The decision to move: response times, neuronal circuits and sensory memory in a simple vertebrate.

All animals use sensory systems to monitor external events and have to decide whether to move. Response times are long and variable compared to reflexes, and fast escape movements. The complexity of adult vertebrate brains makes it difficult to trace the neuronal circuits underlying basic decisions to move. To simplify the problem, we investigate the nervous system and responses of hatchling frog tadpoles which swim when their skin is stimulated. Studying the neuron-by-neuron pathway from sensory to hindbrain neurons, where the decision to swim is made, has revealed two simple pathways generating excitation which sums to threshold in these neurons to initiate swimming. The direct pathway leads to short, and reliable delays like an escape response. The other includes a population of sensory processing neurons which extend firing to introduce noise and delay into responses. These neurons provide a brief, sensory memory of the stimulus, that allows tadpoles to integrate stimuli occurring within a second or so of each other. We relate these findings to other studies and conclude that sensory memory makes a fundamental contribution to simple decisions and is present in the brainstem of a basic vertebrate at a surprisingly early stage in development.

A simple decision to move in response to touch reveals basic sensory memory and mechanisms for variable response times.

KEY POINTS: Short-term working memory and decision-making are usually studied in the cerebral cortex; in many models of simple decision making, sensory signals build slowly and noisily to threshold to initiate a motor response after long, variable delays. When touched, hatchling frog tadpoles decide whether to swim; we define the long and variable delays to swimming and use whole-cell recordings to uncover the neurons and processes responsible. Firing in sensory and sensory pathway neurons is short latency, and too brief and invariant to explain these delays, while recordings from hindbrain reticulospinal neurons controlling swimming reveal a prolonged and variable build-up of synaptic excitation which can reach firing threshold and initiate swimming. We propose this excitation provides a sensory memory of the stimulus and may be generated by small reverberatory hindbrain networks. Our results uncover fundamental network mechanisms that allow animals to remember brief sensory stimuli and delay simple motor decisions. ABSTRACT: Many motor responses to sensory input, like locomotion or eye movements, are much slower than reflexes. Can simpler animals provide fundamental answers about the cellular mechanisms for motor decisions? Can we observe the 'accumulation' of excitation to threshold proposed to underlie decision making elsewhere? We explore how somatosensory touch stimulation leads to the decision to swim in hatchling Xenopus tadpoles. Delays measured to swimming in behaving and immobilised tadpoles are long and variable. Activity in their extensively studied sensory and sensory pathway neurons is too short-lived to explain these response delays. Instead, whole-cell recordings from the hindbrain reticulospinal neurons that drive swimming show that these receive prolonged, variable synaptic excitation lasting for nearly a second following a brief stimulus. They fire and initiate swimming when this excitation reaches threshold. Analysis of the summation of excitation requires us to propose extended firing in currently undefined presynaptic hindbrain neurons. Simple models show that a small excitatory recurrent-network inserted in the sensory pathway can mimic this process. We suggest that such a network may generate slow, variable summation of excitation to threshold. This excitation provides a simple memory of the sensory stimulus. It allows temporal and spatial integration of sensory inputs and explains the long, variable delays to swimming. The process resembles the 'accumulation' of excitation proposed for cortical circuits in mammals. We conclude that fundamental elements of sensory memory and decision making are present in the brainstem at a surprisingly early stage in development.

Modelling Feedback Excitation, Pacemaker Properties and Sensory Switching of Electrically Coupled Brainstem Neurons Controlling Rhythmic Activity.

What cellular and network properties allow reliable neuronal rhythm generation or firing that can be started and stopped by brief synaptic inputs? We investigate rhythmic activity in an electrically-coupled population of brainstem neurons driving swimming locomotion in young frog tadpoles, and how activity is switched on and off by brief sensory stimulation. We build a computational model of 30 electrically-coupled conditional pacemaker neurons on one side of the tadpole hindbrain and spinal cord. Based on experimental estimates for neuron properties, population sizes, synapse strengths and connections, we show that: long-lasting, mutual, glutamatergic excitation between the neurons allows the network to sustain rhythmic pacemaker firing at swimming frequencies following brief synaptic excitation; activity persists but rhythm breaks down without electrical coupling; NMDA voltage-dependency doubles the range of synaptic feedback strengths generating sustained rhythm. The network can be switched on and off at short latency by brief synaptic excitation and inhibition. We demonstrate that a population of generic Hodgkin-Huxley type neurons coupled by glutamatergic excitatory feedback can generate sustained asynchronous firing switched on and off synaptically. We conclude that networks of neurons with NMDAR mediated feedback excitation can generate self-sustained activity following brief synaptic excitation. The frequency of activity is limited by the kinetics of the neuron membrane channels and can be stopped by brief inhibitory input. Network activity can be rhythmic at lower frequencies if the neurons are electrically coupled. Our key finding is that excitatory synaptic feedback within a population of neurons can produce switchable, stable, sustained firing without synaptic inhibition.

Modelling the Effects of Electrical Coupling between Unmyelinated Axons of Brainstem Neurons Controlling Rhythmic Activity.

Gap junctions between fine unmyelinated axons can electrically couple groups of brain neurons to synchronise firing and contribute to rhythmic activity. To explore the distribution and significance of electrical coupling, we modelled a well analysed, small population of brainstem neurons which drive swimming in young frog tadpoles. A passive network of 30 multicompartmental neurons with unmyelinated axons was used to infer that: axon-axon gap junctions close to the soma gave the best match to experimentally measured coupling coefficients; axon diameter had a strong influence on coupling; most neurons were coupled indirectly via the axons of other neurons. When active channels were added, gap junctions could make action potential propagation along the thin axons unreliable. Increased sodium and decreased potassium channel densities in the initial axon segment improved action potential propagation. Modelling suggested that the single spike firing to step current injection observed in whole-cell recordings is not a cellular property but a dynamic consequence of shunting resulting from electrical coupling. Without electrical coupling, firing of the population during depolarising current was unsynchronised; with coupling, the population showed synchronous recruitment and rhythmic firing. When activated instead by increasing levels of modelled sensory pathway input, the population without electrical coupling was recruited incrementally to unpatterned activity. However, when coupled, the population was recruited all-or-none at threshold into a rhythmic swimming pattern: the tadpole "decided" to swim. Modelling emphasises uncertainties about fine unmyelinated axon physiology but, when informed by biological data, makes general predictions about gap junctions: locations close to the soma; relatively small numbers; many indirect connections between neurons; cause of action potential propagation failure in fine axons; misleading alteration of intrinsic firing properties. Modelling also indicates that electrical coupling within a population can synchronize recruitment of neurons and their pacemaker firing during rhythmic activity.

Can simple rules control development of a pioneer vertebrate neuronal network generating behavior?

How do the pioneer networks in the axial core of the vertebrate nervous system first develop? Fundamental to understanding any full-scale neuronal network is knowledge of the constituent neurons, their properties, synaptic interconnections, and normal activity. Our novel strategy uses basic developmental rules to generate model networks that retain individual neuron and synapse resolution and are capable of reproducing correct, whole animal responses. We apply our developmental strategy to young Xenopus tadpoles, whose brainstem and spinal cord share a core vertebrate plan, but at a tractable complexity. Following detailed anatomical and physiological measurements to complete a descriptive library of each type of spinal neuron, we build models of their axon growth controlled by simple chemical gradients and physical barriers. By adding dendrites and allowing probabilistic formation of synaptic connections, we reconstruct network connectivity among up to 2000 neurons. When the resulting "network" is populated by model neurons and synapses, with properties based on physiology, it can respond to sensory stimulation by mimicking tadpole swimming behavior. This functioning model represents the most complete reconstruction of a vertebrate neuronal network that can reproduce the complex, rhythmic behavior of a whole animal. The findings validate our novel developmental strategy for generating realistic networks with individual neuron- and synapse-level resolution. We use it to demonstrate how early functional neuronal connectivity and behavior may in life result from simple developmental "rules," which lay out a scaffold for the vertebrate CNS without specific neuron-to-neuron recognition.

Sensory initiation of a co-ordinated motor response: synaptic excitation underlying simple decision-making.

KEY POINTS: Deciding whether or how to initiate a motor response to a stimulus can be surprisingly slow and the underlying processes are not well understood. The neuronal circuitry that allows frog tadpoles to swim in response to touch is well characterized and includes excitatory reticulospinal neurons that drive swim circuit neurons. Build-up of excitation to reticulospinal neurons is the key decision-making step for swimming. Asymmetry in this build-up between the two sides allows bilateral initiation at the same time as avoiding inappropriate co-activation of motor antagonists. Following stronger stimuli, reticulospinal neurons are excited through a trigeminal nucleus pathway and swimming starts first on the stimulated side. If this pathway fails or is lesioned, swimming starts later on the unstimulated side. The mechanisms underlying initiation of a simple tadpole motor response may share similarities with more complex decisions in other animals, including humans. ABSTRACT: Animals take time to make co-ordinated motor responses to a stimulus. How can sensory input initiate organized movements, activating all necessary elements at the same time as avoiding inappropriate co-excitation of antagonistic muscles? In vertebrates, this process usually results in the activation of reticulospinal pathways. Young Xenopus tadpoles can respond to head-skin touch by swimming, which may start on either side. We investigate how motor networks in the brain are organized, and whether asymmetries in touch sensory pathways avoid co-activation of antagonists at the same time as producing co-ordinated movements. We record from key reticulospinal neurons in the network controlling swimming. When the head skin is stimulated unilaterally, excitation builds up slowly and asymmetrically in these neurons such that those on both sides do not fire synchronously. This build-up of excitation to threshold is the key decision-making step and determines whether swimming will start, as well as on which side. In response to stronger stimuli, the stimulated side tends to 'win' because excitation from a shorter, trigeminal nucleus pathway becomes reliable and can initiate swimming earlier on the stimulated side. When this pathway fails or is lesioned, swimming starts later and on the unstimulated side. Stochasticity in the trigeminal nucleus pathway allows unpredictable turning behaviour to weaker stimuli, conferring potential survival benefits. We locate the longer, commissural sensory pathway carrying excitation to the unstimulated side and record from its neurons. These neurons fire to head-skin stimuli but excite reticulospinal neurons indirectly. We propose that asymmetries in the sensory pathways exciting brainstem reticulospinal neurons ensure alternating and co-ordinated swimming activity from the start.

The role of a trigeminal sensory nucleus in the initiation of locomotion.

While we understand how stimuli evoke sudden, ballistic escape responses, like fish fast-starts, a precise pathway from sensory stimulation to the initiation of rhythmic locomotion has not been defined for any vertebrate. We have now asked how head skin stimuli evoke swimming in hatchling frog tadpoles. Whole-cell recordings and dye filling revealed a nucleus of ∼20 trigeminal interneurons (tINs) in the hindbrain, at the level of the auditory nerve, with long, ipsilateral, descending axons. Stimulation of touch-sensitive trigeminal afferents with receptive fields anywhere on the head evoked large, monosynaptic EPSPs (∼5-20 mV) in tINs, at mixed AMPAR/NMDAR synapses. Following stimuli sufficient to elicit swimming, tINs fired up to six spikes, starting 4-8 ms after the stimulus. Paired whole-cell recordings showed that tINs produce small (∼2-6 mV), monosynaptic, glutamatergic EPSPs in the hindbrain reticulospinal neurons (descending interneurons, dINs) that drive swimming. Modelling suggested that summation of EPSPs from 18-24 tINs can make 20-50% of dINs fire. We conclude that: brief activity in a few sensory afferents is amplified by recruitment of many tINs; these relay summating excitation to hindbrain reticulospinal dINs; dIN firing then initiates activity for swimming on the stimulated side. During fictive swimming, tINs are depolarised and receive rhythmic inhibition but do not fire. Our recordings demonstrate a neuron-by-neuron pathway from head skin afferents to the reticulospinal neurons and motoneurons that drive locomotion in a vertebrate. This direct pathway, which has an important amplifier function, implies a simple origin for the complex routes to initiate locomotion in higher vertebrates.

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.

Skin impulse excitation of spinal sensory neurons in developing Xenopus laevis (Daudin) tadpoles.

Responses to gentle touch in young Xenopus tadpoles are mediated by spinal cord sensory Rohon-Beard neurons. Tadpoles also respond to noxious stimuli that elicit 'skin impulses', which propagate between epithelial cells over the whole body surface, somehow entering the CNS to generate a response. After hatching (~48 h post-fertilization), skin impulse signals enter the CNS only via cranial nerves, but previous evidence suggested the possibility of direct entry to the spinal cord before this (~24 h). We have used behavioural and electrophysiological methods to explore the developmental pattern of skin impulse entry into the spinal cord and the involvement of Rohon-Beard neurons. Lesioning confirmed that skin impulse signals can directly enter the spinal cord in young embryos, but access decreases over ~12 h and disappears soon after hatching. Electrical recordings from central Rohon-Beard axons in young embryos showed firing in response to skin impulses. However, unit recordings from Rohon-Beard somata showed that individuals that responded to touch within a characteristic, localised receptive field did not fire to skin impulses, whereas others from similar locations responded reliably. Developmental loss of skin impulse access to the spinal cord mirrored the known spread of sensitivity to gentle touch as the peripheral mechanosensory endings of Rohon-Beard neurons mature. Together, these results suggest that Rohon-Beard neurons respond to skin impulses only while immature, providing a transitory route for skin impulses to excite the CNS. In this way, Rohon-Beard neurons would mediate responses first to noxious and then to localised, gentle touch stimuli as the neurons developed.

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.

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 approximately 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-beta-GA, carbenoxolone, flufenamic acid and heptanol) in paired recordings. 18-beta-GA at 40-60 mum produced substantial (84%) coupling block but few effects on cellular properties. Swimming episodes in 18-beta-GA were significantly shortened (to approximately 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-beta-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.

Stimulation of Single, Possible CHX10 Hindbrain Neurons Turns Swimming On and Off in Young Xenopus Tadpoles.

Vertebrate central pattern generators (CPGs) controlling locomotion contain neurons which provide the excitation that drives and maintains network rhythms. In a simple vertebrate, the developing Xenopus tadpole, we study the role of excitatory descending neurons with ipsilateral projecting axons (descending interneurons, dINs) in the control of swimming rhythms. In tadpoles with both intact central nervous system (CNS) and transections in the hindbrain, exciting some individual dINs in the caudal hindbrain region could start swimming repeatedly. Analyses indicated the recruitment of additional dINs immediately after such evoked dIN spiking and prior to swimming. Excitation of dINs can therefore be sufficient for the initiation of swimming. These "powerful" dINs all possessed both ascending and descending axons. However, their axon projection lengths were not different from those of other excitatory dINs at similar locations. The dorsoventral position of dINs, as a population, significantly better matched that of cells marked by immunocytochemistry for the transcription factor CHX10 than other known neuron types in the ventral hindbrain and spinal cord. The comparison suggests that the excitatory interneurons including dINs are CHX10-positive, in agreement with CHX10 as a marker for excitatory neurons with ipsilateral projections in the spinal cord and brainstem of other vertebrates. Overall, our results further demonstrate the key importance of dINs in driving tadpole swimming rhythms.

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 Brown's (1911) hypothesis for locomotor rhythmogenesis.

Bifurcations of Limit Cycles in a Reduced Model of the Xenopus Tadpole Central Pattern Generator.

We present the study of a minimal microcircuit controlling locomotion in two-day-old Xenopus tadpoles. During swimming, neurons in the spinal central pattern generator (CPG) generate anti-phase oscillations between left and right half-centres. Experimental recordings show that the same CPG neurons can also generate transient bouts of long-lasting in-phase oscillations between left-right centres. These synchronous episodes are rarely recorded and have no identified behavioural purpose. However, metamorphosing tadpoles require both anti-phase and in-phase oscillations for swimming locomotion. Previous models have shown the ability to generate biologically realistic patterns of synchrony and swimming oscillations in tadpoles, but a mathematical description of how these oscillations appear is still missing. We define a simplified model that incorporates the key operating principles of tadpole locomotion. The model generates the various outputs seen in experimental recordings, including swimming and synchrony. To study the model, we perform detailed one- and two-parameter bifurcation analysis. This reveals the critical boundaries that separate different dynamical regimes and demonstrates the existence of parameter regions of bi-stable swimming and synchrony. We show that swimming is stable in a significantly larger range of parameters, and can be initiated more robustly, than synchrony. Our results can explain the appearance of long-lasting synchrony bouts seen in experiments at the start of a swimming episode.

Structural and functional properties of a probabilistic model of neuronal connectivity in a simple locomotor network.

Although, in most animals, brain connectivity varies between individuals, behaviour is often similar across a species. What fundamental structural properties are shared across individual networks that define this behaviour? We describe a probabilistic model of connectivity in the hatchling Xenopus tadpole spinal cord which, when combined with a spiking model, reliably produces rhythmic activity corresponding to swimming. The probabilistic model allows calculation of structural characteristics that reflect common network properties, independent of individual network realisations. We use the structural characteristics to study examples of neuronal dynamics, in the complete network and various sub-networks, and this allows us to explain the basis for key experimental findings, and make predictions for experiments. We also study how structural and functional features differ between detailed anatomical connectomes and those generated by our new, simpler, model (meta-model).

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.

Studying the role of axon fasciculation during development in a computational model of the Xenopus tadpole spinal cord.

During nervous system development growing axons can interact with each other, for example by adhering together in order to produce bundles (fasciculation). How does such axon-axon interaction affect the resulting axonal trajectories, and what are the possible benefits of this process in terms of network function? In this paper we study these questions by adapting an existing computational model of the development of neurons in the Xenopus tadpole spinal cord to include interactions between axons. We demonstrate that even relatively weak attraction causes bundles to appear, while if axons weakly repulse each other their trajectories diverge such that they fill the available space. We show how fasciculation can help to ensure axons grow in the correct location for proper network formation when normal growth barriers contain gaps, and use a functional spiking model to show that fasciculation allows the network to generate reliable swimming behaviour even when overall synapse counts are artificially lowered. Although we study fasciculation in one particular organism, our approach to modelling axon growth is general and can be widely applied to study other nervous systems.

The spinal interneurons and properties of glutamatergic synapses in a primitive vertebrate cutaneous flexion reflex.

Unlike the monosynaptic "stretch" reflex, the exact neuronal pathway for a simple cutaneous reflex has not yet been defined in any vertebrate. In young frog tadpoles, we made whole-cell recordings from pairs of spinal neurons. We found direct, excitatory, glutamatergic synapses from touch-sensitive skin-sensory neurons to sensory pathway interneurons, and then from these sensory interneurons to motoneurons and premotor interneurons on the other side of the body. We conclude that the minimal pathway for this primitive reflex, in which stroking the skin on one side leads to flexion on the other side, is disynaptic. This detailed circuit information has allowed us to ask whether the properties of glutamatergic synapses during the first day of CNS development are tuned to their function in the tadpole's responses. Stroking the skin excites a few sensory neurons. These activate primarily AMPA receptors producing short, strong excitation that activates many sensory pathway interneurons but only allows temporal summation of closely synchronous inputs. In contrast, the excitation produced in contralateral neurons by the sensory pathway interneurons is weak and primarily mediated by NMDA receptors. As a result, considerable summation is required for this excitation to lead to postsynaptic neuron firing and a contralateral flexion. We conclude that from their early functioning, synapses from sensory neurons are strong and those from sensory pathway interneurons are weak. The distribution of glutamate receptors at synapses in this developing circuit is tuned so that synapses have properties suited to their roles in the whole animal's reflex responses.

A developmental approach to predicting neuronal connectivity from small biological datasets: a gradient-based neuron growth model.

Relating structure and function of neuronal circuits is a challenging problem. It requires demonstrating how dynamical patterns of spiking activity lead to functions like cognitive behaviour and identifying the neurons and connections that lead to appropriate activity of a circuit. We apply a "developmental approach" to define the connectome of a simple nervous system, where connections between neurons are not prescribed but appear as a result of neuron growth. A gradient based mathematical model of two-dimensional axon growth from rows of undifferentiated neurons is derived for the different types of neurons in the brainstem and spinal cord of young tadpoles of the frog Xenopus. Model parameters define a two-dimensional CNS growth environment with three gradient cues and the specific responsiveness of the axons of each neuron type to these cues. The model is described by a nonlinear system of three difference equations; it includes a random variable, and takes specific neuron characteristics into account. Anatomical measurements are first used to position cell bodies in rows and define axon origins. Then a generalization procedure allows information on the axons of individual neurons from small anatomical datasets to be used to generate larger artificial datasets. To specify parameters in the axon growth model we use a stochastic optimization procedure, derive a cost function and find the optimal parameters for each type of neuron. Our biologically realistic model of axon growth starts from axon outgrowth from the cell body and generates multiple axons for each different neuron type with statistical properties matching those of real axons. We illustrate how the axon growth model works for neurons with axons which grow to the same and the opposite side of the CNS. We then show how, by adding a simple specification for dendrite morphology, our model "developmental approach" allows us to generate biologically-realistic connectomes.

A functional scaffold of CNS neurons for the vertebrates: the developing Xenopus laevis spinal cord.

In young and developing amphibians and fish the spinal cord is functional but remarkably simple compared with the adult. Is the pattern of neurons and their connections common across at least these lower vertebrates? Does this basic pattern extend into the brainstem? Could the development of simple functioning neuronal networks depend on very basic rules of connectivity and act as pioneer networks providing a substrate for the development of more complex and subtle networks. In this review of the functional neuron classes in the Xenopus laevis tadpole spinal cord up to hatching, we will consider progress and difficulties in using anatomy, transcription factor expression, physiology, and activity to define spinal neuron types. Even here it is not straightforward and is rarely possible to bring all the different strands of evidence together. But, we think we have a rather complete picture of the hatchling tadpole spinal neuron types and can define clear roles for most of them in behavior. Our present knowledge about the hatchling Xenopus spinal cord should set up many of the problems to be unraveled in the future by more developmentally oriented research.