Octopamine-like immunoreactive neurones in locust genital abdominal ganglia

Using a well characterized anti-serum, the distribution of octopamine-like immunoreactive neurones is described in the locust seventh abdominal (A7) and terminal ganglia (TG), which are associated with genital organs. Apart from 4 paired ventral somata occasionally observed in the TG, all labelled cells could be identified as efferent dorsal- and ventral unpaired median (DUM/VUM) neurones by virtue of the characteristic large size and position of their somata, projections of their primary neurites in DUM-cell tracts, and bifurcating axons which arise from dorsal T-junctions and enter peripheral nerves. For the examined ganglia our data indicate that the whole population of efferent DUM and VUM-cells, defined here as progeny of the segment specific unpaired median neuroblast with peripheral axons, are octopaminergic, and that equal numbers of these cells occur in both sexes: 8 in A7 and 11 in TG. Sex-specific differences are probably restricted to the axonal projections of 5 octopamine-like immunoreactive DUM-somata in A7, and 5 in TG, which in females project into their segment specific sternal nerves, but in males into the genital nerve of the TG. Numerous intersegmentally projecting octopamine-like immunoreactive fibres traverse both ganglia. The majority probably stem from previously described octopamine-like immunoreactive neurones in the thoracic and suboesophageal ganglia.     

Localization of octopaminergic neurones in insects

This paper reviews data on the localization of octopaminergic neurones revealed by immunocytochemistry in insects, primarily the locusts Schistocerca gregaria and Locusta migratoria, cricket Gryllus bimaculatus, and cockroach Periplaneta americana. Supporting evidence for their octopaminergic nature is mentioned where available. In orthopteran ventral ganglia, the major classes of octopamine-like immunoreactive (-LI) neurones include: (1) efferent dorsal and ventral unpaired median (DUM, VUM) neurones; (2) several intersegmentally projecting DUM interneurones in the suboesophageal ganglion; other DUM interneurones are probably GABAergic; (3) a pair of anterior median cells in the prothoracic ganglion; (4) a single pair of ventral cells in most thoracic and some other ganglia; these appear to be plurisegmentally projecting interneurones. Eight categories of octopamine-LI neurones occur in the orthopteran brain. The basic projections of three types are described here: one class project to the optic lobes to form wide field projections. Another type descends to cross into the tritocerebral commissure and may invade the contralateral brain hemisphere. A further class is the median neurosecretory cells with axons in the nervi corpori cardiaci I. Available data for the honey bee Apis mellifera and moth Manduca sexta indicate that the octopamine-LI cell types found in orthopterans also occur in holometabolous insects. Immunocytochemical evidence suggests that some octopaminergic DUM cells contain an FMRFamide-related peptide and the amino acid taurine as putative cotransmitters.     

Dendritic projections of different types of octopaminergic unpaired median neurons in the locust metathoracic ganglion

Octopaminergic dorsal unpaired median (DUM) neurons of locust thoracic ganglia are important components of motor networks and are divided into various sub-populations. We have examined individually stained metathoracic DUM neurons, their dendritic projection patterns, and their relationship to specific architectural features of the metathoracic ganglion, such as longitudinal tracts, transverse commissures, and well-defined sensory neuropils. The detailed branching patterns of individually characterized DUM neurons of various types were analyzed in vibratome sections in which architectural features were revealed by using antibodies against tubulin and synapsin. Whereas DUM3,4,5 and DUM5 neurons (the group innervating leg and "non-wing-power" muscles) had many ventral and dorsal branches, DUM1 and DUM3,4 neurons (innervating "wing-power" muscles) branched extensively only in dorsal areas. The structure of DUM3 neurons differed markedly from that of the other DUM neurons examined in that they sent branches into dorsal areas and had differently structured side branches that mostly extended laterally. The differences between the branching patterns of these neurons were quantified by using currently available new reconstruction algorithms. These structural differences between the various classes of DUM neurons corresponded to differences in their function and biophysical properties.     

Electromyographic characterization of walking behavior initiated spontaneously in crayfish

Crayfish initiate walking behavior not only reflexively in response to external stimuli but also spontaneously in the absence of any specific stimulus. In order to analyze the initiation mechanism underlying these different types of walking, we made simultaneous electromyographic (EMG) recordings from thoracic legs when animals initiated walking, either reflexively or spontaneously, and video recorded their movements synchronously with the EMG recording. Two different stimuli, mechanical and chemical, were used to reflexively induce walking. A non-rhythmic, sustained activation of leg muscles was found to precede the behavioral initiation of either type of walking. The duration of this non-rhythmic muscle activation was significantly longer in the spontaneously initiated walking than in the mechanical stimulus-evoked walking, although no difference was observed between the spontaneous and chemical stimulus-evoked walking. EMG recordings from all eight legs revealed that their non-rhythmic muscle activation occurred almost simultaneously prior to initiation of rhythmical stepping movements. When an animal was suspended without a leg substratum, the timing of muscle activation was more variable among the legs than in the free condition on the substratum. When the circumesophageal commissures were both severed to eliminate signals descending from the brain to the thoracic ganglia, the bilaterally coordinated rhythmic burst activity was not observed in the walking legs. These findings suggest that the spontaneous initiation of walking behavior requires sensory feedback signals from leg proprioceptors, subserved by a different descending activation mechanism from that for stimulus-driven initiation of walking.     

A mathematical modeling study of inter-segmental coordination during stick insect walking

The biomechanical conditions for walking in the stick insect require a modeling approach that is based on the control of pairs of antagonistic motoneuron (MN) pools for each leg joint by independent central pattern generators (CPGs). Each CPG controls a pair of antagonistic MN pools. Furthermore, specific sensory feedback signals play an important role in the control of single leg movement and in the generation of inter-leg coordination or the interplay between both tasks. Currently, however, no mathematical model exists that provides a theoretical approach to understanding the generation of coordinated locomotion in such a multi-legged locomotor system. In the present study, I created such a theoretical model for the stick insect walking system, which describes the MN activity of a single forward stepping middle leg and helps to explain the neuronal mechanisms underlying coordinating information transfer between ipsilateral legs. In this model, CPGs that belong to the same leg, as well as those belonging to different legs, are connected by specific sensory feedback pathways that convey information about movements and forces generated during locomotion. The model emphasizes the importance of sensory feedback, which is used by the central nervous system to enhance weak excitatory and inhibitory synaptic connections from front to rear between the three thorax-coxa-joint CPGs. Thereby the sensory feedback activates caudal pattern generation networks and helps to coordinate leg movements by generating in-phase and out-of-phase thoracic MN activity.     

Properties of descending dorsal unpaired median (DUM) neurons of the locust suboesophageal ganglion

A group of six dorsal unpaired median (DUM) neurons of the suboesophageal ganglion (SOG) of locusts was studied with neuroanatomical and electrophysiological techniques. The neurons are located posteriorly in the SOG and have axons that descend into the ganglia of the ventral nerve cord, some as far as the terminal abdominal ganglion. Within thoracic ganglia the neurons have profuse dendritic ramifications in many neuropiles, including ventral sensory neuropiles. Based on their projection patterns three different morphological types of neurons can be distinguished. These neurons receive excitatory inputs through sensory pathways that ascend from the thoracic ganglia and are activated by limb movements. They may be involved in the modulation of synaptic transmission in thoracic ganglia.     

Responses of efferent octopaminergic thoracic unpaired median neurons in the locust to visual and mechanosensory signals

Insect thoracic ganglia contain efferent octopaminergic unpaired median neurons (UM neurons) located in the midline, projecting bilaterally and modulating neuromuscular transmission, muscle contraction kinetics, sensory sensitivity and muscle metabolism. In locusts, these neurons are located dorsally or ventrally (DUM- or VUM-neurons) and divided into functionally different sub-populations activated during different motor tasks. This study addresses the responsiveness of locust thoracic DUM neurons to various sensory stimuli. Two classes of sense organs, cuticular exteroreceptor mechanosensilla (tactile hairs and campaniform sensilla), and photoreceptors (compound eyes and ocelli) elicited excitatory reflex responses. Chordotonal organ joint receptors caused no responses. The tympanal organ (Müller's organ) elicited weak excitatory responses most likely via generally increased network activity due to increased arousal. Vibratory stimuli to the hind leg subgenual organ never elicited responses. Whereas DUM neurons innervating wing muscles are not very responsive to sensory stimulation, those innervating leg and other muscles are very responsive to stimulation of exteroreceptors and hardly responsive to stimulation of proprioceptors. After cutting both cervical connectives all mechanosensory excitation is lost, even for sensory inputs from the abdomen. This suggests that, in contrast to motor neurons, the sensory inputs to octopaminergic efferent neuromodulatory cells are pre-processed in the suboesophageal ganglion.     

The functional role of octopaminergic neurons in insect motor behavior

The role of efferent, octopaminergic dorsal unpaired median (DUM) neurons in insects is examined by recording from them during motor behaviour. This population of neuromodulatory neurons is divided into sub-populations which are specifically activated or inhibited during ongoing motor behavior. These neurons are always activated in parallel to the respective motor circuits, and in addition to their modulatory effects on synaptic transmission may also cause metabolic changes in their target tissues.     

Activation of DUM cell interneurons by ventral giant interneurons in the cockroach, Periplaneta americana

Dorsal unpaired median (DUM) cells in orthopteran insects are known to contain the neuromodulatory substance octopamine, and DUM cells with peripheral axons augment synaptic activity at neuromuscular junctions. One of the most studied systems in the cockroach is the giant interneuron (GI) system which controls the initial movements of a wind-mediated escape response. Our data demonstrate that DUM cells that are restricted to the central nervous system (DUM interneurons) receive inputs from ventral giant interneurons (vGIs) but not from dorsal giant interneurons (dGIs). In contrast, DUM cells that have peripheral axons consistently fail to be excited by any giant interneurons. The DUM interneurons are excited by vGIs on both sides of the CNS and, when the vGIs are excited in pairs, summation occurs. Wind fields that have been generated for two of the DUM interneurons are omnidirectional. These data, taken along with the known association of DUM cells with the neuromodulatory substance octopamine, suggest that the DUM interneurons may act to modulate central synapses.     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

The function of auditory neurons in cricket phonotaxis

Summary The activity of auditory receptor cells and prothoracic auditory neurons of the cricket,Gryllus bimaculatus, was recorded intracellularly while the animal walked on a sphere or while passive movement was imposed on a foreleg.During walking the responses to simulated calling song is altered since (i) the auditory sensory cells and interneurons discharged impulses in the absence of sound stimuli (Figs. 1, 3) and (ii) the number of action potentials in response to sound is reduced in interneurons (Figs. 2, 3).These two effects occurred in different phases of the leg movement during walking and therefore masked, suppressed or did not affect the responses to auditory stimuli (Figs. 3, 4). Hence there is a time window within which the calling song can be detected during walking (Fig. 5).The extra excitation of receptors and interneurons is probably produced by vibration of the tympanum because (i) the excitation occurred at the same time as the leg placement (Fig. 4), (ii) during walking on only middle and hindlegs, no extra action potentials were observed (Fig. 6), (iii) in certain phases of passive movements receptor cells and interneurons were excited as long as the ipsilateral ear was not blocked (Figs. 8, 9).Suppression of auditory responses seems to be peripheral as well as central in origin because (i) it occurred at particular phases during active and passive leg movements in receptor cells and interneurons (Figs. 1, 4, 9), (ii) it disappeared if the ear was blocked during passive leg movements (Fig. 9) and (iii) it persisted if the animal walked only on the middle and hind legs (Fig. 6).     

Rotational locomotion by the cockroach Blattella germanica

Locomotion on complex substrata can be expressed in a plane by two geometric components of body movement: linear locomotion and rotational locomotion. This study examined pure rotation by analysing the geometry of leg movements and stepping patterns during the courtship turns of male Blattella germanica. Strict rotation or translation by an insect requires that each side of the body cover equal distance with respect to the substrate. There are three mechanisms by which the legs can maintain this equality: frequency of stepping, magnitude of the leg arcs relative to the body and the degree to which legs flex and extend during locomotion. During the courtship behaviour of Blattella germanica selected males executed turns involving body rotation along with leg movements in which the legs on the outside of the turn swung through greater average arcs than those on the inside of the turn. This difference should have resulted in a translation component. However, legs on the inside of the turn compensated by flexion and extension movements which were greater than those of opposing legs. The net effect was that both sides of the body covered equal average ground. These cockroaches used a wide variety of stepping combinations to effect rotation. The frequency of these combinations was compared to an expected frequency distribution of stepping combinations and further to an expected frequency of these stepping combinations used for straight walking. These comparisons demonstrated a similarity between interleg coordination during straight walking and that during turning in place.     

Taurine-like immunoreactivity in octopaminergic neurones of the cockroach,Periplaneta americana (L.)

Taurine (2-aminoethanesulphonic acid) is reported to interact with the octopaminergic system. The distribution of taurine-like immunoreactivity (-LIR) in relation to octopamine-like immunoreactive dorsal unpaired median (DUM) neurones was investigated with the aim of revealing possible colocalization of these two neuromediators. The specificity of the anti-taurine serum used was demonstrated by dot blot immunoassay and by use of preabsorption controls. There was no crossreactivity with octopamine. The specificity of the octopamine antiserum employed has been described elsewhere. Taurine-LIR could be demonstrated in large dorso-median cells in the suboesophageal and the mesothoracic ganglion as well as in the abdominal ganglia. In addition taurine-LIR is distributed in numerous other regions of the ganglia. A comparison of the immunostaining for taurine and octopamine indicates that several of the taurine-like immunoreactive (-LI) neurones are probably members of the octopamine-immunoreactive DUM cell population. These taurine-LI neurones resemble octopamine-LI DUM cells in soma position and size as well as in the projections of their primary neurites. Colocalization of octopamine-LIR and taurine-LIR within the same neuronal element could be shown by alternate immunostaining of consecutive sections. It is probable that all octopamine-LI DUM neurones also exhibit taurine-LIR, and the possible physiological significance of this coexistence is discussed.     

The central connections and actions during walking of tibial campaniform sensilla in the locust

Strain acting on the exoskeleton of insects is monitored by campaniform sensilla. On the tibia of a mesothoracic leg of the locust (Schistocerca gregaria) there are three groups of campaniform sensilla on the proximo-dorsal surface. This study analyses the responses of the afferents from one group, their connections with central neurones and their actions during walking.The afferents of the campaniform sensilla make direct excitatory connections with flexor tibiae motor neurones. They also make direct connections with particular spiking local interneurones that make direct inhibitory output connections with the slow extensor tibiae motor neurone.During walking extension movements of the tibiae during stance produce longitudinal tensile forces on the dorsal tibia that peak during mid stance before returning to zero prior to swing. This decline in tension can activate the campaniform sensilla. In turn this would lead to an inhibition of the extensor tibiae motor neurone and an excitation of the flexor tibiae motor neurones. This, therefore, aids the transition from stance to swing. During turning movements, the tibia is flexed and the dorsal surface is put under compression. This can also activate some of campaniform sensilla whose effect on the flexor motor neurones will reinforce the flexion of the tibia.     

Prothoracic DUM neurons of the cricket Gryllus bimaculatus — responses to natural stimuli and activity in walking behavior

Summary Responses to sensory stimuli and spike activity uring walking were investigated in bilaterally symmetrical dorsal unpaired median (DUM) neurons of the cricket. Intracellular recordings within the prothoracic ganglion were made either in restrained animals or in stationary walking specimens whilst parameters of their intended locomotion were measured. Three types of DUM cells were distinguished morphologically and physiologically. DUMa neurons send axons through segmental nerves. They often generated spontaneously large action potentials with low frequencies. Most DUMa neurons showed multimodal sensitivity, preferentially to cereal wind puffs and 15 kHz sound. Mean latencies ranged from 25 to 349 ms. Their large intraindividual variability could be correlated with behavioral modes during walking. Generally, the spike frequency increased with increased forward speed, while it was not related to turning. DUMb neurons projected either through the anterior or posterior connectives, but seemed physiologically similar to DUMa neurons. DUMc neurons were H-shaped with axons in both pairs of connectives. No external stimulus led to discrete spikes, but the regular spontaneous activity was modulated following cereal wind puffs to a restrained animal. During wind evoked escape the spike activity of another DUMc cell was modulated in phase with the rhythmic running behavior. The possibly different functions of DUMa and DUMc neurons during walking are discussed.     

Interaction between descending input and thoracic reflexes for joint coordination in cockroach. II Comparative studies on tethered turning and searching

Tethered cockroaches turn from unilateral antennal contact using asymmetrical movements of mesothoracic (T2) legs (Mu and Ritzmannin J Comp Physiol A 191:1037–1054, 2005). During the turn, the leg on the inside of the turn (the inside T2 leg) has distinctly different motor patterns from those in straight walking. The transformation from walking to inside leg turning could be triggered by descending commands that alter a few critical reflexes that start a cascade of physical changes in leg movement or posture, leading to further alterations. This hypothesis has two implications: First, the descending activities must be able to influence thoracic reflexes. Second, one should be able to initiate the turning motor pattern in the absence of descending signals by mimicking a point farther down in the reflex cascade. We addressed the first implication in the companion paper. To examine the second implication, we compared kinematics and motor activities of the T2 leg during searching with that of inside leg turning. The reaching movements made during searching were found to be similar to the movements made by the inside leg during turning. Moreover, even after disconnecting the brain from the thoracic ganglia the reaching movements were similar. This observation is consistent with the second implication from the hypothesis.     

Dominance of local sensory signals over inter-segmental effects in a motor system: experiments

Legged locomotion requires that information local to one leg, and inter-segmental signals coming from the other legs are processed appropriately to establish a coordinated walking pattern. However, very little is known about the relative importance of local and inter-segmental signals when they converge upon the central pattern generators (CPGs) of different leg joints. We investigated this question on the CPG of the middle leg coxa?Ctrochanter (CTr)-joint of the stick insect which is responsible for lifting and lowering the leg. We used a semi-intact preparation with an intact front leg stepping on a treadmill, and simultaneously stimulated load sensors of the middle leg. We found that middle leg load signals induce bursts in the middle leg depressor motoneurons (MNs). The same local load signals could also elicit rhythmic activity in the CPG of the middle leg CTr-joint when the stimulation of middle leg load sensors coincided with front leg stepping. However, the influence of front leg stepping was generally weak such that front leg stepping alone was only rarely accompanied by switching between middle leg levator and depressor MN activity. We therefore conclude that the impact of the local sensory signals on the levator?Cdepressor motor system is stronger than the inter-segmental influence through front leg stepping.     

昆虫中枢DUM神经元受体和离子通道电生理学研究进展

背侧不成对中间神经元(DUM)是一类位于多种昆虫腹神经索神经节背侧的神经元,能自发产生内源性超射动作电位。在DUM神经元细胞膜表达多种受体和离子通道,且电生理特性有别于哺乳动物中枢神经元膜上同种类型的受体和离子通道。目前已证实其细胞膜上表达K+通道、电压依赖的Na+通道、Ca2+敏感的Cl-通道、Ca2+通道、氯离子通道、乙酰胆碱受体、谷氨酸受体等多种离子通道和受体。近年来因膜片钳(patch-clamp)技术进展和对受体和离子通道研究的深入,该类神经细胞已用于杀虫剂选择性神经毒性研究和杀虫剂离体筛选。     

Descending interneurones from the brain and suboesophageal ganglia and their role in the control of locust behaviour

Lesion and stimulation experiments suggest that the suboesophageal ganglion (SOG) plays a special role in the control of insect behaviour: in bilateral coordination and by maintaining ongoing motor activity. Anatomical observations indicate that there are descending interneurones (DINs) originating in the SOG in addition to those from the brain. An SOG preparation for sampling both types of DIN intracellularly in walking locusts is described. Forty-three units showing activity changes during leg movements and walking were recorded. Using dye injection six were shown to be through-running axons; one was an SOG ascending interneurone; and eight were SOG DINs, 7 contralateral, one ipsilateral. All fired before or during movements and received various sensory inputs. Many gave complex responses to different modalities, several showing directional preferences. Some SOG neurones showed spontaneous changes in activity; activity outlasting movements; or responses to passive as well as active movements. These preliminary results suggest neuronal substrates for the special functions of the SOG in behaviour. They also indicate that DINs, rather than being simple relays, are part of a dynamic network which includes the motor centres. Regulation of complex and subtle aspects of behaviour may be achieved by dynamic and sequential patterns of activity in groups of DINs, some of which may be multifunctional.     

Reflex modulation in locusts walking on a treadwheel intracellular recordings from motoneurons

Summary In locusts (Locusta migratoria) walking on a treadwheel, afferents of tarsal hair sensilla were stimulated via chronically implanted hook electrodes (Fig. 1). Stimuli applied to the middle leg tarsus elicited avoidance reflexes (Fig. 2). In quiescent animals, the leg was lifted off the ground and the femur adducted. In walking locusts, the response was phase-dependent. During the stance phase, no reaction was observed except occasional, premature triggering of swing movements; stimuli applied near the end of the swing phase were able to elicit an additional, short leg protraction.Central nervous correlates of phase-dependent reflex modulation were observed by recording intracellularly from motoneuron somata in walking animals. As a rule, motoneurons recruited during the swing phase showed excitatory stimulus-related responses around the end of the swing movement, correlated to the triggering of additional leg protractions (Figs. 3, 4, 5). Motoneurons active during the stance phase were often inhibited by tarsal stimulation, some showed only weak responses (Figs. 8, 9, 10). Common inhibitory motoneuron 1 was excited by tarsal stimulation during all phases of the leg movement (Figs. 6, 7). In one type of flexor tibiae motoneuron, a complex response pattern was observed, involving the inversion of stimulus-related synaptic potentials from excitatory, recorded during rest, to inhibitory, observed during long-lasting stance phases (Figs. 11, 12).The results demonstrate how reflex modulation is represented on the level of synaptic input to motoneurons. They further suggest independent gain control in parallel, antagonistic pathways converging onto the same motoneuron as a mechanism for reflex reversal during locomotion.Abbreviations CI ₁ common inhibitory motoneuron (1) - EMG electromyogram - Feti fast extensor muscle of the tibia