A bioinspired autonomous swimming robot as a tool for studying goal-directed locomotion.

The bioinspired approach has been key in combining the disciplines of robotics with neuroscience in an effective and promising fashion. Indeed, certain aspects in the field of neuroscience, such as goal-directed locomotion and behaviour selection, can be validated through robotic artefacts. In particular, swimming is a functionally important behaviour where neuromuscular structures, neural control architecture and operation can be replicated artificially following models from biology and neuroscience. In this article, we present a biomimetic system inspired by the lamprey, an early vertebrate that locomotes using anguilliform swimming. The artefact possesses extra- and proprioceptive sensory receptors, muscle-like actuation, distributed embedded control and a vision system. Experiments on optimised swimming and on goal-directed locomotion are reported, as well as the assessment of the performance of the system, which shows high energy efficiency and adaptive behaviour. While the focus is on providing a robotic platform for testing biological models, the reported system can also be of major relevance for the development of engineering system applications.

A novel autonomous, bioinspired swimming robot developed by neuroscientists and bioengineers.

This paper describes the development of a new biorobotic platform inspired by the lamprey. Design, fabrication and implemented control are all based on biomechanical and neuroscientific findings on this eel-like fish. The lamprey model has been extensively studied and characterized in recent years because it possesses all basic functions and control mechanisms of higher vertebrates, while at the same time having fewer neurons and simplified neural structures. The untethered robot has a flexible body driven by compliant actuators with proprioceptive feedback. It also has binocular vision for vision-based navigation. The platform has been successfully and extensively experimentally tested in aquatic environments, has high energy efficiency and is ready to be used as investigation tool for high level motor tasks.

Ion channels of importance for the locomotor pattern generation in the lamprey brainstem-spinal cord.

The intrinsic function of the spinal network that generates locomotion can be studied in the isolated brainstem-spinal cord of the lamprey, a lower vertebrate. The motor pattern underlying locomotion can be elicited in the isolated spinal cord. The network consists of excitatory glutamatergic and inhibitory glycinergic interneurones with known connectivity. The current review addresses the different subtypes of ion channels that are present in the cell types that constitute the network. In particular the roles of the different subtypes of Ca2+ channels and potassium channels that regulate integrated neuronal functions, like frequency regulation, spike frequency adaptation and properties that are important for generating features of the motor pattern (e.g. burst termination), are reviewed. By knowing the role of an ion channel at the cellular level, we also, based on previous knowledge of network connectivity, can understand which effect a given ion channel may exert at the different levels from molecule and cell to network and behaviour.

The intrinsic function of a motor system--from ion channels to networks and behavior.

The forebrain, brainstem and spinal cord contribution to the control of locomotion is reviewed in this article. The lamprey is used as an experimental model since it allows a detailed cellular analysis of the neuronal network underlying locomotion. The focus is on cellular mechanisms that are important for the pattern generation, as well as different types of pre- and postsynaptic modulation. This experimental model is bridging the gap between the molecular and cellular level to the network and behavioral level.

More porous fibrin gel structure obtained by interaction with Lys-plasminogen than with Glu-plasminogen.

The effect of Glu1- and Lys78-plasminogen on the assembly and structure of fibrin gels was studied in purified fibrinogen-thrombin system and in plasminogen-free plasma, using turbidity, liquid permeation and three-dimensional (3D) confocal laser microscopy methods. In the purified fibrinogen system using the turbidity method, the final optical density of the fibrin gels increased with increasing concentrations of Lys-plasminogen. The fiber mass/length ratio mu increased with increasing concentrations of both Glu1- and Lys78-plasminogen, the effect of Lys78-plasminogen being much stronger. The permeability coefficient (Ks) analyzed with the permeation method revealed that fibrin gels formed in the presence of Lys78-plasminogen were more permeable (porous) than the control gels. The effect on the gel structure was inhibited by the fibrinolytic inhibitor epsilon-aminocaproic acid. The same results were obtained in plasma milieu for both mu and Ks as in the purified system, i.e. the gels became more porous with increasing concentrations of Lys78-plasminogen. 3D microscopy pictures of the gels verified the findings.

Intrinsic function of a neuronal network - a vertebrate central pattern generator.

The cellular bases of vertebrate locomotor behaviour is reviewed using the lamprey as a model system. Forebrain and brainstem cell populations initiate locomotor activity via reticulospinal fibers activating a spinal network comprised of glutamatergic and glycinergic interneurons. The role of different subtypes of Ca2+ channels, Ca2+ dependent K+ channels and voltage dependent NMDA channels at the neuronal and network level is in focus as well as the effects of different metabotropic, aminergic and peptidergic modulators that target these ion channels. This is one of the few vertebrate networks that is understood at a cellular level.

Neural networks that co-ordinate locomotion and body orientation in lamprey.

The networks of the brainstem and spinal cord that co-ordinate locomotion and body orientation in lamprey are described. The cycle-to-cycle pattern generation of these networks is produced by interacting glutamatergic and glycinergic neurones, with NMDA receptor-channels playing an important role at lower rates of locomotion. The fine tuning of the networks produced by 5-HT, dopamine and GABA systems involves a modulation of Ca2+-dependent K+ channels, high- and low-threshold voltage-activated Ca2+ channels and presynaptic inhibitory mechanisms. Mathematical modelling has been used to explore the capacity of these biological networks. The vestibular control of the body orientation during swimming is exerted via reticulospinal neurones located in different reticular nuclei. These neurones become activated maximally at different angles of tilt.

Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA.

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.

Activity-related calcium dynamics in lamprey motoneurons as revealed by video-rate confocal microscopy.

In lamprey spinal cord, intracellular calcium ([Ca2+]i) plays a key role in mechanisms regulating neuronal activity in the segmental network for locomotion. In this report, measurements of [Ca2+]i with fluo-3 in various regions of motoneurons in the intact spinal cord were obtained on a high speed confocal microscope following electrical stimulation. Likewise, rhythmic calcium fluctuations within dendrites and axons were seen during "fictive swimming" and were directly correlated with electrical activity. Antidromic stimulation of motoneuron axons induced large calcium transients and revealed spatially restricted "hot spots," both of which required external calcium and were blocked by nickel, but not by known calcium channel antagonists. These results suggest that lamprey spinal cord axons may possess a pharmacologically novel class of calcium channel.

Sensorimotor integration in the lamprey locomotor system.

The locomotor movements of vertebrates are controlled by rhythm-generating networks in the spinal cord. These circuits are under modulatory influence from different sources, of both supraspinal, propriospinal and sensory origin. This paper reviews the neuronal mechanisms underlying swimming movements in the lamprey, the CNS of which provides a convenient model system for detailed investigations of the cellular bases for vertebrate locomotor behaviour, and which can be studied under in vitro conditions. The principal organization of the spinal, segmental network responsible for the generation of the rhythm, intrinsic membrane properties of importance for burst activity, as well as different mechanisms for modulation of the network activity including its sensory control, will be briefly described. The principles for coordination between segments during locomotion will also be shortly described.

Computer simulations of NMDA and non-NMDA receptor-mediated synaptic drive: sensory and supraspinal modulation of neurons and small networks.

1. The segmental locomotor network in lamprey can generate the rhythmic burst pattern underlying locomotion when it is driven via synaptic glutamate receptors. Lower rates of activity can be evoked by activation of N-methyl-D-aspartate (NMDA) receptors, whereas a rapid activity can only be induced by non-NMDA receptors [kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)]. The reticulospinal and sensory inputs are known to act via both NMDA and non-NMDA receptors, but it is unclear how these inputs can provide an appropriate control of the locomotor rate. We have examined the effects of different types of excitatory synaptic input to neurons of the locomotor network with the use of a computer-simulated electrical neuron model, with Na+, K+, Ca(2+)-dependent K+ channels, and with inherent oscillatory properties linked to the NMDA conductance. Synapses were modeled as a modulated ionic conductance in the membrane of the postsynaptic cell comprising a voltage-dependent NMDA component (Na+, K+, Ca2+ conductances) of long duration, and/or a non-NMDA component (Na+, K+ conductance) of short duration. 2. By using two neurons to drive a postsynaptic cell with non-NMDA-type synapses, a continuous range of firing frequencies could be evoked in the postsynaptic cell, by altering the firing rate of the presynaptic cells. If a single presynaptic neuron was used, there was a tendency toward spike synchronization between the pre- and postsynaptic cells. 3. When a postsynaptic neuron was driven via NMDA synapses, an oscillatory burst activity could be evoked. The rate of the oscillations was, however, little affected by the presynaptic firing rate. When a drive neuron with mixed (NMDA and non-NMDA) synapses was used, the rate of the oscillations could be changed within a limited frequency range by altering the presynaptic firing rate. By adding another larger drive neuron, having a larger rheobase current and mixed synapses with smaller relative NMDA components, the frequency range of the postsynaptic oscillations could be markedly increased. The frequency range depended on the parameters selected for each of the two types of mixed synapses. 4. A small rhythm-generating neuronal network, comprising six cells connected as the principal interneurons of the lamprey spinal locomotor network, was used to test the role of a tonic NMDA and non-NMDA receptor activation to drive the network and produce bursting.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrical coupling of mechanoreceptor afferents in the crayfish: a possible mechanism for enhancement of sensory signal transmission.

1. Central electrical coupling between primary afferent axons was investigated in an in vitro preparation of the crayfish thoracic locomotor system by using intracellular recordings. 2. Intracellular injection of the dye Lucifer yellow in single afferents resulted in staining of one to three additional afferents through dye-coupling. Three-dimensional confocal imaging of dye-coupled axons revealed a large zone of close apposition that may correspond to the gap junction site. 3. A depolarization preceding the spike in one sensory terminal was shown to facilitate the excitatory postsynaptic potential occurring in postsynaptic motoneurons. Further, a spike in one afferent axon can depolarize other, electrically coupled, axons above spike threshold, resulting in an increased number of active afferents. 4. The electrical coupling occurred between sensory afferents of similar function. It may therefore serve to facilitate sensory signal transmission from functionally homologous afferents onto postsynaptic target neurons.

Origin of phasic synaptic inhibition in myotomal motoneurons during fictive locomotion in the lamprey.

The periodic membrane potential fluctuations in motoneurons during fictive locomotion in the lamprey, a primitive vertebrate, involve phasic synaptic excitation and inhibition. This paper investigates the origin of the phasic synaptic input to lamprey myotomal motoneurons in the in vitro spinal cord preparation with regard to the relative contribution of descending propriospinal input from interneurons in the local segment. The synaptic drive to myotomal motoneurons in the most rostral and the most caudal part of the spinal cord preparation are compared before and after selective spinal cord lesions. Current clamp recordings of the same cell before and after lesion showed that neither the excitatory phase nor the inhibitory phase was abolished after interruption of the descending or the ascending ipsilateral input, or after interrupting crossing segmental input by a local longitudinal midline incision. None of these sources thus appears to be alone responsible for the phasic synaptic drive. To quantitatively evaluate these effects, and in particular the contribution from the descending propriospinal fibres to the inhibitory phase, voltage clamp recordings were made in combination with a spinal cord hemisection just rostral to the motoneuron. The input from propriospinal interneurons in approximately 15 rostral segments may be responsible for as much as 70% of the phase of inhibitory current during the locomotor cycle. In accordance with these findings, a similar voltage clamp analysis of rostrally and caudally located motoneurons showed that the average peak-to-peak amplitude of the current fluctuations in rostral cells was approximately 50% of that in caudal cells.

A computer-based model for realistic simulations of neural networks. II. The segmental network generating locomotor rhythmicity in the lamprey.

1. To analyze the function of the spinal interneuronal network generating locomotion in the lamprey CNS, a vertebrate model system, we performed computer simulations with realistic model neurons possessing the essential properties of their biological counterparts. 2. The segmental network has been simulated by modeling experimentally established types of neurons with their specific membrane properties and synaptic interconnections. Fictive locomotor activity, which can be experimentally induced by elevating the background excitability by bath application of excitatory amino acids, was simulated by opening membrane conductances for kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. Kainate/AMPA receptor activation induced a rhythm in the middle and upper part of the physiological burst frequency range, whereas NMDA receptor activation evoked bursting in the lower part of the range, which corresponds well to earlier experimental findings. 3. Several factors contributing to the termination of the burst were studied and their interaction was assessed in simulations of the network. 1) The summation of spike afterhyperpolarizations (late AHPs), leading to adaptation of the discharge, acts as a primary burst-terminating factor at lower rates of kainate/AMPA-induced bursting, and it also interacts with the NMDA-induced oscillatory membrane properties during slow rhythmicity. 2) The termination of the depolarized NMDA plateau is another important factor during NMDA-evoked rhythmicity. 3) The synaptic inhibition from lateral interneurons to the interneurons mediating reciprocal inhibition is important at higher rates of kainate/AMPA-induced bursting. 4. The mechanism of action of 5-hydroxytryptamine (5-HT) on the lamprey segmental network was further investigated by simulation. 5-HT is known to lower the burst frequency during fictive locomotion and also to decrease the conductance through the Ca(2+)-dependent K+ channels, and thereby the size of the late AHP that follows the action potential. Decreasing this conductance in the network simulations resulted in a lesser amount of AHP summation and thereby less frequency adaptation during the burst, longer bursts, and a lower locomotor frequency. Thus the selective action of 5-HT on the Ca(2+)-dependent K+ channels, and hence on the AHP, can account for the modulatory effect on the fictive locomotor rhythm seen experimentally. 5. The results demonstrate that the present simulation of the segmental network can account for essential features of the motor pattern seen experimentally during lamprey locomotion.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding of tissue plasminogen activator to human endothelial cells. Importance of the B-chain as a ligand.

The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.

Two types of motoneurons supplying dorsal fin muscles in lamprey and their activity during fictive locomotion.

The location and dendritic morphology of motoneurons supplying the dorsal fin muscles were studied in the lamprey spinal cord (Ichthyomyzon unicuspis). Motoneurons were retrogradely labelled after injection of HRP into the fin muscles or after its application on the cut ends of the ventral roots. HRP-labelled cells were subsequently reconstructed, in the horizontal and/or transverse planes. Fin motoneurons were also injected intracellularly with Lucifer Yellow and their detailed three-dimensional structure was analysed by confocal laser-scanning microscopy. Unlike myotomal motoneurons, which are closely spaced in the lateral cell column, fin motoneurons were distributed along the spinal cord separately or in pairs. They could be distinguished from motoneurons supplying trunk muscles by having a limited number of dendrites in the lateral part of the spinal cord. In addition, some fin motoneurons extend their dendrites into the dorsal column. The motor cells innervating fin muscles were divided into two types based on their dendritic morphology. Type I have a widespread dendritic tree in the rostrocaudal direction and, with few exceptions, completely restricted to the ipsilateral side. A proportion (25%) of these cells have dendrites extending into the dorsal column. Type II fin motoneurons extended their dendrites both ipsi- and contra-laterally. The contra-lateral dendrites pass below and above the central canal. The dendrites send off branches into the dorsal columns on both the ipsi- and the contra-lateral sides. Electron microscopic analysis showed that both type I and type II fin motoneurons receive numerous synaptic contacts from dorsal column axons. During fictive locomotion both types of motoneurons are active in antiphase in relation to myotomal motoneurons and to the main locomotor burst.

5-Hydroxytryptamine modulates spike frequency regulation in reticulospinal neurons involved in the control of locomotion in lamprey.

To investigate the effects of 5-hydroxytryptamine (5-HT) on reticulospinal neurons involved in the initiation and control of locomotion in lamprey, 5-HT (10 mM) was locally pressure ejected on the dorsal surface of the brainstem during intracellular recordings from identified reticulospinal neurons in the in vitro brainstem-spinal cord preparation. 5-HT induced a reduction of the late afterhyperpolarization (AHP) following the spike due to a reduction of a Ca(2+)-activated K+ current. In addition, 5-HT caused a resting membrane hyperpolarization in a proportion of these cells. Due to the 5-HT induced reduction of the AHP, reticulospinal cells, including those that became hyperpolarized by an application of 5-HT, discharged at a higher rate after 5-HT as a response to the same excitatory drive.

Vestibular control of swimming in lamprey. I. Responses of reticulospinal neurons to roll and pitch.

A method has been developed for recording the response of single neurons in the lamprey brainstem in vitro to natural stimulation of vestibular receptors. The brainstem dissected together with the intact vestibular apparatus could be rotated in space, in two perpendicular planes (transverse, the roll tilt, and sagittal, the pitch tilt), in one of them up to 360 degrees, and in the other one up to +/- 30 degrees. The responses of single reticulospinal (RS) neurons, in all four reticular nuclei of the brainstem, to roll and pitch were recorded extracellularly and, with small inclinations (up to +/- 45 degrees) also intracellularly. Two types of preparations were used, with and without the rostral part of the spinal cord. In the brainstem preparations, most RS neurons responded both to a definite brain orientation in space and to a change of the orientation (static and dynamic reactions). Responses to roll tilt were similar in all reticular nuclei: all cells were excited with roll tilt towards the contralateral side, this reaction was qualitatively preserved when the roll was performed in combination with different pitch inclinations. Responses to pitch tilt were less clearcut; some neurons were activated with nose-up deflection while others responded to nose-down tilt. In preparations including the spinal cord, responses of RS neurons to roll and pitch tilt differed from those in the isolated brainstem in that they were much less specific and stable. Roll and pitch tilts could trigger the spinal locomotor CPG, which, by sending "efference copy" signals back to the brainstem, produced modulation of RS neurons in relation to the locomotor rhythm.

Vestibular control of swimming in lamprey. II. Characteristics of spatial sensitivity of reticulospinal neurons.

1. Experiments were carried out on an in vitro preparation of the lamprey brainstem isolated together with intact labyrinths. Responses of reticulospinal neurons from different brainstem reticular nuclei (mesencephalic, MRN; anterior rhombencephalic, ARRN; middle rhombencephalic, MRRN; and posterior rhombencephalic, PRRN) to rotation of the preparation (0 degrees-360 degrees) either in the sagittal plane (pitch tilt, or nose up-down movement) or in the transverse plane (roll tilt, or left-right inclination) were recorded. 2. Responses to roll tilt were qualitatively similar in all nuclei: contralateral side down tilt (in relation to the location of the neuron in the brain) caused an activation of reticulospinal neurons. The angular thresholds for activation differed, however, between nuclei as well as the angle at which the maximal activity occurred. The maximal response for MRN was at 45 degrees, for MRRN and PRRN at 90 degrees, for ARRN at 180 degrees. Thus, the zones of spatial sensitivity differed in different nuclei, and they covered the whole range of possible inclinations in the transverse plane. 3. Responses to pitch tilt were not uniform in the different nuclei. MRN neurons responded preferentially in the range of 45 degrees-90 degrees nose-up inclinations, but a proportion of the cells responded in the range of 45 degrees-90 degrees nose-down inclinations. The ARRN neurons had their maximal response when the brain was turned to a dorsal side-down position (180 degrees). In the MRRN, three subgroups of neurons could be distinguished, the first responding at around 90 degrees nose-down, the second responding at around 90 degrees nose-up and the third responding in both zones. However, the activation in the nose-up zone was less robust: responses in this zone were present only in approximately one half of the experiments. Finally, the PRRN neurons were found to be very heterogeneous, with their zones of sensitivity being distributed throughout the whole space (0 degrees-360 degrees). Thus, also in the sagittal plane, the zones of spatial sensitivity in the different nuclei covered the whole range of possible inclinations. 4. Long-term recording of MRRN neurons having the zone of sensitivity around 90 degrees nose-up showed that this response was rather unstable. Its amplitude varied considerably and could disappear with time to reappear later. These results, together with the fact that in a part of the experiments the MRRN neurons responded only in the 90 degrees nose-down zone (see above), leads us to suggest that the system of spatial orientation can dynamically re-organize.(ABSTRACT TRUNCATED AT 400 WORDS)