Adult nervous systems are so complex that understanding how they produce behavior remains a real challenge. We chose to study hatchling Xenopus tadpoles where behavior is controlled by a few thousand neurons but there is a very limited number of types of neuron. Young tadpoles can flex, swim away, adjust their trajectory, speed-up and slow-down, stop when they contact support and struggle when grasped. They are sensitive to touch, pressure, noxious stimuli, light intensity and water currents. Using whole-cell recording has led to rapid progress in understanding central networks controlling behavior. Our methods are illustrated by an analysis of the flexion reflex to skin touch. We then define the seven types of neuron that allow the tadpole to swim when the skin is touched and use paired recordings to investigate neuron properties, synaptic connections and activity patterns. Proposals on how the swim network operates are evaluated by experiment and network modeling. We then examine GABAergic inhibitory pathways that control swimming but also produce tonic inhibition to reduce responsiveness when the tadpole is at rest. Finally, we analyze the strong alternating struggling movements the tadpole makes when grasped. We show that the mechanisms for rhythm generation here are very different to those during swimming. Although much remains to be explained, study of this simple vertebrate has uncovered basic principles about the function and organization of vertebrate nervous systems.

Electrical coupling is important in rhythm generating systems. We examine its role in circuits controlling locomotion in a simple vertebrate model, the young Xenopus tadpole, where the hindbrain and spinal cord excitatory descending interneurons (dINs) that drive and maintain swimming have been characterised. Using simultaneous paired recordings, we show that most dINs are electrically coupled exclusively to other dINs (DC coupling coefficients ∼8.5%). The coupling shows typical low-pass filtering. We found no evidence that other swimming central pattern generator (CPG) interneurons are coupled to dINs or to each other. Electrical coupling potentials between dINs appear to contribute to their unusually reliable firing during swimming. To investigate the role of electrical coupling in swimming, we evaluated the specificity of gap junction blockers (18-β-GA, carbenoxolone, flufenamic acid and heptanol) in paired recordings. 18-β-GA at 40-60 μm produced substantial (84%) coupling block but few effects on cellular properties. Swimming episodes in 18-β-GA were significantly shortened (to ∼2% of control durations). At the same time, dIN firing reliability fell from nearly 100% to 62% of swimming cycles and spike synchronization weakened. Because dINs drive CPG neuron firing and are critical in maintaining swimming, the weakening of dIN activity could account for the effects of 18-β-GA on swimming. We conclude that electrical coupling among pre motor reticulospinal and spinal dINs, the excitatory interneurons that drive the swimming CPG in the hatchling Xenopus tadpole, may contribute to the maintenance of swimming as well as synchronization of activity.

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.

Important questions remain about the origin of the excitation that drives locomotion in vertebrates and the roles played by reticulospinal neurons. In young Xenopus tadpoles, paired whole-cell recordings reveal reticulospinal neurons that directly excite swimming circuit neurons in the brainstem and spinal cord. They form part of a column of neurons (dINs) with ipsilateral descending projections which fire reliably and rhythmically in time with swimming. We ask if, at this early stage of development, these reticulospinal neurons are themselves the primary source of rhythmic drive to spinal cord neurons on each cycle of swimming. Loose-patch recordings in the hindbrain and spinal cord from neurons active during fictive swimming distinguished dINs from other neurons by spike shape. These recordings showed that reticulospinal dINs in the caudal hindbrain (rhombomeres 7–8) fire significantly earlier on each swimming cycle than other, ipsilateral, swimming circuit neurons. Whole-cell recordings showed that fast EPSCs typically precede, and probably drive, spikes in most swimming circuit neurons. However, the earliest-firing reticulospinal dINs spike too soon to be driven by underlying fast EPSCs. We propose that rebound following reciprocal inhibition can contribute to early reticulospinal dIN firing during swimming and show rebound firing in dINs following evoked, reciprocal inhibitory PSPs. Our results define reticulospinal neurons that are the source of the primary, descending, rhythmic excitation that drives spinal cord neurons to fire during swimming. These neurons are an integral part of the rhythm generating circuitry. We discuss the origin of these reticulospinal neurons as specialised members of a longitudinally distributed population of excitatory interneurons extending from the brainstem into the spinal cord.

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.