be made, and criteria are established to characterize the three classes of interneuron with ipsilateral axonal projections. With identifying criteria established, the distribution and axonal projection patterns of the four classes of interneuron are described. By using data from gamma-aminobutyric acid immunocytochemistry, the distribution of the population of ascending interneurons is defined. Together with the results from the axonal projection data, this allows the ascending interneuron axon distribution along the spinal cord to be estimated. By making simple assumptions and using existing information about the soma distributions of the other interneurons, estimates of their axon distributions are made. The possible functional roles of the four interneuron classes are discussed.

Functional projection distances of spinal interneurons mediating reciprocal inhibition during swimming in Xenopus tadpoles.

The basis for longitudinal coordination among spinal neurons during locomotion is still poorly understood. We have now examined the functional projection distances for the longitudinal axons of reciprocal inhibitory 'commissural interneurons' in the spinal cord of young Xenopus tadpoles. In quiescent animals, glycinergic inhibitory postsynaptic potentials (IPSPs) were evoked in ventral spinal neurons by stimulating small rostral and caudal groups of commissural interneuron somata at different distances on the opposite side of the hindbrain and spinal cord. Unitary IPSPs, produced by single synaptic contacts, could be distinguished from background noise. Local cord stimulation at different distances revealed maximum functional projection distances up to approximately 0.5 mm for both descending and ascending axons, but with the probability of recording connections falling steeply over this distance. These maximum longitudinal projection distances are smaller than predicted by axonal anatomy (approximately 1.2 mm). We then measured functional projection distances during swimming by examining the synaptic output of a surgically isolated group of rostral commissural interneurons, mapping the occurrence of the mid-cycle, reciprocal IPSPs they produced in more caudal neurons. IPSPs occurred with high probability up to 0.9 mm away, nearly twice the projection distance found in quiescent tadpoles. These results show that synaptic contacts from commissural interneurons could influence longitudinal coupling during swimming at distances of up to 0.9 mm (approximately 4-5 myotome segments or approximately 25% of the spinal cord). They provide direct evidence for functional projection distances of a characterized class of interneurons belonging to a spinal locomotor pattern generator.

Motoneurons of the axial swimming muscles in hatchling Xenopus tadpoles: features, distribution, and central synapses.

Xenopus tadpole motoneurons make cholinergic synapses within the spinal cord. This excitation changes with longitudinal position and contributes to the excitation that controls motor activity and its longitudinal spread during swimming. To explore the anatomic constraints on this excitation, backfilling has been used to examine the anatomy and distribution of the whole population of spinal motoneurons, to define the extent of their central axons and to find where they make synapses. Motoneuron features show considerable variation but do not allow their separation into primary and secondary. Most motoneurons have descending central axons and it is likely that central synapses are made from these axons as longitudinal dendritic extent is very limited. Motoneuron density reaches a broad plateau over the mid-trunk region at 12-13 per 100 microm. Soma size does not change with longitudinal position, but the dorsoventral extent of the dendrites decreases caudally, whereas the central axon length increases. Motoneuron distribution data were used to estimate the longitudinal distribution of central motoneuron axons. This has a broad plateau at 12-14 per 100 microm over much of the trunk and only decreases significantly caudal to the anus. This distribution correlates with cholinergic excitation during swimming. Transmission electron microscopy of motoneurons backfilled with horseradish peroxidase was used to show that central motoneuron axons make en passant synapses with motoneuron dendrites and the dendrites of other unstained neurons. By using measures of synapse frequency and total dendrite length, trunk motoneurons are estimated to each receive 100-200 synapses.

Axon projections of reciprocal inhibitory interneurons in the spinal cord of young Xenopus tadpoles and implications for the pattern of inhibition during swimming and struggling.

We have examined the morphology and longitudinal axon projections of a population of spinal commissural interneurons in young Xenopus tadpoles. We aimed to define how the distribution of axons of the whole population constrains the longitudinal distribution of the inhibition they mediate. Forty-three neurons at different positions were filled intracellularly with biocytin and processed with avidin-conjugated horseradish peroxidase. Soma size did not vary longitudinally and only one ipsilateral axon was found. Contralateral axons ascended, descended, or usually branched to do both. Total axon length and the extent of dendritic arborisation decreased caudally. The distributions of ascending and descending axon lengths were different; there were more long ascending (mean 737 +/- standard deviation 365 microm) than long descending (447 +/- 431 microm) axons. We used the axon length distribution data with existing data on the distribution of commissural interneuron somata to calculate the overall longitudinal density of these inhibitory axons. Axon numbers showed a clear rostrocaudal gradient. Axon length distributions were then incorporated into a simple spatiotemporal model of the forms of inhibition during swimming and struggling motor patterns. The model predicts that the peak of inhibition on each cycle will decrease from head to tail in both motor patterns, a feature already confirmed physiologically for swimming. It also supports a previous proposal that ascending inhibition during struggling shortens cycle period by shortening rostral motor bursts, whereas descending inhibition could delay subsequent burst onset.

Roles of ascending inhibition during two rhythmic motor patterns in Xenopus tadpoles.

We have investigated the effects of ascending inhibitory pathways on two centrally generated rhythmic motor patterns in a simple vertebrate model, the young Xenopus tadpole. Tadpoles swim when touched, but when grasped respond with slower, stronger struggling movements during which the longitudinal pattern of motor activity is reversed. Surgical spinal cord transection to remove all ascending connections originating caudal to the transection (in tadpoles immobilized in alpha-bungarotoxin) did not affect "fictive" swimming generated more rostrally. In contrast, cycle period and burst duration both significantly increased during fictive struggling. Increases were progressively larger with more rostral transection. Blocking caudal activity with the anesthetic MS222 (pharmacological transection) produced equivalent but reversible effects. Reducing crossed-ascending inhibition selectively, either by midsagittal spinal cord division or rostral cord hemisection (1-sided transection) mimicked the effects of transection. Like transection, both operations increased cycle period and burst duration during struggling but did not affect swimming. The changes during struggling were larger with more rostral hemisection. Reducing crossed-ascending inhibition by spinal hemisection also increased the rostrocaudal longitudinal delay during swimming, and the caudorostral delay during struggling. Weakening inhibition globally with low concentrations of the glycine antagonist strychnine (10-100 nM) did not alter swimming cycle period, burst duration, or longitudinal delay. However, strychnine at 10-60 nM decreased cycle period during struggling. It also increased burst duration in some cases, although burst duration increased as a proportion of cycle period in all cases. Strychnine reduced longitudinal delay during struggling, making rostral and caudal activity more synchronous. At 100 nM, struggling was totally disrupted. By combining our results with a detailed knowledge of tadpole spinal cord anatomy, we conclude that inhibition mediated by the crossed-ascending axons of characterized, glycinergic, commissural interneurons has a major influence on the struggling motor pattern compared with swimming. We suggest that this difference is a consequence of the larger, reversed longitudinal delay and the extended burst duration during struggling compared with swimming.

Central circuits controlling locomotion in young frog tadpoles.

The young Xenopus tadpole is a very simple vertebrate that can swim. We have examined its behavior and neuroanatomy, and used immobilized tadpoles to study the initiation, production, coordination, and termination of the swimming motor pattern. We will outline the sensory pathways that control swimming behavior and the mainly spinal circuits that produce the underlying motor output. Our recent work has analyzed the glycinergic, glutamatergic, cholinergic, and electrotonic synaptic input to spinal neurons during swimming. This has led us to study the nonlinear summation of excitatory synaptic inputs to small neurons. We then analyzed the different components of excitation during swimming to ask which components control frequency, and to map the longitudinal distribution of the components along the spinal cord. The central axonal projection patterns of spinal interneurons and motoneurons have been defined in order to try to account for the longitudinal distribution of synaptic drive during swimming.

The pattern of sensory discharge can determine the motor response in young Xenopus tadpoles.

Young Xenopus tadpoles were used to test whether the pattern of discharge in specific sensory neurons can determine the motor response of a whole animal. Young Xenopus tadpoles show two main rhythmic behaviours: swimming and struggling. Touch-sensitive skin sensory neurons in the spinal cord of immobilised tadpoles were penetrated singly or in pairs using microelectrodes to allow precise control of their firing patterns. A single impulse in one Rohon-Beard neuron (= light touch) could sometimes trigger "fictive" swimming. Two to six impulses at 30-50 Hz (= a light stroke) reliably triggered fictive swimming. Neither stimulus evoked fictive struggling. Twenty-five or more impulses at 30-50 Hz (= pressure) could evoke a pattern of rhythmic bursts, distinct from swimming and suitable to drive slower, stronger movements. This pattern showed some or all the characteristics of "fictive" struggling. These results demonstrate clearly that sensory neurons can determine the pattern of motor output simply by their pattern of discharge. This provides a simple form of behavioural selection according to stimulus.

Composition of the excitatory drive during swimming in two amphibian embryos: Rana and Bufo.

It has recently been shown that spinal neurons in Xenopus embryos receive cholinergic and electrotonic excitation during swimming, in addition to the well documented excitatory amino acid (EAA)-mediated excitation. We have now examined the composition of the excitatory drive during swimming in embryos of two further amphibian species, Rana and Bufo, which have somewhat different motor patterns. Localised applications of antagonists show that presumed motoneurons in Rana and Bufo embryos receive both cholinergic and FAA input during swimming. There is also a further chemical component which is blocked by Cd2+ and a small Cd(2+)-insensitive component, which is usually non-rhythmic. Rhythmic Cd(2+)-insensitive components, presumed to be phasic electrotonic potentials, were only seen in a small proportion of Bufo neurons and in no Rana neurons. While EAA and cholinergic inputs therefore appear to be consistent features of excitatory drive for swimming in amphibian embryo motoneurons, electrotonic input apparently occurs less commonly. Antagonist specificity was tested using applied agonists in Rana. Results of these tests also suggested that the further, unidentified Cd(2+)-sensitive component seen during swimming could represent an incomplete block of AMPA receptor-mediated excitation.

Local effects of glycinergic inhibition in the spinal cord motor systems for swimming in amphibian embryos.

1. We have studied the effects of locally applying the glycinergic antagonist strychnine to rhythmically active spinal neurons in amphibian embryos during fictive swimming. Intracellular recordings were made from motoneurons and premotor interneurons in Xenopus laevis, a well-studied model system, and from motoneurons in three other species (Rana temporaria, Bufo bufo, and Triturus vulgaris). Overall, these embryos cover a range of swimming patterns from the short-cycle-period, brief-motor-root bursts of Xenopus, to the long-cycle-period, long-motor-root bursts of Rana, which are more typical of adult patterns. 2. Local strychnine application had no significant effect on the gross pattern of swimming; episode duration and the burst duration in rostral ventral roots away from the application site were unaltered, and left-right alternation was preserved. We have therefore been able to examine the effects of inhibition on individual neurons, uninfluenced by overall changes in the operation of the swimming neural circuitry. 3. In all cases strychnine blocked midcycle inhibition and significantly increased the peak on-cycle depolarization during swimming. In Rana, Bufo, and Triturus motoneurons, and in Xenopus interneurons, strychnine significantly increased the reliability of firing during swimming. In Xenopus motoneurons, where spiking was 100% reliable anyway, the timing of the spikes was advanced relative to rostral ventral root activity. These results do not provide support for postinhibitory rebound as a factor in the spike-generating process during swimming. In addition to midcycle inhibition, Xenopus motoneurons can also show a smaller, additional on-cycle inhibition that is blocked by strychnine. 4. In both Rana and Bufo the duration of caudal ventral root bursts close to the site of drug application was increased by strychnine, showing that the increased motoneuron reliability not only leads to more intense, but also more extensive, ventral root activity. 5. At the level of single neurons, glycinergic inhibition effectively reduces on-cycle excitation and in turn controls the reliability, extent, and precise timing of motoneuron firing. These changes may be the individual components underlying broader effects of inhibition described previously, such as locomotor frequency control. They also show how any modulation of inhibition in localized regions of the spinal cord could produce localized control of neuronal firing properties.