Neuromechanical simulation of the locust jump.

The neural circuitry and biomechanics of kicking in locusts have been studied to understand their roles in the control of both kicking and jumping. It has been hypothesized that the same neural circuit and biomechanics governed both behaviors but this hypothesis was not testable with current technology. We built a neuromechanical model to test this and to gain a better understanding of the role of the semi-lunar process (SLP) in jump dynamics. The jumping and kicking behaviors of the model were tested by comparing them with a variety of published data, and were found to reproduce the results from live animals. This confirmed that the kick neural circuitry can produce the jump behavior. The SLP is a set of highly sclerotized bands of cuticle that can be bent to store energy for use during kicking and jumping. It has not been possible to directly test the effects of the SLP on jump performance because it is an integral part of the joint, and attempts to remove its influence prevent the locust from being able to jump. Simulations demonstrated that the SLP can significantly increase jump distance, power, total energy and duration of the jump impulse. In addition, the geometry of the joint enables the SLP force to assist leg flexion when the leg is flexed, and to assist extension once the leg has begun to extend.

Practical tools for analysing rhythmic neural activity.

This report describes an integrated software package, DataView, which contains a number of tools for analysing rhythmic neural activity. These include simple autocorrelation, a merge-and-drop filter, an enhanced version of the Poisson surprise method and a flexible hill-and-valley analysis tool. The package contains facilities for identifying, examining, and if appropriate, correcting, outliers arising from misidentification or rhythm abnormalities. The package has a full graphical user interface which provides flexible and rapid feedback on the progress of analysis, and the consequences of choices regarding parameters for the various tools. The user can thus easily experiment with different methodologies and tool settings, and tune the analysis to the most appropriate form for the data in question.

Glutamate is a transmitter that mediates inhibition at the rectifying electrical motor giant synapse in the crayfish.

Spike transmission at the electrical synapse between the giant fibres (GFs) and motor giant neurone (MoG) in the crayfish can be blocked by depolarising postsynaptic chemical inhibition, which has previously been shown to be mediated in part by gamma-aminobutyric acid (GABA). The authors show that glutamate applied to the synaptic region of the MoG mimics the depolarisation of the chemical input and can also block spike transmission from the GFs. The glutamate induces an inward current mediated by a conductance increase that is 30-40% of that induced by GABA and that is blocked substantially by picrotoxin. Glutamate has no effect on the presynaptic GF, and the effects in the MoG are maintained in the presence of cadmium, indicating that the glutamate is acting directly on the MoG. Both GABA and glutamate have similar effects on the cell body, where the response reverses 10-20 mV positive to resting potential, is dependent on chloride concentration, and is inhibited by picrotoxin. Joint application of glutamate and GABA induces a nonadditive current under voltage clamp, suggesting that the transmitters can activate the same postsynaptic receptors. Immunocytochemical staining shows that, whereas some synaptic profiles impinging on the MoG contain pleomorphic agranular vesicles and are immunoreactive to GABA and not glutamate (as previously reported), there are at least as many other profiles that contain round, agranular vesicles and that are immunoreactive to glutamate and not to GABA. Thus, the authors conclude that some of the interneurones mediating inhibition of the electrical synapse use glutamate as their neurotransmitter.

Escape behaviour in the stomatopod crustacean Squilla mantis, and the evolution of the caridoid escape reaction.

The mantis shrimp Squilla mantis shows a graded series of avoidance/escape responses to visual and mechanical (vibration and touch) rostral stimuli. A low-threshold response is mediated by the simultaneous protraction of the thoracic walking legs and abdominal swimmerets and telson, producing a backwards 'lurch' or jump that can displace the animal by up to one-third of its body length, but leaves it facing in the same direction. A stronger response starts with similar limb protraction, but is followed by partial abdominal flexion. The maximal response also consists of limb protraction followed by abdominal flexion, but in this case the abdominal flexion is sufficiently vigorous to pull the animal into a tight vertical loop, which leaves it inverted and facing away from the stimulus. The animal then swims forward (away from the stimulus) and rights itself by executing a half-roll. A bilaterally paired, large-diameter, rapidly conducting axon in the dorsal region of the ventral nerve excites swimmeret protractor motoneurons in several ganglia and is likely to be the driver neuron for the limb-protraction response. The same neuron also excites unidentified abdominal trunk motoneurons, but less reliably. The escape response is a key feature of the malacostracan caridoid facies, and we provide the first detailed description of this response in a group that diverged early in malacostracan evolution. We show that the components of the escape response contrast strongly with those of the full caridoid reaction, and we provide physiological and behavioural evidence for the biological plausibility of a limb-before-tail thesis for the evolution of the escape response.

Fifty years of a command neuron: the neurobiology of escape behavior in the crayfish.

Fifty years ago C.A.G. Wiersma established that the giant axons of the crayfish nerve cord drive tail-flip escape responses. The circuitry that includes these giant neurons has now become one of the best-understood neural circuits in the animal kingdom. Although it controls a specialized behavior of a relatively simple animal, this circuitry has provided insights that are of general neurobiological interest concerning matters as diverse as the identity of the neural substrates involved in making behavioral decisions, the cellular bases of learning, subcellular neuronal computation, voltage-gated electrical synaptic transmission and modification of neuromodulator actions that result from social experience. This work illustrates the value of studying a circuit of moderate, but tractable, complexity and known behavioral function.

Central and peripheral control of the trigger mechanism for kicking and jumping in the locust.

A crucial stage of the locust kick motor program is the trigger activity that inhibits the flexor motorneurons at the end of flexor-extensor coactivation and releases the tibia. One source of this inhibition is the M interneuron, which produces a spike burst at the time of the trigger activity. Previous work has suggested that sensory input resulting from extensor muscle tension may contribute to the M spike burst. We find that extensor muscle tension produced during thrusting behavior or by direct electrical stimulation with the tibia held fixed results in the depolarization of M, but this is not of sufficient amplitude to account for the M spike burst during the trigger activity. Furthermore, M still produces a spike burst after ablating the sensory systems that produce the response to the muscle stimulation. It is concluded that the major component of the M trigger activity is central in origin, although sensory feedback from extensor muscle tension makes some contribution. The combination of both central and peripheral paths for M activation may enhance the robustness of the behavior.

Effect of temperature on a voltage-sensitive electrical synapse in crayfish.

The effects of temperature on transmission through the voltage-sensitive giant motor synapse (GMS) were investigated in crayfish both experimentally and in computer simulation. The GMS is part of the fast reflex escape pathway of the crayfish and mediates activation from the lateral giant (LG) command neurone to the motor giant (MoG) flexor motoneurone. The investigation was motivated by an apparent mismatch between the temperature sensitivity of the activation time constant of the GMS, with a Q10 reported to be close to 11, and that of the active membrane properties of LG and MoG, which are thought to have Q10 values close to 3. Our initial hypothesis was that at cold temperatures the very slow activation of the GMS conductance would reduce the effectiveness of transmission compared with higher temperatures. However, the reverse was found to be the case. Effective transmission through the GMS was reliable at low temperatures, but failed at an upper temperature limit that varied between 12 degrees C and 25 degrees C in isolated nerve cord preparations. The upper limit was extended above 30 degrees C in semi-intact preparations where the GMS was less disturbed by dissection. The results of experiments and simulations both indicate that transmission becomes more reliable at low temperatures because the longer-duration presynaptic spikes are able to drive more current through the GMS into the MoG, which is more excitable at low temperatures. Conversely, effective transmission is difficult at high temperatures because the transfer of charge through the GMS is reduced and because the input resistance of MoG is lowered as its current threshold is increased. The effect of the high Q10 of the GMS activation is to help preserve effective transmission through the synapse at high temperatures and so extend the temperature range for effective operation of the escape circuit.

The concerted activity in parallel proprioceptive pathways controls the initiation of co-activation in the locust kick motor programme.

To jump and kick the locust uses a catapult mechanism implemented by a three-stage motor programme: initial flexion of the hind tibiae, co-activation of the antagonist flexor and extensor tibiae motor neurons and trigger inhibition of the flexor motorneurons. The transition from stage 1 to stage 2 thus involves a switch from the normal alternate activation to co-activation of the antagonist tibrae motorneurons. However, co-activation has never been observed when the central nervous system has been isolated from the leg. This led us to investigate the possibility that the transition from stage 1 to stage 2 is controlled by a proprioceptive signal. In the first set of experiments intracellular recordings were made in the flexor and extensor motorneurons while the position of the tendon of the femoral chordotonal organ (FCO), which signals tibial position and movement, was experimentally controlled. In these heavily dissected preparations, stretch of the FCO tendon (signalling tibial flexion) was a necessary condition for co-activation. However, in minimally dissected preparations (in which merely EMG recordings were made), we found that co-activation occurred even when the FCO was signalling tibial extension, suggesting the involvement of other proprioceptors. A series of experiments were then conducted on minimally dissected preparations to determine the relative contributions of each of the three main hind leg proprioceptors which might signal tibial flexion: the FCO, the lump receptor and Brünners organ. When all three proprioceptors were intact the chance of evoking co-activation was largest, when all three were eliminated co-activation could no longer be evoked, irrespective of the level of arousal. Various combinations of partial de-afferentation showed that the FCO plays the major role, with the lump receptor and Bünners organ playing significant, but progressively less important, roles. We conclude that the three receptors act together as a permissive proprioceptive gate for the kick and jump motor programme, but with a hierarchy of the strengths of their effectiveness.

A simple method for the removal of mains interference from pre-recorded electrophysiological data.

MOTIVATION: A common problem with electrophysiological recording is the contamination of the signal of interest with interference generated at mains frequency. Standard filtering techniques are often inappropriate because the signal of interest has components spectrally close to the mains frequency. RESULTS: A digital subtraction method is described for removing mains frequency interference from pre-recorded data. The data are first digitized with a sample rate that is some direct multiple of mains frequency. Next a 20 ms (for UK mains frequency) data set is constructed containing the average interference pattern. This is subtracted from each 20 ms window of the raw data. Finally, the mean value of the interference is added back to the raw data to restore the DC component. AVAILABILITY: A Windows program which implements the method for the EGAA data acquisition system (R.C.Electronics, Santa Barbara, CA) is available from the author. CONTACT: wjh@st-andrews.ac.uk

Quasi-reversible photo-axotomy used to investigate the role of extensor muscle tension in controlling the kick motor programme of grasshoppers.

The jump and kick of the grasshopper are behaviours which are potentially critical for the survival of the animal, and whose maximal performance depends upon optimizing the rate and level of tension development in the extensor tibiae muscle of the hind legs. In experimental conditions extensor tension control can be reduced to a single motoneuron, the fast extensor tibiae (FETi). The axon of FETi can be cut using dye-mediated laser photoaxotomy without damaging the central or peripheral portions of that neuron or any other neuron innervating the leg. The axotomy can be functionally reversed (i.e. the cut axon repaired) by an electronic axonal bypass which detects FETi spikes on the proximal side of the cut and stimulates the axon on the distal side of the cut. In this way motor spikes can either be allowed to reach the muscle or prevented from doing so (by switching the bypass on or off), and the motor programmes produced with and without extensor tension can be compared. The jump and kick are normally produced by a three-stage motor programme: (i) initial flexion brings the tibia into the fully flexed position; (ii) coactivation of extensor and flexor muscles allows the extensor muscle to develop maximal tension almost isometrically, while the simultaneous contraction of the flexor muscle holds the tibia flexed; (iii) sudden trigger inhibition of the flexor system (motoneurons and muscle) releases the tibia and allows the behaviour to be expressed. The grasshopper can produce fictive kicks with motor programmes which show each of these three major structural features of a normal kick, but without any extensor tension whatsoever. There is no significant difference in the frequency of FETi spikes, the duration of coactivation or the maximum depolarization of the flexor motoneurons between fictive and quasi-normal (i.e. reversed axotomy) kicks. The trigger inhibition of flexor motoneurons is shallower in fictive than in quasi-normal kicks. The significance of this is discussed in relation to the activity of the interneuron M, which is known to mediate trigger inhibition onto FITi motoneurons. There are two main conclusions from this study. First, the CNS does not need feedback from ETi muscle tension in order to produce the three-stage motor programme of the kick (and, by implication, the jump). Second, the CNS does not adjust the frequency or duration of FETi activity in response to unexpected changes in ETi tension. ETi tension appears to be under open-loop control in the kick motor programme.

Two classes of vesicles are present and change in relative proportion during post-embryonic development of rectifying electrical synapses in the crayfish.

The size and shape of vesicles at junctional appositions of the rectifying electrical synapses between the medial giant fibre and motor giant neurone of the crayfish were measured during the first 2 months after hatching. Summed data over this period reveal a bimodal distribution in vesicle diameter. From the day of hatching until about 7 days of age, small vesicles (circa 25 nm diameter) predominate. From day 7 onwards, larger vesicles (circa 55 nm diameter) occur in increasing numbers, until at day 56 they constitute about 85% of the population at any one junctional apposition. At intermediate ages (day 7-28) individual junctional appositions may show the same bimodal distribution in size as does the age group as a whole, indicating that large and small vesicles occur together at the same junction. The larger vesicles are mainly circular, while the small vesicles are pleomorphic, with shapes ranging from almost circular down to a shape factor of about 0.6.

Structural and functional post-embryonic development of a non-rectifying electrical synapse in the crayfish.

The post-embryonic development of the non-rectifying septate synapse between homologous lateral giant (LG) fibre segments has been investigated using electron microscopy and electrophysiology. In adults, the LG-LG synapse is characterized by closely apposed membranes (approximately 4 nm separation) traversed by regularly spaced particles, and large (60-80 nm) spherical vesicles on both sides of the junction. In newly hatched crayfish the junction between lateral giant fibre segments comprises regions of close membrane apposition as seen in the adult along with non-specialized areas of wide (10-15 nm) membrane separation. Vesicles associated with these junctions are small (25-40 nm) and pleomorphic. The number of vesicles is low by comparison with adult junctions; in most sections of hatchling junctions there are normally fewer than five vesicles, although as many as 30 have occasionally been seen. During development the non-specialized areas of wide membrane separation become rare and the vesicle population changes to a mixture of small pleomorphic forms and larger (60-80 nm) spherical ones. However even at two months the number of large spherical vesicles is markedly less than that at the adult synapse, while small pleomorphic vesicles are still abundant. Despite the difference between the adult and hatchling vesicle populations, intracellular recordings have shown that the synapse is fully functional as a non-rectifying electrical junction on hatching and that the intracellular marker Lucifer Yellow can pass between adjacent lateral giant fibre neurons.

Different types of rectification at electrical synapses made by a single crayfish neurone investigated experimentally and by computer simulation.

The rectification properties of electrical synapses made by the segmental giant (SG) neurone of crayfish (Pacifastacus leniusculus) were investigated. The SG acts as an interneurone, transmitting information from the giant command fibres (GFs) to the abdominal fast flexor (FF) motoneurones. The GF-SG (input) synapses are inwardly-rectifying electrical synapses, while the SG-FF (output) synapses are outwardly rectifying electrical synapses. This implies that a single neurone can make gap junction hemichannels with different rectification properties. The coupling coefficient of these synapses is dependent upon transjunctional potential. There is a standing gradient in resting potential between the GFs, SG and FFs, with the GFs the most hyperpolarized, and the FFs the most depolarized. The gradient thus biases each synapse into the low-conductance state under resting conditions. There is functional double rectification between the bilateral pairs of SGs within a single segment, such that depolarizing membrane potential changes of either SG pass to the other SG with less attenuation than do hyperpolarizing potential changes. Computer simulation suggests that this may result from coupling through the intermediary FF neurones.

Postsynaptic modulation of rectifying electrical synaptic inputs to the LG escape command neuron in crayfish.

The lateral giant (LG) tail-flip escape system of crayfish is organized to provide a massive convergence of mechanosensory inputs onto the LG command neuron through electrical synapses from both mechanosensory afferents and interneurons. We used electrophysiological techniques to show that the connections between three major mechanosensory interneurons and LG rectify, and that their inputs to LG can be reduced by postsynaptic depolarization and increased by postsynaptic hyperpolarization. The mechanosensory afferents and interneurons are excited by sensory nerve shock, and the components of the resulting LG PSP can be similarly modulated by the same postsynaptic potential changes. Because these inputs are all made through electrical synapses, we conclude that they are rectifying connections, as well. To test the physical plausibility of this conclusion, we developed an electrical model of the rectifying connection between a mechanosensory interneuron and LG, and found that it can reproduce all the qualitative features of the orthodromic and antidromic experimental responses. The ability of postsynaptic membrane potential to modulate inputs through rectifying electrical synapses is used in the escape system to enhance LG's relative sensitivity to novel, phasic stimuli. Postsynaptic depolarization of LG produced by earlier inputs "reverse-biases" the rectifying input synapses and reduces their strength relative to times when LG is at rest.

Postembryonic development of rectifying electrical synapses in crayfish: physiology.

In a previous paper we showed that the ultrastructure of the giant fibre to motor giant synapse of crayfish changes in the first few weeks after hatching from having predominantly the appearance of a chemical synapse to having the appearance of an electrical synapse. This is paralleled by a behavioural change from non-giant fibre-mediated to giant fibre-mediated tailflips. In this paper we describe the physiology of the giant fibre to motor giant synapse over this period. We find the following: (1) The giant fibre to motor giant synapse usually transmits spikes 1:1 from the day of hatching. (2) The synapse operates by electrical transmission from the day of hatching, when no connexons are apparent at the ultrastructural level. (3) The synapse has no detectable chemical component, even at an age when the predominant type of junctional apposition has the ultrastructural appearance of a chemical synapse. (4) Inhibitory chemical synapses occur onto the motor giant at the day of hatching, and these show similar physiological characteristics to those which occur onto the motor giant in adults. (5) In some preparations, the giant fibre to motor giant electrical synapse shows rectification similar to that in the adult, but in most cases both depolarizing and hyperpolarizing current injected into the medial giant spreads to the motor giant. (6) Current spread from the medial giant to the motor giant is increased by hyperpolarizing the motor giant neuron, even when medial giant to motor giant transmission is apparently non-rectifying. (7) Both the giant fibre and the motor giant have resting potentials of about -90 mV. There is no standing difference in resting potential as there is in the adult. This may explain the apparent lack of medial giant to motor giant rectification observed in most preparations.

Photoinactivation of the crayfish segmental giant neuron reveals a direct giant-fiber to fast-flexor connection with a chemical component.

The escape tail flip of the crayfish is "commanded" by 2 sets of giant-fiber (GF) interneurons. In each hemisegment, these drive the motor giant (MoG) abdominal flexor motor-neuron through a monosynaptic electrical connection, but the remaining 8 or 9 fast-flexor (FF) motorneurons receive most of their input via a disynaptic electrical pathway through the segmental giant (SG) neuron. We have investigated a monosynaptic GF-FF pathway, which operates in parallel to the disynaptic GF-SG-FF pathway, by using dye-mediated photoinactivation to remove the SGs from the tail-flip circuit. SG photoinactivation involves an initial broadening of the spike, leading to a long-duration, massively depolarized plateau. This is followed by loss of spike capability, a gradual reduction in the resting potential, and eventual total loss of electrical responsiveness. After bilateral photoinactivation of the SGs, a spike in one set of GFs, the medial giants (MGs), produces little if any effect in FFs in any ganglion. A spike in the other set, the lateral giants (LGs), produces an EPSP in FFs with a declining anterior-to-posterior segmental gradient in amplitude. These differences in LG and MG outputs, which are obscured in the intact circuit by the common MG/LG-SG-FF pathway, give clues to a probable early evolutionary form of the circuit. The LG-FF connection in anterior ganglia has a significant electrical component. However, it also has an apparent monosynaptic chemical component, as revealed by the response to saline containing cadmium ions, and to cooling the preparation. This is the first physiological evidence for chemical output from a crayfish GF.