Stephen R. Soffe

How neurons generate behaviour in a hatchling amphibian tadpole: an outline

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.

Persistent responses to brief stimuli: feedback excitation among brainstem neurons

The ability of brief stimuli to trigger prolonged neuronal activity is a fundamental requirement in nervous systems, common to motor responses and short-term memory. Bistable membrane properties and network feedback excitation have both been proposed as suitable mechanisms to sustain such persistent responses. There is now good experimental evidence for membrane bistability. In contrast, the long-standing hypotheses based on positive feedback excitation have yet to be supported by direct evidence for mutual excitatory connections between appropriate neurons. In young frog tadpoles (Xenopus), we show that a small region of caudal hindbrain and rostral spinal cord is sufficient to generate prolonged swimming in response to a brief stimulus. We used paired whole-cell patch recordings to identify hindbrain neurons in this region that actively excite spinal neurons to drive sustained swimming. We show directly that some of these hindbrain neurons make reciprocal excitatory connections with each other. We use a population model of the hindbrain network to illustrate how feedback excitation can provide a robust mechanism to generate persistent responses. Our recordings provide direct evidence for feedback excitation among neurons within a network that drives a prolonged response. Its presence in a lower brain region early in development suggests that it is a basic feature of neuronal network design.

Reconfiguration of a Vertebrate Motor Network: Specific Neuron Recruitment and Context-Dependent Synaptic Plasticity

Motor networks typically generate several related output patterns or gaits where individual neurons may be shared or recruited between patterns. We investigate how a vertebrate locomotor network is reconfigured to produce a second rhythmic motor pattern, defining the detailed pattern of neuronal recruitment and consequent changes in the mechanism for rhythm generation. Hatchling Xenopus tadpoles swim if touched, but when held make slower, stronger, struggling movements. In immobilized tadpoles, a brief current pulse to the skin initiates swimming, whereas 40 Hz pulses produce struggling. The classes of neurons active during struggling are defined using whole-cell patch recordings from hindbrain and spinal cord neurons during 40 Hz stimulation of the skin. Some motoneurons and inhibitory interneurons are active in both swimming and struggling, but more neurons are recruited within these classes during struggling. In addition, and in contrast to a previous study, we describe two new classes of excitatory interneuron specifically recruited during struggling and define their properties and synaptic connections. We then explore mechanisms that generate struggling by building a network model incorporating these new neurons. As well as the recruitment of new neuron classes, we show that reconfiguration of the locomotor network to the struggling central pattern generator (CPG) reveals a context-dependent synaptic depression of reciprocal inhibition: the result of increased inhibitory neuron firing frequency during struggling. This provides one possible mechanism for burst termination not seen in the swimming CPG. The direct demonstration of depression in reciprocal inhibition confirms a key element of Browns (1911) hypothesis for locomotor rhythmogenesis.

Locomotor rhythm maintenance: electrical coupling among premotor excitatory interneurons in the brainstem and spinal cord of young Xenopus tadpoles

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.

Roles for multifunctional and specialized spinal interneurons during motor pattern generation in tadpoles, zebrafish larvae, and turtles.

The hindbrain and spinal cord can produce multiple forms of locomotion, escape, and withdrawal behaviors and (in limbed vertebrates) site-specific scratching. Until recently, the prevailing view was that the same classes of central nervous system neurons generate multiple kinds of movements, either through reconfiguration of a single, shared network or through an increase in the number of neurons recruited within each class. The mechanisms involved in selecting and generating different motor patterns have recently been explored in detail in some non-mammalian, vertebrate model systems. Work on the hatchling Xenopus tadpole, the larval zebrafish, and the adult turtle has now revealed that distinct kinds of motor patterns are actually selected and generated by combinations of multifunctional and specialized spinal interneurons. Multifunctional interneurons may form a core, multipurpose circuit that generates elements of coordinated motor output utilized in multiple behaviors, such as left-right alternation. But, in addition, specialized spinal interneurons including separate glutamatergic and glycinergic classes are selectively activated during specific patterns: escape-withdrawal, swimming and struggling in tadpoles and zebrafish, and limb withdrawal and scratching in turtles. These specialized neurons can contribute by changing the way central pattern generator (CPG) activity is initiated and by altering CPG composition and operation. The combined use of multifunctional and specialized neurons is now established as a principle of organization across a range of vertebrates. Future research may reveal common patterns of multifunctionality and specialization among interneurons controlling diverse movements and whether similar mechanisms exist in higher-order brain circuits that select among a wider array of complex movements.

Defining the excitatory neurons that drive the locomotor rhythm in a simple vertebrate: insights into the origin of reticulospinal control

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.

Specific Brainstem Neurons Switch Each Other into Pacemaker Mode to Drive Movement by Activating NMDA Receptors

Rhythmic activity is central to brain function. In the vertebrate CNS, the neuronal circuits for breathing and locomotion involve inhibition and also neurons acting as pacemakers, but identifying the neurons responsible has proven difficult. By studying simple hatchling Xenopus laevis tadpoles, we have already identified a population of electrically coupled hindbrain neurons (dINs) that drive swimming. During rhythm generation, dINs release glutamate to excite each other and activate NMDA receptors (NMDARs). The resulting depolarization enables a network mechanism for swimming rhythm generation that depends on reciprocal inhibition between antagonistic right and left sides. Surprisingly, a surgically isolated hemi-CNS without inhibition can still generate swimming-like rhythms. We have now discovered that activation of NMDARs transforms dINs, which normally fire singly to current injection, into pacemakers firing within the normal swimming frequency range (10–25 Hz). When dIN firing is blocked pharmacologically, this NMDAR activation produces 10 Hz membrane potential oscillations that persist when electrical coupling is blocked but not when the voltage-dependent gating of NMDARs by Mg2+ is removed. The NMDA-induced oscillations and pacemaker firing at swimming frequency are unique to the dIN population and do not occur in other spinal neurons. We conclude that NMDAR-mediated self-resetting switches critical neurons that drive swimming into pacemaker mode only during locomotion where it provides an additional, parallel mechanism for rhythm generation. This allows rhythm generation in a half-CNS and raises the possibility that such concealed pacemaker properties may be present underlying rhythm generation in other vertebrate brain networks.

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 γ‐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. J. Comp. Neurol. 441:248–265, 2001.

Axon and dendrite geography predict the specificity of synaptic connections in a functioning spinal cord network

BackgroundHow specific are the synaptic connections formed as neuronal networks develop and can simple rules account for the formation of functioning circuits? These questions are assessed in the spinal circuits controlling swimming in hatchling frog tadpoles. This is possible because detailed information is now available on the identity and synaptic connections of the main types of neuron.ResultsThe probabilities of synapses between 7 types of identified spinal neuron were measured directly by making electrical recordings from 500 pairs of neurons. For the same neuron types, the dorso-ventral distributions of axons and dendrites were measured and then used to calculate the probabilities that axons would encounter particular dendrites and so potentially form synaptic connections. Surprisingly, synapses were found between all types of neuron but contact probabilities could be predicted simply by the anatomical overlap of their axons and dendrites. These results suggested that synapse formation may not require axons to recognise specific, correct dendrites. To test the plausibility of simpler hypotheses, we first made computational models that were able to generate longitudinal axon growth paths and reproduce the axon distribution patterns and synaptic contact probabilities found in the spinal cord. To test if probabilistic rules could produce functioning spinal networks, we then made realistic computational models of spinal cord neurons, giving them established cell-specific properties and connecting them into networks using the contact probabilities we had determined. A majority of these networks produced robust swimming activity.ConclusionSimple factors such as morphogen gradients controlling dorso-ventral soma, dendrite and axon positions may sufficiently constrain the synaptic connections made between different types of neuron as the spinal cord first develops and allow functional networks to form. Our analysis implies that detailed cellular recognition between spinal neuron types may not be necessary for the reliable formation of functional networks to generate early behaviour like swimming.

Role of type-specific neuron properties in a spinal cord motor network

Recent recordings from spinal neurons in hatchling frog tadpoles allow their type-specific properties to be defined. Seven main types of neuron involved in the control of swimming have been characterized. To investigate the significance of type-specific properties, we build models of each neuron type and assemble them into a network using known connectivity between: sensory neurons, sensory pathway interneurons, central pattern generator (CPG) interneurons and motoneurons. A single stimulus to a sensory neuron initiates swimming where modelled neuronal and network activity parallels physiological activity. Substitution of firing properties between neuron types shows that those of excitatory CPG interneurons are critical for stable swimming. We suggest that type-specific neuronal properties can reflect the requirements for involvement in one particular network response (like swimming), but may also reflect the need to participate in more than one response (like swimming and slower struggling).