Central nervous sensitization and dishabituation of reflex action in an insect by the neuromodulator octopamine

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.     

The structure and function of serially homologous leg motor neurons in the locust. II. Physiology

Simultaneous intracellular recordings were made from pairs of motor neurons in the pro- or mesothoracic ganglion of the locust. Though central connections were sought between pairs of motor neurons, none were found. This is in sharp contrast to the findings that flexor and extensor tibiae neurons in the metathoracic ganglion make certain connections between themselves (Hoyle and Burrows, 1973; Heitler and Burrows, 1977a). As the previously mentioned authors believed that the metathoracic flexor-extensor connections were used as part of the motor program for jumping and kicking, the present results strongly support their hypothesis. Common PSPs have been found in a variety of pairs of motor neurons. Of note are common PSPs of the same sign to antagonists. Different innervation patterns have been found for the flexor and extensor muscles. It is proposed that serially homologous motor neurons serving similar functions are, to a first approximation, similar in the locust. Serially homologous motor neurons serving different functions will, in most cases, have altered structures and/or functions.     

Organization of motor neurons to a multiply innervated insect muscle

Nine excitatory motor neurons have been identified as innervating the locust metathoracic flexor tibiae. The anatomical organization of the flexor motor neurons within the ganglion was examined with both light and electron microscopy. Flexor motor neurons were physiologically identified prior to intracellular staining with Procion or cobalt. Some of the cobalt-stained neurons were then silver intensified. The reliability of soma location and variability of neurite branching were examined. While the position of a soma could vary within its cluster by up to one radius, the anterior, posterior, and lateral soma clusters bore a consistent relationship to each other. The density of neurite branching varied greatly for any particular flexor. The ultrastructure of the tract containing the flexor neurites revealed the individual neurites to be glial wrapped, while the tract itself was isolated from the neuropil by additional glia. The hypothesis that subsets of the flexor motor neuron pool are recruited for different behaviors is discussed in light of the last two findings.     

The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy

Twenty-one prothoracic and 17 mesothoracic motor neurons innervating leg muscles have been identified physiologically and subsequently injected with dye from a microelectrode. A tract containing the primary neurites of motor neurons innervating the retractor unquis, levator and depressor tarsus, flexor tibiae, and reductor femora is described. All motor neurons studied have regions in which their dendritic branches overlap with those of other leg motor neurons. Identified, serially homologous motor neurons in the three thoracic ganglia were found to have: (1) cell bodies at similar locations and morphologically similar primary neurites (e.g., flexor tibiae motor neurons), (2) cell bodies at different locations in each ganglion and morphologically different primary neurites in each ganglion (e.g., fast retractor unguis motor neurons), or (3) cell bodies at similar locations and morphologically similar primary neurites but with a functional switch in one ganglion relative to the function of the neurons in the other two ganglia. As an example of the latter, the morphology of the metathoracic slow extensor tibiae (SETi) motor neurons was similar to that of pro- and mesothoracic fast extensor tibiae (FETi) motor neurons. Similarly the metathoracic FETi bears a striking resemblance to the pro- and the mesothoracic SETi. It is proposed that in the metathoracic ganglion the two extensor tibiae motor neurons have switched functions while retaining similar morphologies relative to the structure and function of their pro- and mesothoracic serial homologues.     

Octopaminergic modulation of locust motor neurones

The effect of octopamine on the fast extensor and the flexor tibiae motor neurones in the locust (Schistocerca gregaria) metathoracic ganglion, and also on synaptic transmission from the fast extensor to the flexor motor neurones, was examined. Bath application or ionophoresis of octopamine depolarized and increased the excitability of the flexor tibiae motor neurones. 1 mM octopamine reduced the amplitude of the fast extensor-evoked EPSP in the slow but not the fast flexor motor neurones, whereas 10 mM octopamine could reduce the EPSP amplitude in both. Octopamine broadened the fast extensor action potential and reduced the amplitude of the afterhyperpolarization, the modulation requiring feedback resulting from movement of the tibia. Octopamine also increased the frequency of synaptic inputs onto the tibial motor neurones, and could cause rhythmic activity in the flexor motor neurones, and reciprocal activity in flexor and extensor motor neurones. Octopamine also increased the frequency of spontaneous spiking in the octopaminergic dorsal unpaired median neurones. Repetitive stimulation of unidentified dorsal unpaired median neurones could mimic some of the effects of octopamine. However, no synaptic connections were found between dorsal unpaired median neurones and the tibial motor neurones. The diverse effects of octopamine support its role in mediating arousal.     

昆虫神经生物学研究技术:细胞内记录

细胞内记录是昆虫神经生物学研究中的常用技术。它用来获得神经元兴奋和抑制过程及神经脉冲产生机制的信息。该技术的特点是把一根微电极的顶尖插入到神经细胞内进行电生理记录 ,这根电极还能用于向膜内输入电流。作者以对蝗虫Schistocercagregaria后胸神经节内的 2个运动神经元的活性记录为例介绍了这一技术     

Structural comparison of a homologous neuron in gryllid and acridid insects

The fast extensor tibiae (FETi) motor neuron is responsible for exciting the extensor tibiae muscle to produce most of the force for jumping in acridids. Because of its relatively large size and crucial role in jumping, FETi has been studied in an ever-increasing number of orthopteran species. Here we describe the structure of the metathoracic FETi neuron in six species of acridids and in two species of gryllids. The morphology of FETi within the respective groups is essentially equivalent, but marked differences are apparent between acridid and gryllid FETis. There are similarities in the size and location of the cell body and the course of the neurite through the ganglion. Differences are found in the number of large branches, density of branching, and the volume of neuropil receiving branches. We propose that the gryllid FETi is an intermediate form between slow extensor tibiae motor neurons involved in walking and acridid fast extensor tibiae motor neurons specialized for jumping.     

Effects of temperan a central synapse between identified motor neurons in the locust

Summary Changing the temperature from 10–40 °C modifies the transmission at an established monosynaptic connection between the fast extensor tibiae (FETi) and flexor tibiae motor neurons in the metathoracic ganglion of the locustSchistocerca gregaria (Forskål). Striking changes occur to the shape of the spikes, to membrane resistance, to the synaptic delay, and to the evoked synaptic potentials.In the presynaptic FETi motor neuron, raising the temperature reduces the amplitude of an antidromic spike recorded in the soma by a factor of 10 (40 mV to 4 mV), reduces the time taken to reach peak amplitude by 5 (3.5 to 0.7 ms) and decreases the duration at half maximum amplitude by 0.5. The conduction velocity of the spike in the axon is increased by 50% from 10 °C to 40 °C. Orthodromic spikes are affected by temperature in a similar way to the antidromic spikes.The membrane resistance of both pre- and postsynaptic motor neurons falls as the temperature is raised. The membrane resistance of FETi falls by a factor of 4 (about 4 M at 10 °C to 1 M at 40 °C). A contributory component to this fall could be the increase in the frequency of synaptic potentials generated as a result of inputs from other neurons. No temperature dependence could be demonstrated on the voltage threshold relative to resting potential for evoking orthodromic spikes, but because the resistance changes, the current needed to achieve this voltage must be increased at higher temperatures.The latency measured from the peak of the spike in the soma of FETi to the start of the EPSP in the soma of a flexor motor neuron decreases by a factor of 20 (10 ms at 10 °C to 0.5 ms at 40 °C).In a postsynaptic flexor tibiae motor neuron, the amplitude of the evoked synaptic potential increases by a factor of 3.4 (5 mV to 17 mV), its duration at half maximum amplitude decreases by 3 (7 ms at 12 °C to 2.3 ms at 32 °C) and its rate of rise increases by 3. An increased likelihood that spikes will occur in the flexor contributes to the enhanced amplitude of the compound EPSP at temperatures above 20 °C.Abbreviation FETi fast extensor tibiae motor neuron     

Local innervation patterns of the metathoracic flexor and extensor tibiae motor neurons in the cricket Gryllus bimaculatus

To elucidate neural mechanisms underlying walking and jumping in insects, motor neurons supplying femoral muscles have been identified mainly in locusts and katydids, but not in crickets. In this study, the motor innervation patterns of the metathoracic flexor and extensor tibiae muscles in the cricket, Gryllus bimaculatus were investigated by differential back-fills and nerve recordings. Whereas the extensor tibiae muscle has an innervation pattern similar to that of other orthopterans, the flexor has an innervation unique to this species. The main body of the flexor muscle is divided into the proximal, middle and distal regions, which receive morphologically unique terminations from almost non-overlapping sets of motor neurons. The proximal region is innervated by about 12 moderate-sized excitatory motor neurons and two inhibitory neurons while the middle and distal regions are innervated by three and four large excitatory motor neurons, respectively. The most-distally located accessory flexor muscle, inserting on a common flexor apodeme with the main muscle, is innervated by at least four small excitatory (slow-type) and two common inhibitory motor neurons. The two excitatory and two inhibitory motor neurons that innervate the accessory flexor muscle also innervate the proximal bundles of the main flexor muscle. This suggests that the most proximal and distal parts of the flexor muscle participate synergistically in fine motor control while the rest participates in powerful drive of tibial flexion movement.     

Peptidergic activation of locust dorsal unpaired median neurons: Depolarization induced by locustatachykinins may be mediated by cyclic AMP

Four tachykinin-related peptides, locustatachykinin 1–4 (LomTK 1–4) are distributed in interneurons throughout the central nervous system of the locust Locusta migratoria and may have important roles as neurotransmitters or neuromodulators. In search of the central actions of LomTKs, we analyzed the response of the efferent dorsal unpaired median (DUM) neurons in the locust metathoracic ganglion. Immunocytochemistry, using an antiserum against LomTK 1, combined with intracellular filling of efferent DUM neurons with Lucifer yellow, revealed that LomTK-immunoreactive fibers are in close proximity to dendritic arborizations of the DUM neurons. Hence, LomTKs may act on DUM neurons by releasing locally in the metathoracic ganglion. Intracellular recordings were made from somata of DUM neurons, and LomTKs were either bath-applied to an isolated metathoracic ganglion or pressure-ejected onto the DUM neuron soma. LomTK 1 at concentrations of 0.1 mM–0.1 μM caused a relatively slow, reversible depolarization with a subsequent increase in the frequency of action potential firing. Amino-terminally truncated forms of LomTK 1 were applied to DUM neurons. The heptapeptide [3–9]-LomTK 1 had a substantially reduced activity, and bioactivity was lost after further truncation. Spantide 1, an antagonist of mammalian tachykinin receptors, reversibly blocked the effect of LomTK 1. The effect of LomTK 1 was clearly reduced in the presence of GDP-β-S, a stable analog of GDP that inactivates G-proteins. The action of LomTK 1 was potentiated by both IBMX and theophylline, two cyclic AMP (cAMP) phosphodiesterase inhibitors. The action of LomTK 1 was mimicked by pressure-ejecting 8-bromo-cAMP, a membrane permeable analog of cAMP, and by forskolin, an adenylate cyclase activator. Furthermore, cAMPS, a blocker of protein kinase A activity, reduced the effect of LomTK 1. These findings indicate that cAMP is involved in mediating DUM neuron depolariztion. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 297–315, 1997     

Motor patterns during kicking movements in the locust

Locusts (Schistocerca gregaria) use a distinctive motor pattern to extend the tibia of a hind leg rapidly in a kick. The necessary force is generated by an almost isometric contraction of the extensor tibiae muscle restrained by the co-contraction of the flexor tibiae (co-contraction phase) and aided by the mechanics of the femoro-tibial joint. The stored energy is delivered suddenly when the flexor muscle is inhibited. This paper analyses the activity of motor neurons to the major hind leg muscles during kicking, and relates it to tibial movements and the resultant forces.During the co-contraction phase flexor tibiae motor neurons are driven by apparently common sources of synaptic inputs to depolarized plateaus at which they spike. The two excitatory extensor motor neurons are also depolarized by similar patterns of synaptic inputs, but with the slow producing more spikes at higher frequencies than the fast. Trochanteral depressors spike at high frequency, the single levator tarsi at low frequency, and common inhibitors 2 and 3 spike sporadically. Trochanteral levators, depressor tarsi, and a retractor unguis motor neuron are hyperpolarized.Before the tibia extends all flexor motor neurons are hyperpolarized simultaneously, two common inhibitors, and the levator trochanter and depressor tarsi motor neurons are depolarized. Later, but still before the tibial movement starts, the extensor tibiae and levator tarsi motor neurons are hyperpolarized. After the movement has started, the extensor motor neurons are hyperpolarized further and the depressor trochanteris motor neurons are also hyperpolarized, indicating a contribution of both central and sensory feedback pathways.Variations in the duration of the co-contraction of almost twenty-fold, and in the number of spikes in the fast extensor tibiae motor neuron from 2–50 produce a spectrum of tibial extensions ranging from slow and weak, to rapid and powerful. Flexibility in the networks producing the motor pattern therefore results in a range of movements suited to the fluctuating requirements of the animal.     

Neuronal connections between central and enteric nervous system in the locust, Locusta migratoria

The number and location of neurons, in the central nervous system, that project into the frontal connective was studied in the locust by using retrograde neurobiotin staining. Staining one frontal connective revealed some 70 neurons in the brain. Most of these were located within both tritocerebral lobes. Additional groups of neurons were located within the deutocerebrum and protocerebrum. Some 60 neurons were labelled in the suboesophageal ganglion. These formed nine discernable populations. In addition, two neurons were located in the prothoracic ganglion and two neurons in the first abdominal neuromere of the metathoracic ganglion. Thus, some 250 neurons located within the head ganglia, and even neurons in thoracic ganglia, project into the ganglia of the enteric nervous system. This indicates that the coordination between the central and enteric ganglia is much more complex than previously thought. With the exception of some previously described dorsal unpaired median neurons and a few motor neurons in the head ganglia, the identity and function of most of these neurons is as yet unknown. Possible functions of the neurons in the thoracic ganglia are discussed.     

Generation of specific behaviors in a locust by local release into neuropil of the natural neuromodulator octopamine

The natural insect neuromodulator octopamine (OCT) was released iontophoretically into regions of neuropil in locust metathoracic ganglia. A narrowly-defined site was found on one side of the ganglion at which release caused a prolonged bout of repetitive flex-extend-flex movements of the tibia on the injected side, at a frequency of from 2-3.5 Hz. When a bout had terminated, repetition of the OCT release caused an extremely similar bout to occur, and again with further treatments, indefinitely. OCT iontophoresis at the equivalent site on the contralateral side caused the contralateral flexor to make stepping movements. Two sites were found, in each half of the ganglion, at which similar OCT release evoked a bout of flight motor activity at 10 Hz. The flight bout involved both sides synchronously and nearly equally, except for a slightly greater motor output on the injected side. Evoked bouts lasted from 20 sec to 25 min depending on the preparation and amount of OCT released. At a site in the 6th abdominal ganglion of mature female locusts OCT release suppressed ongoing rhythmic oviposition digging evoked by severing the ventral nerve cord. A number of previously undescribed DUM neurons was encountered and their dendritic patterns, which are distinctive, determined following dye injection. A hypothesis, termed the Orchestration Hypothesis is presented, which considers how modulator neurons such as locust octopaminergic neurons, might be involved in the generation of specific behaviors.     

Presynaptic contributions to response shape in an auditory neuron of the grasshopper

Neuron 714 is morphologically one of the most prominent neurons in the central auditory pathway of the grasshopper with arborizations extending from the abdominal neuromeres of the metathoracic ganglion to the brain. The aim of this study is to explore auditory information flow involving neuron 714 at the level of the ventral nerve cord. Paired intracellular recordings were made from neuron 714 in the mesothorax on the one hand, and from candidate presynaptic auditory neurons of the metathorax on the other. Electrical stimulation of the tympanal nerves provides an estimate of the synaptic distance between these interneurons and auditory afferents. Four, including neuron 714, are monosynaptically connected to afferents, the remainder disynaptically. Current-injection and spike-triggered averaging reveal that of nine neurons examined, seven make either monosynaptic, disynaptic or polysynaptic connections onto neuron 714. All connections are excitatory. Paired recordings show that response duration and response amplitude in synaptically linked cells vary according to the frequency of the stimulus. Measurements of the latency of the first excitatory post-synaptic potential evoked in neuron 714 by afferents and by metathoracic interneurons show how the synaptic drive from these sources shapes the auditory response of neuron 714. Accepted: 14 October 1998     

Locomotory activity in the extensor and flexor tibiae of the cockroach, Periplaneta americana

Electromyograms (EMGs) were recorded from the metathoracic extensor and flexor tibiae of cockroaches when the animals were: walking on a level surface, walking on a ball, or producing rhythmic leg movements while being restrained ventral surface upward. In the rapidly walking (> 2 steps/s) and restrained animals, there was reciprocity between EMGs from the extensor and flexor tibiae. In slowly walking (<-2 steps/s) animals there was a conspicuous overlap in the flexor and extensor EMGs. The overlap was due to an increase in duration in the activity of the flexor. In experiments in which distal portions of a limb were amputated, the overlap observed in slow walking was either reduced greatly or lost entirely. These results are in agreement with recent locomotory models which state that the motor output is produced by a central pattern generator but can be modified by peripheral sensory inputs.     

Lobula plate and ocellar interneurons converge onto a cluster of descending neurons leading to neck and leg motor neuropil in Calliphora erythrocephala

Summary In the fly, Calliphora erythrocephala, a cluster of three Y-shaped descending neurons (DNOVS 1–3) receives ocellar interneuron and vertical cell (VS4–9) terminals. Synaptic connections to one of them (DNOVS 1) are described. In addition, three types of small lobula plate vertical cell (sVS) and one type of contralateral horizontal neuron (Hc) terminate at DNOVS 1, as do two forms of ascending neurons derived from thoracic ganglia. A contralateral neuron, with terminals in the opposite lobula plate, arises at the DNOVS cluster and is thought to provide heterolateral interaction between the VS4–9 output of one side to the VS4–9 dendrites of the other. DNOVS 2 and 3 extend through pro-, meso-, and metathoracic ganglia, branching ipsilaterally within their tract and into the inner margin of leg motor neuropil of each ganglion. DNOVS 1 terminates as a stubby ending in the dorsal prothoracic ganglion onto the main dendritic trunks of neck muscle motor neurons. Convergence of VS and ocellar interneurons to DNOVS 1 comprises a second pathway from the visual system to the neck motor, the other being carried by motor neurons arising in the brain. Their significance for saccadic head movement and the stabilization of the retinal image 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.     

Glutamatergic transmission between antagonistic motor neurones in the locust

The pharmacology of the direct central connections between the fast extensor and flexor motor neurones of a locust (Schistocerca gregaria) hind leg was studied. A spike in the fast extensor produces an EPSP in the flexor motor neurones. Glutamate depolarized the flexor motor neurones when injected into the neuropil. Quisqualate, but not by kainate or NMDA, also depolarized the flexor motor neurones. The fast extensor was also depolarized by glutamate, and also by kainate, but not by quisqualate, AMPA or NMDA. The glutamate response in the flexor motor neurones and the EPSP evoked by a spike in FETi both had similar reversal potentials. The FETi-evoked EPSP was blocked by bath application of the glutamate antagonist glutamic acid diethyl ester. The responses of extrasynaptic somata receptors to glutamate were compared to the neuropil responses. Glutamate usually hyperpolarized the somata of FETi and the flexor motor neurones. The response of a flexor motor neurone to glutamate was abolished at potentials less negative than -90 mV. The results provide evidence for glutamate transmission at central synapses in the locust, and show that presumed synaptic receptors in the neuropil differ to the extrasynaptic soma response