Hans-Joachim Pflüger

The unpaired median neurons of insects.

Publisher Summary Unpaired median neurons have received much attention from insect neurobiologists because of their unique morphological, physiological, and ontogenetic characteristics. The large efferent unpaired median cells of the insect ventral nerve cord use the biogenic amine octopamine as their transmitter and/or modulatory substance, and these neurons project into the skeletal or visceral musculature where they modulate neuromuscular transmission. This chapter discusses the distribution of unpaired median cells, embryonic and postembryonic development of unpaired median neurons, and morphology of unpaired median neurons. The chapter examines that the release of octopamine depends on unpaired median neurons being activated and, equally important, deactivated in a behaviorally relevant fashion by presynaptic neuronal networks. As for other arthropod neurosecretory and neuromodulatory cells, there is no information about the neurons that form direct synaptic contacts with unpaired median neurons of insects. It is reasonable to assume that neurons activating modulatory and humoral systems are to be found in higher levels of integrative networks.

Positive feedback loops from proprioceptors involved in leg movements of the locust

Summary1.Two campaniform sensilla (CS) on the proximal tibia of a hindleg monitor strains set up when a locust prepares to kick, or when a resistance is met during locomotion. The connections made by these afferents with interneurones and leg motor neurones have been investigated and correlated with their role in locomotion.2.When flexor and extensor tibiae muscles cocontract before a kick afferents from both campaniform sensilla spike at frequencies up to 650 Hz. They do not spike when the tibia is extended actively or passively unless it encounters a resistance. The fast extensor tibiae motor neurone (FETi) then produces a sequence of spikes in a thrusting response with feedback from the CS afferents maintaining the excitation. Destroying the two campaniform sensilla abolishes the re-excitation of FETi.3.Mechanical stimulation of a single sensillum excites extensor and flexor tibiae motor neurones. The single afferent from either CS evokes EPSPs in the fast extensor motor neurone and in certain fast flexor tibiae motor neurones which follow each sensory spike with a central latency of 1.6 ms that suggests direct connections. The input from one receptor is powerful enough to evoke spikes in FETi. The slow extensor motor neurone does not receive a direct input, although it is excited and slow flexor tibiae motor neurones are unaffected.4.Some nonspiking interneurones receive direct connections from both afferents in parallel with the motor neurones. One of these interneurones excites the slow and fast extensor tibiae motor neurones probably by disinhibition. Hyperpolarization of this interneurone abolishes the excitatory effect of the CS on the slow extensor motor neurone and reduces the excitation of the fast. The disinhibitory pathway may involve a second nonspiking interneurone with direct inhibitory connections to both extensor motor neurones. Other nonspiking interneurones distribute the effects of the CS afferents to motor neurones of other joints.5.The branches of the afferents from the campaniform sensilla and those of the motor neurones and interneurones in which they evoke EPSPs project to the same regions of neuropil in the metathoracic ganglion.6.The pathways described will ensure that more force is generated by the extensor muscle when the tibia is extended against a resistance. The excitatory feedback to the extensor and flexor motor neurones will also contribute to their co-contraction when generating the force necessary for a kick.

The control of the rocking movements of the phasmidCarausius morosus Br.

Summary1.Recordings are made from the flexor tibiae muscle (electromyogram) and from the extensor tibiae nerve of freely moving stick insects.2.During rocking of intact animals the flexor and extensor tibiae burst in an alternating, rhythmical pattern (Fig. 5).3.The following sense organs are eliminated: The receptor tendon which is part of the transducer of the femur-tibia-control system is cut. The coxal and trochanteral hair fields, some of which control the coxa-trochanter joint are shaved. The campaniform sensillae which are thought to be involved in controlling the coxa-trochanter joint are destroyed. These operations, which are done in one, three or in all six legs of a stick insect, in general do not prevent the insect from rocking (Fig. 8). But the rocking periods are shorter than in intact animals. A rhythmical alternating bursting pattern can always be observed even during short rocking periods. The frequencies of the oscillations during the rocking periods are changed significantly by these operations. Intact animals rock with a mean frequency of 1.85 Hz. Stick insects with receptor tendons cut, hair fields shaved and campaniform sensillae destroyed in all six legs possess a mean rocking frequency of 3.6 Hz (Fig. 9).4.Changing the normal negative feedback of the femur-tibia-control system into a positive one can be done by transfering the attachment of the receptor tendon from a dorsal to a ventral position relative to the femur-tibia joint axis. This increases the duration of the flexor bursts.5.From the results it is concluded, that a central program underlies the rocking behaviour. The central oscillator can be influenced by sensory feedback. Its eigen frequency (natural frequency) must be high as it is damped by input from the afferences until the frequency observed lies in the range of the “resonance frequency” of the femur-tibia-control system. Perhaps also the control system of the coxa-trochanter joint possesses a resonance frequency within this range.

Distribution and activation of different types of octopaminergic DUM neurons in the locust

The first part of this study describes the distribution of all different types of octopaminergic, efferent dorsal unpaired median (DUM) neurons in the first two thoracic ganglia by immunocytochemistry, retrograde labeling, and intracellular staining. The prothoracic ganglion contains five different types of 10 DUM neurons. The mesothoracic ganglion has 21 octopaminergic somata in the DUM neuron cluster. Retrograde labeling and intracellular staining show that 19 of these 21 somata belong to five different types of efferent DUM neurons. In both ganglia, the number and the distribution of all types of DUM neurons are completely described. Differences in the distribution of efferent DUM neurons between the thoracic ganglia are discussed as functional segmental specializations.

DUM neurons in locust flight: a model system for amine-mediated peripheral adjustments to the requirements of a central motor program

Abstract In both vertebrates and invertebrates, multiple effects of biogenic amines on neuromuscular transmission, muscle contraction kinetics and metabolism have been described. Nevertheless, it is not yet known whether and how these different effects work in concert during the performance of a specific behavior. In the locust flight system, the biogenic amine octopamine is released as a neurohormone into the haemolymph, and also delivered directly onto specific target muscles by individually identified dorsal unpaired median neurons. Determining the connectivity of these neurons and their activation during behavior, we show for the first time that different types of dorsal unpaired median neurons are differentially connected to certain components of the flight circuitry. During flight, all types of pterothoracic dorsal unpaired median neurons innervating flight muscles receive inhibitory inputs from tegula proprioceptive afferents and from the central flight circuitry, whereas all other types of dorsal unpaired median neurons are excited by wind-sensitive pathways and by the central pattern generator. Considering the results of other studies which investigated metabolic effects of octopamine, we propose a model in which the differential activation of dorsal unpaired median neurons during flight may lead to an adequately controlled release or removal of octopamine to adjust metabolic processes to the requirements of a specific motor program.

A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods

BackgroundThe composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain - key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda - remain unclear.ResultsUsing immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords.ConclusionsOur results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.

Tyramine as an independent transmitter and a precursor of octopamine in the locust central nervous system: An immunocytochemical study

Octopamine and its precursor tyramine are biogenic amines that are found ubiquitously in insects, playing independent but opposite neuromodulatory roles in a wide spectrum of behaviors, ranging from locomotion and aggression to learning and memory. We used recently available antibodies to octopamine and tyramine to label the distribution of immunoreactive profiles in the brain and ventral nerve cord of the locust. In the brain and all ventral cord ganglia all known octopaminergic neurons were labeled with both the tyramine and octopamine antisera. In the brain the subesophageal ganglion and all fused abdominal ganglia we found somata that were only labeled by the tyramine antibody. Some prominent architectural features of the brain, like the protocerebral bridge, the central body, and associated neuropils, also contain intensely labeled tyramine‐immunoreactive fibers. In addition, tyraminergic fibers occur in all ganglia of the ventral cord. For known octopaminergic neurons of the thoracic ganglia, octopamine‐immunoreactivity was confined to the cell body and to the varicosities or boutons, whereas fiber processes always expressed tyramine‐immunoreactivity. The distribution of the tyramine and octopamine content within these neurons turned out to be dependent on how the animal was handled before fixation for immunocytochemistry. We conclude that tyramine is an independent transmitter in locusts, and that in octopaminergic neurons the ratio between octopamine and its precursor tyramine is highly dynamic. J. Comp. Neurol. 512:433–452, 2009.

Colocalization of octopamine and FMRFamide related peptide in identified heart projecting (DUM) neurones in the locust revealed by immunocytochemistry

Immunocytochemical techniques are employed to reveal colocalization of octopamine with FMRFamide related peptide in the locust ventral nervous system. In each unfused pregenital abdominal ganglia (A4-A6) there are 3 octopamine-like immunoreactive neurones. By combining intracellular Lucifer yellow staining with subsequent immunocytochemistry these are individually identified as the efferent dorsal unpaired median (DUM) neurones DUM-1 and DUM-2, which innervate abdominal tergal and respectively sternal skeletal muscles, and DUM heart-1, an FMRFamide-like immunoreactive neurone which projects to the heart and associated alary muscles. Colocalization of octopamine- and FMRFamide-like immunoreactivity in DUM heart-1 is verified by alternate staining of consecutive sections. With respect to locust ventral ganglia, this investigation shows that colocalization of octopamine with an FMRFamide related peptide is restricted to a single DUM cell occurring in each abdominal ganglion 2-7, which most likely corresponds to segmental homologues of DUM heart-1.

Selective neuronal staining in tardigrades and onychophorans provides insights into the evolution of segmental ganglia in panarthropods

BackgroundAlthough molecular analyses have contributed to a better resolution of the animal tree of life, the phylogenetic position of tardigrades (water bears) is still controversial, as they have been united alternatively with nematodes, arthropods, onychophorans (velvet worms), or onychophorans plus arthropods. Depending on the hypothesis favoured, segmental ganglia in tardigrades and arthropods might either have evolved independently, or they might well be homologous, suggesting that they were either lost in onychophorans or are a synapomorphy of tardigrades and arthropods. To evaluate these alternatives, we analysed the organisation of the nervous system in three tardigrade species using antisera directed against tyrosinated and acetylated tubulin, the amine transmitter serotonin, and the invertebrate neuropeptides FMRFamide, allatostatin and perisulfakinin. In addition, we performed retrograde staining of nerves in the onychophoran Euperipatoides rowelli in order to compare the serial locations of motor neurons within the nervous system relative to the appendages they serve in arthropods, tardigrades and onychophorans.ResultsContrary to a previous report from a Macrobiotus species, our immunocytochemical and electron microscopic data revealed contralateral fibres and bundles of neurites in each trunk ganglion of three tardigrade species, including Macrobiotus cf. harmsworthi, Paramacrobiotus richtersi and Hypsibius dujardini. Moreover, we identified additional, extra-ganglionic commissures in the interpedal regions bridging the paired longitudinal connectives. Within the ganglia we found serially repeated sets of serotonin- and RFamid-like immunoreactive neurons. Furthermore, our data show that the trunk ganglia of tardigrades, which include the somata of motor neurons, are shifted anteriorly with respect to each corresponding leg pair, whereas no such shift is evident in the arrangement of motor neurons in the onychophoran nerve cords.ConclusionsTaken together, these data reveal three major correspondences between the segmental ganglia of tardigrades and arthropods, including (i) contralateral projections and commissures in each ganglion, (ii) segmentally repeated sets of immunoreactive neurons, and (iii) an anteriorly shifted (parasegmental) position of ganglia. These correspondences support the homology of segmental ganglia in tardigrades and arthropods, suggesting that these structures were either lost in Onychophora or, alternatively, evolved in the tardigrade/arthropod lineage.

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.