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.

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.

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.

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.

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.

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

Motor programme switching in the crayfish swimmeret system.

Intracellular and extracellular recordings have been made from neurones of the swimmeret system in the semi-isolated abdominal ganglion of the crayfish during rhythmic activity. Extracellular recordings commonly reveal a motor programme (MP1) consisting of low-amplitude symmetrical power and return stroke activity with phase-constant posterior-to-anterior intersegmental coordination. Occasionally a different motor programme (MP2) is expressed. MP2 has higher amplitude episodic activity, with return stroke duration greater than power stroke, and with latency-constant anterior-to-posterior or near synchronous intersegmental coordination. Preparations may switch spontaneously between the two motor programmes. Intracellular recordings show that interneurones whose membrane potentials oscillate during MP1 and which can reset its rhythm usually cease to oscillate during MP2. During production of MP1, current injected into any one of a small number of interneurones can induce MP2. The polarity of current required is usually such as to drive the membrane potential towards the level normally associated with return stroke during MP1. During MP1 many motor neurones receive synaptic input with approximately sinusoidal waveform. During MP2 they may receive an episodic input with approximately sawtooth waveform, and/or input consisting of large, unitary EPSPs. The unitary EPSPs drive a 'bursty' mode of MP2 activity that is sometimes seen. The bursts of unitary EPSPs in MP2 appear to derive from a different source to that of the sinusoidal input in MP1. These sources are probably caudally-conducting through-interneurones and non-spiking local interneurones respectively. Thus experimental perturbation of a single neurone can induce a motor programme switch such as to change the activity of some hundreds of neurones in at least three ganglia. Neurones with this property would be convenient targets for controlling influences in the intact animal.

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.

Ultrastructure of the phasic stretch receptor in the crayfish abdominal nerve cord.

The ultrastructure of the abdominal ganglionic cord stretch receptor of the crayfish Pacifastacus leniusculus is described. This bilaterally-paired, segmentally-repeating phasic receptor monitors stretch applied to the central nervous system itself. It consists of a connective tissue mass closely applied to the medial margin of each medial giant fibre, into which ramifies a collection of specialized terminal dendrites originating from branches (primary dendrites) of a single axon. The connective tissue consists of an electron-opaque matrix in which are embedded many short, electron-lucent, tubular structures whose lumens are continuous with the matrix. Some filamentous material penetrates the connective tissue from its boundaries, and glial cells are present. The primary dendrites are irregular in size and orientation, and contain many microtubules and much filamentous material. The terminal dendrites are of consistent diameter and longitudinal orientation, containing very regularly-spaced microtubules with no microfilaments. The terminal dendrites contain a well-defined cytoskeletal 'tube' or lamina 6 nm thick, evenly spaced about 25 nm below the plasma membrane and connected to it by filamentous material 5 nm in diameter, which is deposited in rings or helices. This lamina arises just at the point where the primary dendrites gave rise to the terminal dendrites. Its function is not known, but it shows some similarities to the subaxolemmal lamina found in some regions of spike initiation.

The locust jump. I. The motor programme.

A motor programme is described for defensive kicking in the locust which is also probably the programme for jumping. The method of analysis has been to make intracellular recordings from the somata of identified motornuerones which control the metathoracic tibiae while defensive kicks are made in response to tactile stimuli. Three stages are recognized in the programme. (1) Initial flexion of the tibiae results from the low spike threshold of tibial flexor motorneurones to tactile stimulation of the body. (2) Co-contraction of flexor and extensor muscles followa in which flexor and extensor excitor motoneurones spike at high frequency for 300-600 ms. the tibia flexed while the extensor muscle develops tension isometrically to the level required for a kick or jump. (3) Trigger activity terminates the co-contraction by inhibiting the flexor excitor motorneurones and simultaneously exciting the flexor inhibitors. This causes relaxation of the flexor muscle and allows the tibiae to extend. If the trigger activity does not occur, the jump or kick is aborted, and the tibiae remain flexed.

The locust jump. II. Neural circuits of the motor programme.

1. Neural circuits which co-ordinate the motorneurones of the meta-thoracic tibiae of the locust in jumping and kicking have been investigated. 2. The fast extensor motorneurone is reflexly excited by the subgenual organ, by a network of cuticle strain receptors, and by Brunner's organ. The subgenual organ and the cuticle strain receptors are excited by tension in the extensor muscle and mediate a positive feedback which could help to produce the burst of fast extensor spikes which precedes a jump or kick. Brunner's organ is stimulated by pressure from the flexed tibia, and will be excited by the initial flexion and throughout the co-contraction phase of a kick. 3. A central excitatory connexion from the fast extensor to the slow extensor ensures that extensor muscle tension is as great as possible early in the co-contraction phase of a kick. 4. A central excitatory connexion from the fast extensor to flexor motorneurones is confirmed. This ensures that flexor muscle tension is great enough to keep the tibia flexed when the extensor muscle tension starts to develop before a jump or kick. 5. Reflex excitation of flexor motorneurones occurs in response to an extensor muscle twitch when the tibia is flexed. This helps to maintain the flexor connexion. 6. A receptor, the 'lump receptor', which is stimulated by flexor muscle tension only when the tibia is flexed, can inhibit the flexor motorneurones and may activate the trigger system which allows the extension of the tibia in a jump or kick. 7. Recptors in the suspensory ligaments of the joint inhibit the fast extensor when the tibia extends.

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.

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.

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.